Francesca Ruggieri

Nina Compagne

Kevin Antraygues

Maxime Eveque

Marion Flipo

Nicolas Willand
email: nicolas.willand@univ-lille.fr

Antibiotics with novel mode of action as new weapons to fight antimicrobial resistance

Keywords: Antimicrobial resistance -New drugs -Novel mode of action -Novel target Abbreviations: 3-MBA, 3-methoxybenzamide, ABSSSI, acute bacterial skin and skin structure infection, ACP, acyl carrier protein, ADME, absorption, distribution, metabolism, excretion, AE, adverse event, AMR, antimicrobial resistance, ATP, adenosine triphosphate, AUC, area under curve, BCS, biopharmaceutics classification system, CABP, community-acquired bacterial pneumonia, CDAD, Clostridioides difficile-associated diarrhea, CDI, 1,1'-carbonyldiimidazole / C. difficile infections, CFU, colony-forming unit, dGTP, deoxyguanosine triphosphate, DISARM, developing faecium, VRSA, vancomycin-resistant

Antimicrobial resistance (AMR) is a major public health issue, causing 5 million deaths per year. Without any action plan, AMR will be in a near future the leading cause of death ahead of cancer. AMR comes from the ability of bacteria to rapidly develop and share resistance mechanisms towards current antibiotics, rendering them less effective. To circumvent this issue and avoid the phenomenon of cross-resistance, new antibiotics acting on novel targets or with new modes of action are required. Today, the pipeline of potential new treatments with these characteristics includes promising compounds such as gepotidacin, zoliflodacin, ibezapolstat, MGB-BP-3, CRS-3123, afabicin and TXA-709, which are currently in clinical trials, and lefamulin, which has been recently approved by FDA and EMA. In this review, we report the chemical synthesis, mode of action, structure-activity relationships, in vitro and in vivo activities as well as clinical data of these eight small molecules listed above.

Introduction

Antimicrobial resistance (AMR) is one of the major public health problems of this century, with an estimated 10 million people killed annually by 2050 [START_REF] O'neill | Tackling drug-resistant infections globally: final report and recommendations[END_REF]. Recently, predictive statistical models have estimated 5 million AMR-related deaths in the year 2019 [START_REF] Murray | [END_REF]. This phenomenon is mainly due to the extensive misuse of antibiotics which leads to the selection of resistant bacteria [START_REF] Harbarth | Antimicrobial resistance: one world, one fight![END_REF][START_REF] Annunziato | Strategies to Overcome Antimicrobial Resistance (AMR) Making Use of Non-Essential Target Inhibitors: A Review[END_REF]. The resulting infections become more difficult to treat, causing serious illness, with the risk of spreading the disease and facing death.

Drug resistance in bacteria can be classified into four different mechanisms: (1) enzymatic inactivation of the drug, (2) export of the drug by efflux pumps, (3) limitation of drug uptake by changes in membrane permeability, and (4) modification of the drug target [START_REF] Annunziato | Strategies to Overcome Antimicrobial Resistance (AMR) Making Use of Non-Essential Target Inhibitors: A Review[END_REF]. Due to the lack of lipopolysaccharide in the outer membrane, Gram-positive bacteria are more likely to enzymatically inactivate the drug or modify its target rather than limit the uptake or express efflux pumps. This is not the case for Gram-negative bacteria, which can acquire resistance through all the mechanisms described above [START_REF] Reygaert | An overview of the antimicrobial resistance mechanisms of bacteria[END_REF].

Considering the different types of mechanisms that confer drug resistance, the alteration of the drug target is a key feature. Therefore, efforts have been made to develop drugs acting on a novel target and/or with a new mode of action (MOA), especially in academia or in small biotech companies [START_REF] Everett | Academic drug discovery: current status and prospects[END_REF][START_REF] Frearson | Drug discovery in academia: the third way?[END_REF]. Interestingly, the identification of novel drug targets may come with the discovery and development of first-in-class drugs [START_REF] Emmerich | Improving target assessment in biomedical research: the GOT-IT recommendations[END_REF].

In this review, we focus on new drugs in clinical development or recently approved, characterized by novel targets and/or MOA, that are of interest for the treatment of infections caused by multidrug-resistant bacteria (Figure 1 and Table 1). We have deliberately chosen not to discuss compounds at the pre-clinical stage. Currently, DNA topoisomerases are targeted by fluoroquinolones, a well-known class of antibiotics. However, more and more fluoroquinolone-resistant strains are being reported. This critical point urges the need to develop topoisomerase inhibitors with a distinct mode of action to avoid cross-resistance. Two of these antibiotics have reached clinical trials: gepotidacin and zoliflodacin.

Gepotidacin

Gepotidacin

Gepotidacin (GSK2140944) is a triazaacenaphtylene developed by GSK and belongs to the class of Novel Bacterial Topoisomerase Inhibitors (NBTI). This new antibiotic is currently being investigated in three phase 3 clinical trials.

Gepotidacin is derived from the analogue GSK299423 described by Bax et al. [START_REF] Bax | Type IIA topoisomerase inhibition by a new class of antibacterial agents[END_REF], which results from a medicinal chemistry program initiated after an unbiased antibacterial screening [START_REF] Coates | Quinoline derivatives as antibacterials[END_REF].

Chemical synthesis

The synthesis of gepotidacin has been described in two patents in 2008 and 2016 and comprises 11 steps (Figure 2) [START_REF] Davies | Tricyclic nitrogen containing compounds as antibacterial agents[END_REF][START_REF] Widdowson | Tricyclic nitrogen containing compounds for treating neisseria gonorrhoea infection[END_REF]. First, 2-chloro-6-methoxy-3-nitro-pyridine reacts with 2-amino-propane-1,3-diol through nucleophilic aromatic substitution (S N Ar). The resulting diol is then protected with 2,2-dimethoxypropane in presence of p-toluenesulfonic acid (PTSA) followed by the reduction of the nitro group with hydrogen and 10% Pd/C. The aniline thus formed is then alkylated with ethyl bromoacetate. Cyclization is performed in basic conditions using sodium hydride, followed by oxidation using manganese dioxide. The acetal is then cleaved and the released diol reacts with methanesulfonic anhydride to form the third cycle of the triazaacenaphtylene core. Substitution with Boc-amino-piperidine, followed by deprotection and subsequent purification by chiral chromatography affords the primary amine derivative, which can be condensed by reductive amination with the corresponding aldehyde to give the free base of gepotidacin. The mono-hydrochloride salt is obtained by reaction with one equivalent of HCl 1M in diethylether [START_REF] Barfoot | The design of efficient and selective routes to pyridyl analogues of 2,3-dihydro-1,4-benzodioxin-6-carbaldehyde[END_REF]. [START_REF] Davies | Tricyclic nitrogen containing compounds as antibacterial agents[END_REF][START_REF] Widdowson | Tricyclic nitrogen containing compounds for treating neisseria gonorrhoea infection[END_REF].

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Target and mechanism of action

Gepotidacin inhibits both DNA gyrase and topoisomerase IV. More specifically, gepotidacin binds to the GyrA subunit of DNA gyrase and to the ParC subunit of topoisomerase IV. With its novel chemical scaffold, gepotidacin can form a ternary complex with DNA and two GyrA or ParC subunits (Figure 3) [START_REF] Bax | Type IIA topoisomerase inhibition by a new class of antibacterial agents[END_REF][START_REF] Gibson | Mechanistic and Structural Basis for the Actions of the Antibacterial Gepotidacin against Staphylococcus aureus Gyrase[END_REF]. The structure of gepotidacin can be divided in three parts: the left-hand side (LHS), the linker and the right-hand side (RHS) (Figure 4). The polar tricyclic core of the LHS intercalates between DNA bases. Its position and structure can affect the spatial orientation of the linker and of the RHS. The linker possesses a basic amine able to interact with both GyrA/ParC subunits of DNA gyrase/topoisomerase IV. Lastly, the RHS binds to a hydrophobic pocket of the bacterial gyrase/topoisomerase IV [START_REF] Kokot | The Structural Features of Novel Bacterial Topoisomerase Inhibitors That Define Their Activity on Topoisomerase IV[END_REF].

With this new binding mode, gepotidacin shows potent antibacterial activity against a broad spectrum of Gram-positive and Gram-negative pathogenic bacteria, including clinical isolates with fluoroquinolone resistance mediated by DNA gyrase and topoisomerase IV mutations. Gepotidacin was also found active against different levofloxacin-resistant strains of S. aureus and S. pneumoniae, with MIC 90 of 0.5 µg/mL. This compound was also potent against levofloxacin-resistant Gram-negative bacteria such as E. coli with MIC 90 of 4 µg/mL [START_REF] Biedenbach | In Vitro Activity of Gepotidacin, a Novel Triazaacenaphthylene Bacterial Topoisomerase Inhibitor, against a Broad Spectrum of Bacterial Pathogens[END_REF][START_REF] Flamm | Gepotidacin (GSK2140944) In Vitro Activity against Gram-Positive and Gram-Negative Bacteria[END_REF].

In vivo experiments

The potency of gepotidacin against MRSA was demonstrated in a neutropenic murine lung infection model. Mice received different doses of gepotidacin, ranging from 1.56 to 400 mg/kg, administered every 6 h and starting 3 h post inoculation with six different clinical isolates of MRSA. The maximum decreases in CFU observed after 24 h were 1.4 to 3.1 log10 CFU compared to the initial lung bacterial load in the neutropenic mice (between 5.6 and 6.3 log10 CFU) [START_REF] So | Pharmacodynamic Profile of GSK2140944 against Methicillin-Resistant Staphylococcus aureus in a Murine Lung Infection Model[END_REF].

Using neutropenic mouse models of thigh and lung infection, the pharmacokinetic-pharmacodynamic (PK-PD) parameters of gepotidacin against S. aureus and S. pneumoniae were characterized. The free plasma AUC/MIC ratio was identified as the PK-PD index most closely associated with efficacy, implying that gepotidacin activity was concentration dependent [START_REF] Bulik | Pharmacokinetic-Pharmacodynamic Evaluation of Gepotidacin against Gram-Positive Organisms Using Data from Murine Infection Models[END_REF].

In a recent study, the predicted human exposure was used to assess the efficacy in a rat pyelonephritis model with four multidrug-resistant (MDR) E. coli isolates. Overall, treatment with gepotidacin resulted in a significant reduction in CFU in the kidney and bladder [START_REF] Hoover | Efficacy of Human Exposures of Gepotidacin (GSK2140944) against Escherichia coli in a Rat Pyelonephritis Model[END_REF].

Regarding toxicity, gepotidacin did not demonstrate arthropathy in a juvenile rat model, contrary to fluoroquinolones [START_REF] Fishman | Novel Bacterial Topoisomerase Inhibitor Gepotidacin Demonstrates Absence of Fluoroquinolone-Like Arthropathy in Juvenile Rats[END_REF].

Phase 1

Since 2012, gepotidacin has been evaluated in thirteen phase 1 clinical trials [START_REF] Glaxosmithkline | Randomized, Double-Blinded, Placebo-and Moxifloxacin-Controlled, 4-Period Crossover Study to Evaluate the Effect of GSK2140944 on Cardiac Conduction as Assessed by 12-lead Electrocardiogram in Healthy Volunteers (NCT02257398)[END_REF][START_REF] Glaxosmithkline | Single Blind, Placebo Controlled Study to Evaluate Safety, Tolerability, and Pharmacokinetics of Single Escalating Oral Doses of GSK2140944 in Healthy Adult Subjects (NCT02202187), clinicaltrials[END_REF][START_REF] Glaxosmithkline | A Single-Center, Three-Part, Open Label Study to Evaluate the Relative Bioavailability of Two Formulations, Food Effect, and Interaction With Itraconazole Following Single Dose of GSK2140944[END_REF][START_REF] Glaxosmithkline | A Two-Part Study to Evaluate Safety, Tolerability, and Pharmacokinetics of Single and Repeat IV Doses of GSK2140944 in Healthy Adult Subjects[END_REF][START_REF] Glaxosmithkline | An Open-label Study To Evaluate Plasma and Pulmonary Pharmacokinetics Following Intravenous Administration of GSK2140944 in Healthy Adult Subjects (NCT01934205), clinicaltrials[END_REF][START_REF] Glaxosmithkline | An Open-Label, Non-Randomized, Two-Period, Cross-Over, Mass Balance Study to Investigate the Recovery, Excretion and Pharmacokinetics of 14C-GSK2140944 Administered as a Single Intravenous and Single Oral Dose to Healthy Adult Male Subjects (NCT02000765), clinicaltrials[END_REF][START_REF] Glaxosmithkline | Single Blind, Placebo-Controlled Study to Evaluate Safety, Tolerability, and Pharmacokinetics of Repeat Escalating Oral Doses of GSK2140944 in Healthy Adult Subjects[END_REF][START_REF] Glaxosmithkline | Double-Blind, Two-Part, Sequential Study to Evaluate the Pharmacokinetics of Gepotidacin Tablets in Healthy Adult and Adolescent Participants (NCT04079790), clinicaltrials.gov[END_REF][START_REF] Glaxosmithkline | Open-Label, Single-Dose, Multi-Part Study to Assess the Pharmacokinetics of Gepotidacin (GSK2140944) in Male and Female Adult Subjects With Varying Degrees of Renal Impairment and in Matched Control Subjects With Normal Renal Function (NCT02729038), clinicaltrials.gov[END_REF][START_REF] Glaxosmithkline | Multi-Center; Open-Label (Parts 1 and 2); Randomized, Double-Blind, Placebo-Controlled (Part 3); Single-Dose; 3-Part Study to Evaluate the Relative Bioavailability of Three Formulations in Healthy Subjects, Food Effect on Tablet Formulation in Healthy Subjects, and Pharmacokinetics of Gepotidacin (GSK2140944) in Japanese Subjects in Fasted and Fed States (NCT02853435)[END_REF][START_REF]Penetration of the Innovative Antibiotic Gepotidacin Into Prostate and Tonsillar Tissue[END_REF][START_REF] Glaxosmithkline | Multi-cohort Study in Healthy Adult Subjects to Assess Gepotidacin as Victim and as Perpetrator of Drug-Drug Interactions Via CYP450, Renal and Intestinal Transporters, and to Assess Gepotidacin Pharmacokinetics in Japanese Healthy Adults (NCT04493931), clinicaltrials[END_REF][START_REF] Glaxosmithkline | Open-Label, Single-Dose, Two-Part Study to Assess the Pharmacokinetics of Gepotidacin (GSK2140944) in Male and Female Adult Participants With Varying Degrees of Hepatic Impairment and in Matched Control Participants With Normal Hepatic Function (NCT03562117), clinicaltrials.gov[END_REF], assessing the safety and tolerability of single and repeated oral or IV doses (100 mg to 6 g per day) in adults and adolescents. In these studies, the authors also evaluated the PK parameters in plasma and lungs, cardiac conduction, food effect, drug-drug interactions and penetration into prostate and tonsil tissue. Finally, the PK was established in patients with various renal and hepatic insufficiencies. Recently, another phase 1 study started to compare the PK properties of the powder for oral suspension to the existing gepotidacin tablets [START_REF] Glaxosmithkline | Open-Label, Single-Dose, 2-Period, Crossover Study to Compare Gepotidacin Powder for Oral Suspension With the Adult Tablet Formulation in Healthy Male and Female Participants Aged 18 to 50 Years (NCT05271799)[END_REF].

Phase 2

Gepotidacin was tested against three types of bacterial infections in phase 2 clinical trials. It was studied as a treatment for Gram-positive Acute Bacterial Skin and Skin Structure Infections (ABSSSI), for uncomplicated urogenital gonorrhoea caused by Neisseria gonorrhoeae, as well as for uncomplicated Urinary Tract Infections (UTI).

In the ABSSSI phase 2 trial, 122 patients with wound infection, major cutaneous abscess or cellulitis were enrolled. Three dose regimens were evaluated: 750 mg every 12 h (q12h), 1000 mg every 12 h and 1000 mg every 8 h (q8h). Success was observed for the 750 mg q12h and the 1000 mg q8h regimens. The majority of adverse events (AE) declared were gastrointestinal events, principally nausea and diarrhoea [START_REF] O'riordan | Efficacy, Safety, and Tolerability of Gepotidacin (GSK2140944) in the Treatment of Patients with Suspected or Confirmed Gram-Positive Acute Bacterial Skin and Skin Structure Infections[END_REF].

In a second phase 2 trial, 69 participants with urogenital gonorrhoea received a single oral dose of 1500 or 3000 mg. 96% of patients achieved microbiological cure (bacterial eradication of N. gonorrhoeae). Of note, 2 out of 3 failures were due to a high-level resistance. AEs were mostly gastrointestinal events (diarrhoea, flatulence, abdominal pain, nausea). Two patients described severe gastrointestinal AE (flatulence or dizziness) but did not discontinue the study. One treatment-related cardiovascular AE (mild tachycardia) was reported by a patient with no underlying cardiovascular history [START_REF] Taylor | Gepotidacin for the Treatment of Uncomplicated Urogenital Gonorrhea: A Phase 2, Randomized, Dose-Ranging, Single-Oral Dose Evaluation[END_REF].

The goal of the third phase 2 trial was to explore the PK properties in UTI patients. 22 women with uncomplicated UTI received 1500 mg gepotidacin orally twice daily for 5 days. The test of cure was performed from days 10 to 13 and the follow-up visits were planned at day 28 +/-3. With a dose of 1500 mg twice daily, the maximum plasma concentration was reached 1 h to 1.5 h after dosing.

Steady state was reached on day 3. Clinical success was validated for 18 patients (82%) at follow-up visits. 21 patients (95%) experienced mild to moderate AEs, primarily gastrointestinal events. Two patients (9%) reported vulvovaginal mycotic infection after treatment, but no AE prevented treatment [START_REF] Overcash | Phase 2a Pharmacokinetic, Safety, and Exploratory Efficacy Evaluation of Oral Gepotidacin (GSK2140944) in Female Participants with Uncomplicated Urinary Tract Infection (Acute Uncomplicated Cystitis)[END_REF].

Current advances

Gepotidacin is currently being evaluated in three phase 3 clinical trials. Two studies aim to compare gepotidacin (1500 mg orally twice a day for 5 days) and nitrofurantoin (100 mg orally twice a day for 5 days) for the treatment of uncomplicated UTI in approximately 2500 patients each [START_REF] Glaxosmithkline | Randomized, Multicenter, Parallel-Group, Double-Blind, Double-Dummy Study in Adolescent and Adult Female Participants Comparing the Efficacy and Safety of Gepotidacin to Nitrofurantoin in the Treatment of Uncomplicated Urinary Tract Infection (Acute Cystitis) (NCT04187144), clinicaltrials[END_REF][START_REF] Glaxosmithkline | Randomized, Multicenter, Parallel-Group, Double-Blind, Double-Dummy Study in Adolescent and Adult Female Participants Comparing the Efficacy and Safety of Gepotidacin to Nitrofurantoin in the Treatment of Uncomplicated Urinary Tract Infection (Acute Cystitis) (NCT04020341), clinicaltrials[END_REF].

The third trial will assess the efficacy of gepotidacin (3000 mg orally twice) compared with the combination ceftriaxone/azithromycin (single doses of 500 mg intramuscular and 1000 mg orally, respectively) for the treatment of uncomplicated urogenital gonorrhoea on an estimated 600 patients [START_REF] Glaxosmithkline | Randomized, Multicenter, Open-Label Study in Adolescent and Adult Participants Comparing the Efficacy and Safety of Gepotidacin to Ceftriaxone Plus Azithromycin in the Treatment of Uncomplicated Urogenital Gonorrhea Caused by Neisseria Gonorrhoeae (NCT04010539), clinicaltrials[END_REF].

In a recent study, gepotodacin was also found efficacious in an inhalational model of pneumonic plague in the African green monkey, a non-human primate, at exposures of the drug equivalent to those achieved in humans. In this experiment, gepotidacin provided a 75 to 100% survival benefit with doses ranging from 28 to 48 mg/kg. These data open up the possibility of also using gepotidacin as a treatment for pneumonic plague caused by Yersinia pestis [START_REF] Jakielaszek | Gepotidacin is efficacious in a nonhuman primate model of pneumonic plague[END_REF].

Zoliflodacin

Zoliflodacin (previously known as ETX0914 or AZD0914) is a novel spiropyrimidinetrione antibiotic being developed as single oral dose for the treatment of uncomplicated gonorrhoea [START_REF] Unemo | Sexually transmitted infections: challenges ahead[END_REF][START_REF] Basarab | Responding to the challenge of untreatable gonorrhea: ETX0914, a first-in-class agent with a distinct mechanism-of-action against bacterial Type II topoisomerases[END_REF][START_REF] Miller | Determination of MIC Quality Control Ranges for the Novel Gyrase Inhibitor Zoliflodacin[END_REF][START_REF] Newman | Thorough QT Study To Evaluate the Effect of Zoliflodacin, a Novel Therapeutic for Gonorrhea, on Cardiac Repolarization in Healthy Adults[END_REF].

Spectrum of activity

Zoliflodacin displays activity against Gram-positive bacteria including S. pneumoniae, Streptococcus pyogenes, and methicillin-resistant fluoroquinolone-resistant S. aureus, as well as against fastidious Gram-negative bacteria including Haemophilus influenzae and N. gonorrhea [START_REF] Basarab | Discovery of Novel DNA Gyrase Inhibiting Spiropyrimidinetriones: Benzisoxazole Fusion with N-Linked Oxazolidinone Substituents Leading to a Clinical Candidate (ETX0914)[END_REF].

Chemical synthesis

The synthesis of zoliflodacin described below was reported in 2015 [START_REF] Basarab | Discovery of Novel DNA Gyrase Inhibiting Spiropyrimidinetriones: Benzisoxazole Fusion with N-Linked Oxazolidinone Substituents Leading to a Clinical Candidate (ETX0914)[END_REF]. The first step, starting from 2,3,4-trifluorobenzaldehyde, consists of the protection of the aldehyde function to an acetal group. After deprotonation using n-BuLi, formylation is performed with DMF to introduce an aldehyde group, which is then converted to oxime using hydroxylamine. Chlorination with N-chlorosuccinimide (NCS), followed by reaction with L-alaninol and intramolecular S N Ar allows the formation of the benzisoxazole ring. The oxazolidinone moiety is obtained using 1,1'-carbonyldiimidazole (CDI). The deprotection of the aldehyde is then preformed in acidic conditions followed by another S N Ar at the ortho position of the aldehyde using (2R,6S)-2,6-dimethylmorpholine. Finally, a Knoevenagel condensation between the aldehyde and hexahydropyrimidine-2,4,6-trione is performed, followed by an intramolecular rearrangement consisting in an [START_REF] O'neill | Tackling drug-resistant infections globally: final report and recommendations[END_REF][START_REF] Murray | [END_REF][START_REF] Harbarth | Antimicrobial resistance: one world, one fight![END_REF][START_REF] Annunziato | Strategies to Overcome Antimicrobial Resistance (AMR) Making Use of Non-Essential Target Inhibitors: A Review[END_REF][START_REF] Reygaert | An overview of the antimicrobial resistance mechanisms of bacteria[END_REF] hydride shift and then intramolecular cyclization leading to zoliflodacin (Figure 5).

Target and mechanism of action

Zoliflodacin is a topoisomerase II inhibitor that presents a new mode of action compared with previous inhibitors, such as fluoroquinolones [START_REF] Bradford | Zoliflodacin: An Oral Spiropyrimidinetrione Antibiotic for the Treatment of Neisseria gonorrheae , Including Multi-Drug-Resistant Isolates[END_REF]. Zoliflodacin binds to the GyrB subunit of the bacterial DNA gyrase whereas fluoroquinolones target the GyrA subunit [START_REF] Jacobsson | Pharmacodynamic Evaluation of Dosing, Bacterial Kill, and Resistance Suppression for Zoliflodacin Against Neisseria gonorrhoeae in a Dynamic Hollow Fiber Infection Model[END_REF]. Briefly, the binding enables to stop the cleaved covalent complex of gyrase which thus blocks the religation of the double-strand cleaved DNA to form fused circular DNA. This process leads to the inhibition of DNA biosynthesis and the accumulation of double-strand cleavages in bacteria [START_REF] Huband | In Vitro Antibacterial Activity of AZD0914, a New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-Positive, Fastidious Gram-Negative, and Atypical Bacteria[END_REF]. Interestingly, there is no cross-resistance with the existing fluoroquinolones [START_REF] Taylor | Single-Dose Zoliflodacin (ETX0914) for Treatment of Urogenital Gonorrhea[END_REF].

Structure-activity relationship (SAR) studies

After a high-throughput screening at Pharmacia & Upjohn, QPT-1 was found as the first spiropyrimidinetrione inhibiting bacterial DNA gyrase and topoisomerase IV with no cross-resistance with fluoroquinolones (Figure 6). A benzisoxazole scaffold was then introduced by scientists at AstraZeneca [START_REF] Shi | Design, Synthesis, and Biological Evaluation of Novel DNA Gyrase-Inhibiting Spiropyrimidinetriones as Potent Antibiotics for Treatment of Infections Caused by Multidrug-Resistant Gram-Positive Bacteria[END_REF]. However, due to toxicity and genotoxicity other variations were studied:  Spirocyclic pyrimidinetrione: the pyrimidinetrione was kept constant in all the synthesized analogues.

 Oxazolidinone introduction: the oxazolidinone ring leading to zoliflodacin was introduced replacing the methyl substituent of the previous lead to mitigate mammalian toxicity and to obtain compounds characterized by a greater safety [START_REF] Basarab | Responding to the challenge of untreatable gonorrhea: ETX0914, a first-in-class agent with a distinct mechanism-of-action against bacterial Type II topoisomerases[END_REF]. Highest potency was found for derivatives with substituents at both the 4-and 5-positions orientated below the plane of the oxazolidinone ring [START_REF] Basarab | Discovery of Novel DNA Gyrase Inhibiting Spiropyrimidinetriones: Benzisoxazole Fusion with N-Linked Oxazolidinone Substituents Leading to a Clinical Candidate (ETX0914)[END_REF].

 Benzisoxazole substitution: the replacement of the oxazolidinone ring with an ethyloxadiazole and triazole rings led to analogues with higher activity than zoliflodacin. Moreover, the introduction of heterocycles linked to the benzisoxazole through an ether oxygen in a two-atom bridge and the replacement of the fluorine atom with a chlorine resulted in compounds with a similar activity to zoliflodacin [START_REF] Basarab | Responding to the challenge of untreatable gonorrhea: ETX0914, a first-in-class agent with a distinct mechanism-of-action against bacterial Type II topoisomerases[END_REF][START_REF] Govender | Spiropyrimidinetrione DNA Gyrase Inhibitors with Potent and Selective Antituberculosis Activity[END_REF]. In contrast, pyrrolidine benzisoxazole substituents led to derivatives 200-fold less active against S. aureus.

 Morpholine modifications: overall, the morpholine ring showed little scope for variations and the modifications resulted in less active compounds. The tested analogues were synthesized replacing the morpholine with substituted piperidines and piperazines. However, the introduction of a N-cyano piperazine resulted in a S. aureus active compound [START_REF] Govender | Spiropyrimidinetrione DNA Gyrase Inhibitors with Potent and Selective Antituberculosis Activity[END_REF]. Furthermore, the biological evaluation of the corresponding enantiomers and diastereoisomers showed that the specific stereochemistry and the two methyl groups around the morpholine are crucial for the antibacterial activity [START_REF] Basarab | Responding to the challenge of untreatable gonorrhea: ETX0914, a first-in-class agent with a distinct mechanism-of-action against bacterial Type II topoisomerases[END_REF][START_REF] Basarab | Discovery of Novel DNA Gyrase Inhibiting Spiropyrimidinetriones: Benzisoxazole Fusion with N-Linked Oxazolidinone Substituents Leading to a Clinical Candidate (ETX0914)[END_REF].

In vitro activity

Zoliflodacin is a potent and very selective inhibitor of bacterial topoisomerases. Indeed, in vitro assays measuring the growth inhibition of myeloid and erythroid cell lines in mammals showed no genotoxicity or bone marrow toxicity for zoliflodacin at the highest concentrations tested. These data indicate that zoliflodacin presents reduced risks of hematologic toxicity compared with some other fluoroquinolones (such as gemifloxacin, for instance), which have been associated with anemia, leukopenia and bone marrow suppression [START_REF] Bradford | Zoliflodacin: An Oral Spiropyrimidinetrione Antibiotic for the Treatment of Neisseria gonorrheae , Including Multi-Drug-Resistant Isolates[END_REF].

Zoliflodacin is bactericidal, with a low frequency of resistance and a potent in vitro activity against N. gonorrhoeae (MIC 90 of 0.125 µg/mL) [START_REF] Miller | Determination of MIC Quality Control Ranges for the Novel Gyrase Inhibitor Zoliflodacin[END_REF][START_REF] Bradford | Zoliflodacin: An Oral Spiropyrimidinetrione Antibiotic for the Treatment of Neisseria gonorrheae , Including Multi-Drug-Resistant Isolates[END_REF]. To date, no clinical zoliflodacin-resistant N. gonorrhoeae isolates have been found [START_REF] Bradford | Zoliflodacin: An Oral Spiropyrimidinetrione Antibiotic for the Treatment of Neisseria gonorrheae , Including Multi-Drug-Resistant Isolates[END_REF]. However, zoliflodacin resistant mutants have been selected in vitro, and all of them contained nonsynonymous mutations in amino acid codons D429 or K450 of GyrB, which leads to increased MICs of zoliflodacin in the mutants [START_REF] Jacobsson | Pharmacodynamic Evaluation of Dosing, Bacterial Kill, and Resistance Suppression for Zoliflodacin Against Neisseria gonorrhoeae in a Dynamic Hollow Fiber Infection Model[END_REF]. Zoliflodacin also presented potent in vitro activity against Chlamydia pneumoniae, certain Gram-positive organisms (S. aureus and S. pneumoniae; including fluoroquinolone-and vancomycin-resistant staphylococci), fastidious Gram-negative organisms (Moraxella catarrhalis and Haemophilus spp., in particular H. influenzae), and atypical anaerobic organisms [START_REF] Basarab | Responding to the challenge of untreatable gonorrhea: ETX0914, a first-in-class agent with a distinct mechanism-of-action against bacterial Type II topoisomerases[END_REF][START_REF] Taylor | Single-Dose Zoliflodacin (ETX0914) for Treatment of Urogenital Gonorrhea[END_REF].

Zoliflodacin also displayed potent in vitro activity against Mycoplasma genitalium and 11 azithromycin-and fluoroquinolone-susceptible isolates with MICs < 1 mg/L [START_REF] Damião Gouveia | In vitro activity of zoliflodacin (ETX0914) against macrolide-resistant, fluoroquinolone-resistant and antimicrobial-susceptible Mycoplasma genitalium strains[END_REF]. Furthermore, zoliflodacin showed activity against Chlamydia trachomatis with the highest zoliflodacin MIC of 0.5 mg/L among 10 strains.

Zoliflodacin was also fully active against laboratory strains of S. aureus and S. pneumoniae resistant to novobiocin. It was also more active against S. aureus (including MRSA) than linezolid, which is the current standard of care for skin infections caused by this pathogen [START_REF] Basarab | Discovery of Novel DNA Gyrase Inhibiting Spiropyrimidinetriones: Benzisoxazole Fusion with N-Linked Oxazolidinone Substituents Leading to a Clinical Candidate (ETX0914)[END_REF].

More recently, zoliflodacin demonstrated potent in vitro antibacterial activity against a collection of clinical gonococcal isolates from China, including isolates with a high-level of resistance to ciprofloxacin, azithromycin, and extended-spectrum cephalosporins [START_REF] Le | Susceptibility Trends of Zoliflodacin against Multidrug-Resistant Neisseria gonorrhoeae Clinical Isolates in Nanjing, China, 2014 to 2018[END_REF]. Due to a distinct mechanism of action, cross-resistance between zoliflodacin and fluoroquinolones has not been observed [START_REF] Alm | Characterization of the Novel DNA Gyrase Inhibitor AZD0914: Low Resistance Potential and Lack of Cross-Resistance in Neisseria gonorrhoeae[END_REF].

In vivo experiments

In vitro and in vivo studies across species showed that zoliflodacin was extensively metabolized in humans via the cytochrome P450 and non-P450-mediated pathways [START_REF] O'donnell | Single-Dose Pharmacokinetics, Excretion, and Metabolism of Zoliflodacin, a Novel Spiropyrimidinetrione Antibiotic, in Healthy Volunteers[END_REF]. The assessment of the in vivo efficacy of zoliflodacin was defined using a murine thigh infection model in immunocompetent and neutropenic mice using a methicillin-susceptible strain of S.aureus [START_REF] Basarab | Novel DNA Gyrase Inhibiting Spiropyrimidinetriones with a Benzisoxazole Scaffold: SAR and in Vivo Characterization[END_REF]. This study showed that the compound was efficient in a dose-dependent manner in both mouse models and a 3-log reduction in CFU was observed when the exposure (AUC) achieved 70 µg.h/mL in immunocompetent mice and 120 µg.h/mL in immunosuppressed mice.

Phase 1

Two phase 1 studies have been conducted with single oral doses in healthy volunteers [START_REF] O'donnell | Single-Dose Pharmacokinetics, Excretion, and Metabolism of Zoliflodacin, a Novel Spiropyrimidinetrione Antibiotic, in Healthy Volunteers[END_REF]. The first study determined the safety, tolerability and PK parameters of zoliflodacin in both fed and fasted states [START_REF] Astrazeneca | Randomized, Placebo-controlled, Single-center Study to Assess the Safety, Tolerability, and Pharmacokinetics of AZD0914 After a Single Oral Administration and to Assess the Effect of Food in Healthy Adult Volunteers (NCT01929629), clinicaltrials.gov[END_REF], whereas the second study assessed the PK and ADME properties using [ 14 C] zoliflodacin [START_REF]A Phase I, Open-Label Study to Assess the Absorption, Metabolism, Excretion, Safety and Tolerability of a Single Oral Dose of Radiolabeled [14C]-AZD0914 in Healthy Male Subjects (NCT02298920)[END_REF].

In the first study, zoliflodacin showed a rapid absorption, with a T max observed between 1.5 and 2.3 h, and a T 1/2 range of 5.3 to 6.3 h. Interestingly, the absorption of the drug was delayed (T max at 4 h) in subjects who were dosed following ingestion of a high-fat meal. Finally, no significant AEs were observed in this study.

In the second study, the ADME properties were defined after a single oral dose of 3000 mg of [ 14 C] zoliflodacin. The data demonstrated that the PK profile of zoliflodacin (AUC and C max ) was similar to the one obtained in the first phase 1 study. Overall, both phase 1 studies showed linear PK data, good oral bioavailability and no significant safety findings.

Phase 2

The safety and efficacy of zoliflodacin was then assessed in a randomized phase 2 study on 179 participants with uncomplicated urogenital gonorrhea [START_REF]A Randomized, Open-label Phase 2 Study to Evaluate the Efficacy and Safety of a Single Dose of Oral AZD0914 Compared to Intramuscular Ceftriaxone in the Treatment of Male and Female Subjects With Uncomplicated Gonorrhea (NCT02257918), clinicaltrials[END_REF]. The subjects were randomized to receive a single oral dose of zoliflodacin (either 2 or 3 g) or a single 500 mg intramuscular dose of ceftriaxone. In summary, oral zoliflodacin has successfully treated most uncomplicated urogenital and rectal gonococcal infections, but has shown little efficacy in treating pharyngeal infections.

Phase 3

Following promising results from one of the phase 2 clinical trials, a phase 3 for the treatment of uncomplicated urogenital gonorrhea is currently underway [START_REF]Global Antibiotics Research and Development Partnership, A Multi-center, Randomized, Open-label, Non Inferiority Trial to Evaluate the Efficacy and Safety of a Single, Oral Dose of Zoliflodacin Compared to a Combination of a Single Intramuscular Dose of Ceftriaxone and a Single Oral Dose of Azithromycin in the Treatment of Patients With Uncomplicated Gonorrhoea (NCT03959527), clinicaltrials[END_REF]. The study is expected to include approximately 1000 adults with urogenital gonorrhea from different sites (USA, The Netherlands, Thailand, and South Africa) and results are now expected in 2023 (current status). This open-label trial compares a single 3 g oral dose of zoliflodacin to a combination of ceftriaxone and azithromycin (2:1 randomization). Recently, a parallel study [START_REF]A Phase 1, Randomized, Double-blinded, Four-period Crossover, Thorough QT/QTC (TQT) Clinical Trial to Evaluate the Effect of Zoliflodacin on Cardiac Repolarization in Healthy Male and Female Subjects (NCT03613649), clinicaltrials[END_REF] demonstrated that a single oral dose of zoliflodacin was positively evaluated for cardiac safety, supporting further clinical experiments with single 3 g doses in patient with gonorrhea [START_REF] Newman | Thorough QT Study To Evaluate the Effect of Zoliflodacin, a Novel Therapeutic for Gonorrhea, on Cardiac Repolarization in Healthy Adults[END_REF].

DNA polymerase as a drug target

Introduction

DNA replication is carried out by the DNA polymerase, a massive enzymatic complex highly organized and therefore susceptible to disruption. Inhibitors of DNA polymerase are widely used to treat viral infections, such as aciclovir for the treatment of herpes virus. However, there is currently no antibiotic in clinic which targets the bacterial DNA polymerase, whereas human and bacterial DNA polymerases have many differences [START_REF] Santos | Novel Antibiotics Targeting Bacterial Replicative DNA Polymerases[END_REF]. To date, ibezapolstat is the first bacterial specific DNA polymerase inhibitor in clinical development.

Ibezapolstat

Ibezapolstat (formerly ACX-362E) is a member of the 6-anilinouracil family of dGTP analogues [START_REF] Tarantino | 6-Anilinouracil-based inhibitors of Bacillus subtilis DNA polymerase III: antipolymerase and antimicrobial structure-activity relationships based on substitution at uracil N3[END_REF]. Developed by Acurx Pharmaceuticals, it selectively inhibits DNA polymerase IIIC of several Gram-positive bacteria, through competition with dGTP [START_REF] Daly | In vitro antimicrobial activities of novel anilinouracils which selectively inhibit DNA polymerase III of gram-positive bacteria[END_REF]. In 2012, Dvoskin et al. demonstrated that ibezapolstat was highly effective against Clostridioides difficile on in vitro and in vivo models [START_REF] Dvoskin | A novel agent effective against Clostridium difficile infection[END_REF]. The compound is currently being evaluated in a phase 2 clinical study [START_REF]E [Ibezapolstat] for Oral Treatment of Clostridioides Difficile Infection: A Phase 2A Open-Label Segment Followed by a Phase 2B Double-Blind Vancomycin-Controlled Segment, clinicaltrials[END_REF].

Chemical synthesis

Ibezapolstat was first synthesized in 2010 in a 3-step procedure, following a protocol described by Wright et al. (Figure 7) [START_REF] Wright | Selective antibacterials for clostridium difficile infections[END_REF]. Firstly, 2-chloro-6-methoxypurine is alkylated with 1,2-dibromoethane to afford a mixture of 7-and 9-isomers. After isolation of the 7-isomer, a S N 2 reaction with morpholine leads to the corresponding intermediate, which is then subjected to reaction with 3,4-dichlorobenzylamine to give ibezapolstat after the concomitant demethylation due to HCl release.

Target and mechanism of action

Ibezapolstat inhibits the DNA polymerase IIIC (DNA pol IIIC), an enzyme essential for bacterial DNA replication [START_REF] Murray | In vitro activity of the novel antibacterial agent ibezapolstat (ACX-362E) against Clostridioides difficile[END_REF], from low G + C Gram-positive germs (DNA containing less than 50 % of cytosine and guanine). Ibezapolstat is also an analogue of dGTP, allowing the compound to pair with dCTG of DNA.

In addition, the aryl part of the molecule can bind to DNA pol IIIC. The combination of these two pro-perties leads to the formation of an inactive ternary complex between DNA, ibezapolstat and DNA pol IIIC (Figure 8). To note, the compound inhibits the DNA pol IIIC of C. difficile with a K i of 0.325 µM [START_REF] Torti | Clostridium difficile DNA polymerase IIIC: basis for activity of antibacterial compounds[END_REF]. Moreover, there is only little homology between DNA pol IIIC and mammalian polymerase [START_REF] Xu | Discovery and development of DNA polymerase IIIC inhibitors to treat Gram-positive infections[END_REF].

Structure-activity relationship studies

Ibezapolstat is a 6-anilinouracil derived from 6-(p-hydroxyphenylhydrazino)-uracil (H2-HPUra), itself identified in the late 1970's as an inhibitor of DNA pol IIIC. In order to improve potency, structure-activity relationship studies were performed [START_REF] Butler | Development of novel inhibitor probes of DNA polymerase III based on dGTP analogs of the HPUra type: base, nucleoside and nucleotide derivatives of N 2 -(3,4-dichlorobenzyl)guanine[END_REF][START_REF] Wright | Active site directed inhibitors of replication-specific bacterial DNA polymerases[END_REF] (Figure 9):

• Replacement of the uracile core by a guanine core: H2-HPUra binds to cytosine via its uracil core. The latter was replaced by the natural DNA base guanine. A loss of activity was observed, except when the 3,4-dichlorobenzyl moiety was introduced.

• Modification of the 3,4-dichlorobenzyl moiety: replacement by other groups and/or modification of the position of the two chlorine atoms led to less potent compounds.

• Alkylation of the guanine: the alkylation of the nitrogen at position 7 resulted in more potent derivatives. On the contrary, the alkylation of the nitrogen at position 9 led to a total loss of potency.

• Modification of the linker: even if compounds with a longer linker are active, the ethylene linker results in the most potent compound.

• Modification of the morpholino part: replacement of the morpholino part by esters or alcohols is tolerated. However, the presence of a protonated nitrogen at physiological pH probably explains why the morpholine ring was kept. for C. difficile infections (CDI) [START_REF] Peng | Advances in the diagnosis and treatment of Clostridium difficile infections[END_REF]. More recently, ibezapolstat was evaluated against 104 isolates of C. difficile [START_REF] Murray | In vitro activity of the novel antibacterial agent ibezapolstat (ACX-362E) against Clostridioides difficile[END_REF]. MIC range of the molecule (1-8 µg/mL) was higher than metronidazole (0.25-16 µg/mL), vancomycin (0.5-4 µg/mL) and fidaxomicin (0.015-1 µg/mL). However, due to the mechanism of action of ibezapolstat targeting DNA pol IIIC, which is not expressed by Gram-negative bacteria, the compound has a narrow spectrum compared to metronidazole and vancomycin. Indeed, ibezapolstat has low activity against representatives of the normal colonic flora, such as Bacteroides fragilis, while metronidazole is active against some of them [START_REF] Torti | Clostridium difficile DNA polymerase IIIC: basis for activity of antibacterial compounds[END_REF]. Overall, ibezapolstat exhibits an interesting spectrum profile, as the compound is unlikely to disrupt the endogenous bacterial flora, a property that is preferable for avoiding recurrence of CDI [START_REF] Rea | Effect of broad-and narrow-spectrum antimicrobials on Clostridium difficile and microbial diversity in a model of the distal colon[END_REF][START_REF] Tsigrelis | Recurrent Clostridioides difficile infection: Recognition, management, prevention, Cleve[END_REF].

In vivo experiments

An efficacy study was performed in a golden Syrian hamster model by calculating the percentage of survivors after 5 days. Animals were completely protected at a dose of 12.5 mg/kg administered twice daily for 3 days. However, 67 % of the animals died after 15 days, due to recurrence of the infection. The recurrence was less pronounced with animals treated for more than 3 days. Indeed, only 40 % of the animals died after 7 days of treatment. Administration of ibezapolstat during 14 days did not result in recurrence in any of the animals infected during the 34 days of the experiment.

In the hamster model of Clostridioides difficile-associated diarrhea (CDAD), ibezapolstat showed no signs of toxicity at an oral dose of 1 g/kg [START_REF] Dvoskin | A novel agent effective against Clostridium difficile infection[END_REF]. Moreover, at the oral dose of 75 mg/kg, only 5 % is absorbed. Because the site of CDI is exclusively located in the colon, a lower absorption results in higher concentrations in the intestine.

Phase 1

Ibezapolstat has completed a phase 1 study in the United States [START_REF] Garey | A randomized, double-blind, placebo-controlled, single and multiple ascending dose Phase 1 study to determine the safety, pharmacokinetics and food and faecal microbiome effects of ibezapolstat administered orally to healthy subjects[END_REF]. The randomized, double-bind, placebo-controlled clinical trial involved 62 healthy adults and was divided into three parts.

The first consisted of oral administration of a single ascending dose of ibezapolstat at doses of 150, 300, 600 and 900 mg. In the second part, a single dose of 300 mg was given with a meal. Finally, the third part was a multi-day ascending dose study of ibezapolstat 300 mg and 450 mg given twice daily or vancomycin 125 mg given four times daily.

The drug candidate was found to be safe and well-tolerated. Consistent with the hamster pharmacokinetics, ibezapolstat was poorly absorbed, resulting in no systemic accumulation. Of note, faecal concentrations of ibezapolstat after 4 days of multiple ascending doses were higher than those of vancomycin. Vancomycin also decreased the diversity of the microbiome while ibezapolstat targeted only low G+C organisms. Moreover, vancomycin increased the proportion of proteobacteria, which is associated with susceptibility to CDI [START_REF] Winston | Impact of microbial derived secondary bile acids on colonization resistance against Clostridium difficile in the gastrointestinal tract[END_REF].

Phase 2

A phase 2a study was conducted in 10 adults with CDI, as a single-arm, open-label multicenter clinical trial [START_REF] Garey | Pharmacokinetics, and Microbiome Changes of Ibezapolstat in Adults with Clostridioides difficile Infection: A Phase 2a Multicenter Clinical Trial[END_REF]. Ibezapolstat was orally given 450 mg twice daily, during 10 days. Safety and pharmacokinetic profiles were consistent with the data from the phase 1 study. C. difficile was eradicated after 3 days, resulting in 100 % cure of all 10 patients. In addition, none of the patients experienced a recurrence during the 28-day study.

Regarding the microbiome, an increased proportion of healthy microbiota was observed, including bacteria capable of metabolizing primary bile acids into secondary bile acids [START_REF] Ridlon | Bile salt biotransformations by human intestinal bacteria[END_REF]. This change in the microbiome resulted in an increased proportion of secondary bile acids, which is associated with protection against CDI [START_REF] Allegretti | Recurrent Clostridium difficile Infection Associates with Distinct Bile Acid and Microbiome Profiles[END_REF]. Ibezapolstat is currently being evaluated in phase 2b study to compare efficacy of the drug candidate against vancomycin [START_REF]E [Ibezapolstat] for Oral Treatment of Clostridioides Difficile Infection: A Phase 2A Open-Label Segment Followed by a Phase 2B Double-Blind Vancomycin-Controlled Segment, clinicaltrials[END_REF].

DNA minor groove as a drug target

Introduction

Small molecules can manifest their effect on DNA through various binding mechanisms such as intercalation, alkylation as well as major and minor groove binding [START_REF] Khan | Chemistry of DNA minor groove binding agents[END_REF]. These interactions can cause a variety of important biological responses. Minor groove binders (MGBs) are well-known for their applications as anticancer drugs, although their importance as anti-infective agents is growing [START_REF] Hlaka | Evaluation of minor groove binders (MGBs) as novel anti-mycobacterial agents and the effect of using non-ionic surfactant vesicles as a delivery system to improve their efficacy[END_REF].

The majority of synthetic MGBs are linked to the two natural occurring antibiotics, netropsin and distamycin A, and are generally characterized by the presence of an alkene, a non-polar N-terminal amide isostere, and N-alkyl chains on the pyrrole rings [START_REF] Khalaf | Amide isosteres in structure-activity studies of antibacterial minor groove binders[END_REF].

MGB-BP-3

MGB-BP-3 is a synthetic polyamide belonging to the large family of minor groove binders, small molecules capable of binding to the minor groove of DNA. Hundreds of MGBs were synthesized and tested for various biological activities by Khalaf et  Amino tail group: substituents such as piperazines, piperidines, pyrrolidines were introduced and led to significantly active but more toxic compounds.

 Aromatic head group: aromatic head groups such as meta-methoxybenzene and para-methoxybenzene led to active compounds. However, the introduction of a naphthalene and isoquinoline head groups resulted in less active compounds.

 Linker: to increase further the non-polar interactions an alkene between the benzene ring and the quinoline was successfully introduced and opened the way for the discovery of the lead compound MGB-BP-3. In contrast, when an alkene was introduced between the two pyrroles the activity was totally lost.

 N-substitution: the introduction of isopropyl or bulkier groups on the nitrogen of pyrrole rings led to active compounds. MGB-BP-3 is highly active against C. difficile, killing the vegetative bacterium cell before sporulation. In particular, MGB-BP-3 exhibits strong bactericidal activity against BI/NAP1/027, the most virulent strain that is highly resistant to current therapies [START_REF] Kullar | Investigational Treatment Agents for Recurrent Clostridioides difficile Infection (rCDI)[END_REF].

In addition, S. aureus was grown at sub-MIC 80 concentrations of MGB-BP-3 or rifampicin (used as a control) for 80 generations. No resistant strain towards MGB-BP-3 was observed, contrary to rifampicin [START_REF] Suckling | Multitargeted anti-infective drugs: resilience to resistance in the antimicrobial resistance era[END_REF].

MGB-BP-3 did not show significant activity against Gram-negative bacteria. Interestingly, significant activity was found when Pseudomonas aeruginosa bacteria were treated with MGB-BP-3 in association with an efflux pump inhibitor (PAβN, Phenylalanine-Arginine-β-naphthylamide) or a membrane permeabilizer (polymyxin B nonapeptide). These results suggest that the major obstacle to the antibacterial activity of MGB-BP-3 is the expression of efflux pumps [START_REF] Suckling | Multitargeted anti-infective drugs: resilience to resistance in the antimicrobial resistance era[END_REF]. Microorganisms), which addresses the lack of financial incentives for pharmaceutical companies to develop new antibiotics [START_REF] Kullar | Investigational Treatment Agents for Recurrent Clostridioides difficile Infection (rCDI)[END_REF]. In addition, a phase 3 head-to-head trial has been agreed with the FDA, comparing MGB-BP-3 to vancomycin [START_REF] Suckling | Multitargeted anti-infective drugs: resilience to resistance in the antimicrobial resistance era[END_REF].

tRNAs and ribosomes as drug targets

Introduction

tRNAs are small non-coding RNAs that play a key role in the translation process by delivering the amino acids to the ribosome for incorporation into a growing polypeptide chain during the protein synthesis [START_REF] Avcilar-Kucukgoze | Hijacking tRNAs From Translation: Regulatory Functions of tRNAs in Mammalian Cell Physiology[END_REF]. Any component of this essential process is thus an interesting target for the development of new antibiotics. This will be highlighted with the development of CRS-3123 and lefamulin.

CRS-3123

CRS-3123 (formerly REP-3123) targets the methionyl-tRNA synthetase of Gram-positive bacteria, in particular C. difficile [START_REF] Citron | Comparative in vitro activity of REP3123 against Clostridium difficile and other anaerobic intestinal bacteria[END_REF]. The drug candidate is being developed by Creston Inc. and has completed two phase 1 studies in 2022 [START_REF] Nayak | Safety, Tolerability, Systemic Exposure, and Metabolism of CRS3123, a Methionyl-tRNA Synthetase Inhibitor Developed for Treatment of Clostridium difficile, in a Phase 1 Study[END_REF][START_REF] Lomeli | Multiple-Ascending-Dose Phase 1 Clinical Study of the Safety, Tolerability, and Pharmacokinetics of CRS3123, a Narrow-Spectrum Agent with Minimal Disruption of Normal Gut Microbiota[END_REF]. Interestingly, the R-enantiomer of CRS-3123 is the most potent stereoisomer against the C. difficile MetRS1, with a K i of 0.02 nM. CRS-3123 inhibits also MetRS1 of other Gram-positive bacteria, such as S. aureus (K i = 0.017 nM) and S. pneumoniae (K i = 0.08 nM). Notably, the compound is 1000 times less potent on the human mitochondrial MetRS1 (K i = 28 nM). CRS-3123 is less potent against MetRS2, which is present among Gram-negative bacteria, such as H. influenzae (K i = 178 nM) and E.

coli (K i = 1900 nM). To note, the human cytoplasmic type 2 enzyme is not inhibited (K i > 20000 nM).

In vitro activity

In agreement with the inhibition data of MetRS1 described above, CRS-3123 is potent against more than 100 isolates of C. difficile with MIC ranging from 0.5 to 1 µg/mL [START_REF] Citron | Comparative in vitro activity of REP3123 against Clostridium difficile and other anaerobic intestinal bacteria[END_REF][START_REF] Critchley | Spectrum of activity and mode of action of REP3123, a new antibiotic to treat Clostridium difficile infections[END_REF]. Moreover, the compound is as potent as vancomycin (MIC = 0.25-4 µg/mL). CRS-3123 is also active against other Gram-positive bacteria, such as MRSA (MIC = 0.015-0.5 µg/mL) or vancomycin-resistant Enterococcus faecium (MIC < 0.004 µg/mL). However, the drug is not potent against Gram-negative bacteria, with MICs > 32 µg/mL for E. coli, Klebsiella pneumoniae, P. aeruginosa or Acinetobacter baumannii.

Moreover, CRS-3123 is not active against various Lactobacillus spp., Actinomyces spp. and Bifidobacterium spp. (MIC > 32 mg/mL for each) which are Gram-positive anaerobic bacteria of gut microbiota. Even if no pre-existing resistance was observed for C. difficile, mutants with decreased susceptibility towards CRS-3123 were selected, with spontaneous resistance frequencies of 10 -9 -10 -8 at 16x MIC and 4x MIC respectively [START_REF] Rinninella | What is the Healthy Gut Microbiota Composition? A Changing Ecosystem across Age[END_REF].

Contrary to vancomycin and metronidazole, CRS-3123 is able to decrease production of toxins A and B from C. difficile [START_REF] Ochsner | Inhibitory effect of REP3123 on toxin and spore formation in Clostridium difficile, and in vivo efficacy in a hamster gastrointestinal infection model[END_REF], which are at the origin of the pathogenicity of the bacterium [START_REF] Leffler | Clostridium difficile Infection[END_REF].

Finally, CRS-3123 shows reduction of spore production after 4 days, even at 0.25x MIC [START_REF] Ochsner | Inhibitory effect of REP3123 on toxin and spore formation in Clostridium difficile, and in vivo efficacy in a hamster gastrointestinal infection model[END_REF]. In contrast, metronidazole and vancomycin are not able to decrease the formation of spores, which is even promoted for some strains. The inhibition of spore production observed for CRS-3123 is an interesting property, as spores are very difficult to eliminate and are the main source of new infections and recurrences [START_REF] Leffler | Clostridium difficile Infection[END_REF].

In vivo experiments

CRS-3123 was shown more effective than vancomycin in vivo [START_REF] Ochsner | Inhibitory effect of REP3123 on toxin and spore formation in Clostridium difficile, and in vivo efficacy in a hamster gastrointestinal infection model[END_REF]. In a hamster model of CDI, CRS-3123 at daily doses of 0.5 mg/kg and 5 mg/kg greatly reduced the mortality of the animal, with 62 % and 75 % survival after 33 days, respectively. In this experiment, CRS-3123 was also superior to vancomycin, where no animal survived after 25 days. The increased survival of CRS-3123 is likely due to the inhibition of spore formation, preventing re-infection of animals during the study.

Toxicity and pharmacokinetics

In healthy uninfected hamsters, CRS-3123 displayed a bioavailability of 0.6 %, leading to low systemic exposure and maximal concentrations in the gut [START_REF] Ochsner | Inhibitory effect of REP3123 on toxin and spore formation in Clostridium difficile, and in vivo efficacy in a hamster gastrointestinal infection model[END_REF]. The bioavailability is also low in Sprague-Dawley rats (1 to 10.5 %) and in beagle dogs (< 1 to 7.3 %). CRS-3123 safety was sufficient for progression of the drug into clinical studies [START_REF] Nayak | Safety, Tolerability, Systemic Exposure, and Metabolism of CRS3123, a Methionyl-tRNA Synthetase Inhibitor Developed for Treatment of Clostridium difficile, in a Phase 1 Study[END_REF].

Phase 1

CRS-3123 has completed two phase 1 clinical studies. A double-bind, single-center, placebo-controlled single-ascending dose study was conducted in 40 healthy adults, with a dose of 100, 200, 400, 800 or 1200 mg [START_REF] Nayak | Safety, Tolerability, Systemic Exposure, and Metabolism of CRS3123, a Methionyl-tRNA Synthetase Inhibitor Developed for Treatment of Clostridium difficile, in a Phase 1 Study[END_REF]. The drug candidate was found safe and well tolerated, with the same frequency and severity of AEs between the drug group and the placebo group. Although bioavailability has not been precisely determined, it was found that plasma concentration at higher dose of CRS-3123 were above MICs of Gram-positive pathogenic bacteria. By LC-MS analysis, the N-glucuronide metabolite of CRS-3123 was observed in plasma and urine, indicating that the metabolite is excreted in urine. This is also the case for the non-metabolized CRS-3123.

The second phase 1 trial was a double-bind, single-center, placebo-controlled multiple-ascending dose study, conducted in 30 healthy adults with doses of 200, 400 and 600 mg administered twice daily during 10 days [START_REF] Lomeli | Multiple-Ascending-Dose Phase 1 Clinical Study of the Safety, Tolerability, and Pharmacokinetics of CRS3123, a Narrow-Spectrum Agent with Minimal Disruption of Normal Gut Microbiota[END_REF]. As for the first phase 1 study, CRS-3123 was safe and well tolerated. In particular, no prolongation of the QT interval was noticed. After administration, a large proportion of the drug was retained in the gut and excreted in feces. Indeed, the percentage of free drug and glucuronide metabolite in urine was inferior to 2 %. The low absorption of CRS-3123 leads to no sys-temic accumulation. Moreover, high concentrations of the drug are recovered in feces at any doses (2115 -8280 µg/g), which are superior to the MIC 90 value against C. difficile (1 µg/mL). However, even with high concentrations, CRS-3123 has a minimal effect on the normal microbiota and does not increase the proportion of Proteobacteria. With this narrow spectrum of activity, CRS-3123 is likely to reduce recurrences after treatment of CDI, as it was demonstrated for fidaxomicin [START_REF] Louie | Fidaxomicin preserves the intestinal microbiome during and after treatment of Clostridium difficile infection (CDI) and reduces both toxin reexpression and recurrence of CDI[END_REF].

Current advances

With a promising pharmacokinetic and bacterial selectivity profile for therapeutic use, CRS-3123 is currently evaluated in a phase 2 study, to compare efficacy of the drug against vancomycin in adults with CDI [START_REF] Crestone | A Phase 2, Randomized, Double-Blind, Comparator-Controlled, Multicenter Study to Evaluate the Safety and Efficacy of CRS3123 Compared With Oral Vancomycin in Adults With Clostridioides Difficile Infection, clinicaltrials[END_REF].

Lefamulin

Lefamulin is a semisynthetic derivative of the naturally occurring tricyclic diterpenoid pleuromutilin class of antibiotics [START_REF] Falcó | An overview of lefamulin for the treatment of community acquired bacterial pneumonia[END_REF][START_REF] Mercuro | Clinical Utility of Lefamulin: If Not Now, When?[END_REF]. The compound (formerly known as BC-3781) was developed by Nabriva Therapeutics and is currently marketed under the brand name Xenleta. The drug was approved by the FDA and the EMA in 2019 and 2020, respectively, as a short-course monotherapy for the treatment of community-acquired bacterial pneumonia (CABP) in adults [START_REF] Wu | In vitro Activity of Lefamulin Against the Common Respiratory Pathogens Isolated From Mainland China During 2017-2019[END_REF][START_REF] Covvey | Clinical use of lefamulin: A first-in-class semisynthetic pleuromutilin antibiotic[END_REF][START_REF] Wicha | Pharmacokinetics and safety of lefamulin after single intravenous dose administration in subjects with impaired-hepatic function[END_REF]. This is the first agent within the Pleuromutilin derivative class to be developed for systemic use in humans by oral and intravenous administration [START_REF] Falcó | An overview of lefamulin for the treatment of community acquired bacterial pneumonia[END_REF][START_REF] Perry | Therapeutic potential of lefamulin in the treatment of community-acquired pneumonia[END_REF].

Chemical synthesis

The synthetic route was patented in 2008 by Mang et al. [START_REF] Mang | Pleuromutilin derivatives for the treatment of diseases mediated by microbes[END_REF]. Lefamulin is synthesized in two step from pleuromutilin thiol [START_REF] Nagarajan | Pleuromutilin glycoside derivatives[END_REF] and syn-3,4-epoxycyclohexyl-carbamic acid tert-butyl ester [START_REF] Gómez-Sánchez | Synthesis and transformations of alkyl N-(1-cyclohex-3-enyl)carbamates prepared from cyclohex-3-ene carboxylic acid via Curtius rearrangement[END_REF].

After a S N 2 reaction leading to a mixture of diastereomers (1R, 2R, 4R) and (1S, 2S, 4S), the Boc protecting group is cleaved in acidic conditions. The mixture is then separated by chiral column chromatography to afford lefamulin (Figure 13).

Target and mechanism of action

Lefamulin selectively inhibits bacterial ribosomal translation without affecting the eukaryotic process through a unique mechanism of tight-fit binding to the peptidyl transferase center (PTC) on the 50S ribosomal subunit [START_REF] Falcó | An overview of lefamulin for the treatment of community acquired bacterial pneumonia[END_REF][START_REF] Rodvold | Introduction: lefamulin and pharmacokinetic/pharmacodynamic rationale to support the dose selection of lefamulin[END_REF]. More precisely, the tricyclic core and the side chain of lefamulin interact respectively with the A (aminoacyl) and P (peptidyl) sites of the subunit, through hydrophobic and hydrogen bond interactions (Figure 14) [START_REF] Falcó | An overview of lefamulin for the treatment of community acquired bacterial pneumonia[END_REF][START_REF] Rodvold | Introduction: lefamulin and pharmacokinetic/pharmacodynamic rationale to support the dose selection of lefamulin[END_REF][START_REF] Eraikhuemen | Treatment of Community-Acquired Pneumonia: A Focus on Lefamulin[END_REF]. A binding pocket is then formed and the bacterial ribosome closes around the tricyclic core, preventing the correct positioning and binding of tRNA at the A and P sites, which leads to protein synthesis inhibition. However, once elongation has started, lefamulin is ineffective [START_REF] Mercuro | Clinical Utility of Lefamulin: If Not Now, When?[END_REF][START_REF] Perry | Therapeutic potential of lefamulin in the treatment of community-acquired pneumonia[END_REF].

Structure-activity relationship studies

Lefamulin was designed after a comprehensive study of the possible modifications at the C14-side chain of pleuromutilins [START_REF] Adhikary | Lefamulin: a New Hope in the Field of Community-Acquired Bacterial Pneumonia[END_REF] (Figure 15).

 R side chain: Replacement of the cyclohexyl moiety with an aromatic or non-aromatic heterocycle was tolerated. The thioether function resulted in a gain in potency and water solubility [START_REF] Li | Design, synthesis, and biological activity evaluation of a series of pleuromutilin derivatives with novel C14 side chains[END_REF].

 Linker: the ester linker was favorable for the antibacterial activity. Indeed, the replacement with an amide or a methoxy linker resulted in less potent compounds.

 Tricyclic core: the tricyclic moiety together with its hydroxyl and vinyl groups are essential for the antimicrobial activity.

Pleuromutilin Lefamulin

Tricyclic core essential for activity

Linker Ester favorable Thioether increases potency R-chain (C14)

Figure 15. Summary of the SARs of Lefamulin starting from pleuromutilin.

In vitro activity

Lefamulin selectively inhibits prokaryotic ribosomal protein synthesis [START_REF] Rodvold | Introduction: lefamulin and pharmacokinetic/pharmacodynamic rationale to support the dose selection of lefamulin[END_REF]. This selectivity is due to the double layer nucleotides around the pleuromutilin tricyclic core, which creates a structurally and chemically different binding pocket between the prokaryotes and the eukaryotes/archaea. [START_REF] Eyal | A novel pleuromutilin antibacterial compound, its binding mode and selectivity mechanism[END_REF] Lefamulin has a potent activity against Gram-positive (S. [START_REF] Falcó | An overview of lefamulin for the treatment of community acquired bacterial pneumonia[END_REF][START_REF] Covvey | Clinical use of lefamulin: A first-in-class semisynthetic pleuromutilin antibiotic[END_REF][START_REF] Rodvold | Introduction: lefamulin and pharmacokinetic/pharmacodynamic rationale to support the dose selection of lefamulin[END_REF][START_REF] Dillon | Lefamulin: a promising new pleuromutilin antibiotic in the pipeline[END_REF][START_REF] Veve | Lefamulin: Review of a Promising Novel Pleuromutilin Antibiotic[END_REF]. Lefamulin is also fully active against MRSA, β-hemolytic streptococci (group A: Streptococcus pyogenes and group B: Streptococcus agalactiae) and other drug-resistant organisms such as macrolide-resistant M. pneumoniae, for instance [START_REF] Covvey | Clinical use of lefamulin: A first-in-class semisynthetic pleuromutilin antibiotic[END_REF][START_REF] Rodvold | Introduction: lefamulin and pharmacokinetic/pharmacodynamic rationale to support the dose selection of lefamulin[END_REF]. Other potent activities against coagulase-negative staphylococci and viridans group streptococci have also been demonstrated [START_REF] Rodvold | Introduction: lefamulin and pharmacokinetic/pharmacodynamic rationale to support the dose selection of lefamulin[END_REF][START_REF] Dillon | Lefamulin: a promising new pleuromutilin antibiotic in the pipeline[END_REF]. Pleuromutilins suppress bacterial protein translation with excellent specificity, but has no effect on eukaryotic protein synthesis, as shown by in vitro transcription/translation assays using bacterial ribosomes

In vitro studies revealed that lefamulin is both a substrate and an inhibitor of the CYP3A isoenzyme [START_REF] Wicha | Pharmacokinetics and tolerability of lefamulin following intravenous and oral dosing[END_REF]. Lefamulin has shown in vitro activity against aerobic Gram-positive bacteria, a limited number of fastidious Gram-negative bacteria, and atypical bacteria [START_REF] Dillon | Lefamulin: a promising new pleuromutilin antibiotic in the pipeline[END_REF][START_REF] Chahine | Lefamulin: The First Systemic Pleuromutilin Antibiotic[END_REF]. Interestingly, the compound is bactericidal against S. pneumoniae, H. influenzae and M. pneumoniae at concentrations of 0.06, 0.5 and 0.008 µg/ml, respectively, and bacteriostatic against S. aureus and S. pyogenes [START_REF] Eraikhuemen | Treatment of Community-Acquired Pneumonia: A Focus on Lefamulin[END_REF].

In addition, lefamulin exhibits excellent activity against drug-resistant strains of S. pneumoniae, the most prevalent bacterial cause of CABP. The drug-resistant strains include isolates resistant to penicillins, cephalosporins, clarithromycin, doxycycline, and trimethoprim-sulfamethoxazole, as well as MDR isolates [START_REF] Taylor | In vitro susceptibility of common bacterial pathogens causing respiratory tract infections in Canada to lefamulin, a new pleuromutilin[END_REF]. Lefamulin also displayed potent in vitro activity against Gram-positive organisms commonly associated with ABSSSIs, which include S. pyogenes, S. aureus and MRSA strains [START_REF] Paukner | Antimicrobial Activity of the Pleuromutilin Antibiotic BC-3781 against Bacterial Pathogens Isolated in the SENTRY Antimicrobial Surveillance Program in 2010[END_REF][START_REF] Sader | Antimicrobial Activity of the Investigational Pleuromutilin Compound BC-3781 Tested against Gram-Positive Organisms Commonly Associated with Acute Bacterial Skin and Skin Structure Infections[END_REF][START_REF] Pollack | Acute Bacterial Skin and Skin Structure Infections (ABSSSI): Practice Guidelines for Management and Care Transitions in the Emergency Department and Hospital[END_REF]. Although not indicated for this use, lefamulin has in vitro activity against C. trachomatis, susceptible and MDR M. genitalium and susceptible and resistant N. gonorrhoeae, which are the most relevant bacterial pathogens that cause sexually transmitted infections (STI) [START_REF] Paukner | In Vitro Activity of Lefamulin against Sexually Transmitted Bacterial Pathogens[END_REF].

In vivo experiments

Lefamulin has shown a potent in vivo efficacy in pulmonary infection mouse model [START_REF] Wicha | Pharmacokinetics/pharmacodynamics of lefamulin in a neutropenic murine pneumonia model with Staphylococcus aureus and Streptococcus pneumoniae[END_REF]. The study showed that the compound is not affected by pulmonary surfactant and is able to quickly penetrate and accumulate in J774 mouse macrophages, with a plateau reached in 3 h. The PK parameters were assessed in mice (35 to 70 mg/kg), and demonstrated a fast distribution of lefamulin from the plasma to the target tissue (the epithelial lining fluid (ELF) in lungs), with a 2-fold increase in the drug exposure in the ELF during the 5.5 h period. Moreover, the study displayed desirable PD data in the neutropenic lung infections experiments, where mice received cyclophosphamide before being infected with single S. pneumoniae or S. aureus strains, and subcutaneous lefamulin (1.25-160 mg/kg) given twice daily post-infection.

The same authors reported that lefamulin exhibits time-and concentration-dependent killing in a neutropenic murine thigh infection model [START_REF] Wicha | In vivo pharmacodynamics of lefamulin, the first systemic pleuromutilin for human use, in a neutropenic murine thigh infection model[END_REF]. In this study, free drug values (~20% free fraction)

were also established, ranging from 1.98-6.42 and 8.04-16.5 for the S. pneumoniae or S. aureus strains, respectively.

Phase 1

A four-period crossover study was performed to assess the bioavailability of lefamulin [START_REF] Therapeutics | A Study to Assess the Bioavailability and Pharmacokinetics of an Immediate Release Tablet Formulation of Lefamulin When Administered to Fed and Fasted Healthy Subjects in Comparison to an Intravenous Formulation and a Capsule Formulation (NCT02557789)[END_REF].

Moreover, a single-dose study was performed to evaluate the penetration of lefamulin into the ELF.

The latter was performed on healthy human volunteers and demonstrated the good penetration into the ELF (ƒAUC 0-24 of 8978 ng•h/mL and C max 706 ng/mL) but also in other tissues such as the skeletal muscle and adipose tissue [START_REF] Zeitlinger | Simultaneous assessment of the pharmacokinetics of a pleuromutilin, lefamulin, in plasma, soft tissues and pulmonary epithelial lining fluid[END_REF]. Last but not least, the PK parameters of IV lefamulin and its major metabolite BC-8041 were assessed in subjects with different degrees of hepatic impairment as well as healthy individuals. Overall, lefamulin was well tolerated, and no significant pharmacokinetic difference was observed between the compound and its metabolite [START_REF] Wicha | Pharmacokinetics and safety of lefamulin after single intravenous dose administration in subjects with impaired-hepatic function[END_REF].

Phase 2

A double-blind, parallel group, multicenter phase 2 study was performed to compare the efficacy of IV lefamulin to IV vancomycin in 210 adults with ABSSIs. Similar clinical success rates across pathogens were observed between the two groups, including methicillin-susceptible S. aureus (MSSA)

and MRSA [START_REF] Prince | Phase II Clinical Study of BC-3781, a Pleuromutilin Antibiotic, in Treatment of Patients with Acute Bacterial Skin and Skin Structure Infections[END_REF]. To date, no subsequent phase 3 trials have been pursued for the treatment of ABSSIs.

Phase 3

Although no phase 2 study for the indication of CABP was performed, two multicentre, randomized, double-blind, double-dummy phase 3 trials were conducted for this infectious disease: the Lefamulin Evaluation Against Pneumonia 1 and 2 (LEAP 1 and LEAP 2). The LEAP 1 trial assessed the efficacy and safety of IV lefamulin compared to IV moxifloxacin +/-linezolid in 551 adults patients with moderate to severe CABP [START_REF] Therapeutics | A Phase 3, Randomized, Double-Blind, Double-Dummy Study to Compare the Efficacy and Safety of Lefamulin (BC 3781) Versus Moxifloxacin (With or Without Adjunctive Linezolid) in Adults With Community-Acquired Bacterial Pneumonia (NCT02559310), clinicaltrials[END_REF][START_REF] File | Efficacy and Safety of Intravenous-to-oral Lefamulin, a Pleuromutilin Antibiotic, for the Treatment of Community-acquired Bacterial Pneumonia: The Phase III Lefamulin Evaluation Against Pneumonia (LEAP 1) Trial[END_REF]. The LEAP 2 study compared the safety and efficacy of 600mg oral lefamulin twice daily for 5 days versus 400 mg of oral moxifloxacin once daily for 7 days in 738 adults patients with moderate CABP [START_REF] Therapeutics | A Phase 3, Randomized, Double-Blind, Double-Dummy Study to Compare the Efficacy and Safety of Oral Lefamulin (BC 3781) Versus Oral Moxifloxacin in Adults With Community-Acquired Bacterial Pneumonia (NCT02813694), clinicaltrials[END_REF][START_REF] File | Lefamulin efficacy and safety in a pooled phase 3 clinical trial population with community-acquired bacterial pneumonia and common clinical comorbidities[END_REF]. Data from both trials have shown noninferior efficacy with short course therapy compared with moxifloxacin in the treatment of CABP [START_REF] Perry | Therapeutic potential of lefamulin in the treatment of community-acquired pneumonia[END_REF].

Safety and tolerability

Intravenous and oral lefamulin was generally well tolerated in phase 1 and phase 2 clinical studies [START_REF] Wicha | Pharmacokinetics and tolerability of lefamulin following intravenous and oral dosing[END_REF][START_REF] Zeitlinger | Simultaneous assessment of the pharmacokinetics of a pleuromutilin, lefamulin, in plasma, soft tissues and pulmonary epithelial lining fluid[END_REF]. From the phase 2 trial, the most frequently reported AEs were headache, nausea, and diarrhea [START_REF] Prince | Phase II Clinical Study of BC-3781, a Pleuromutilin Antibiotic, in Treatment of Patients with Acute Bacterial Skin and Skin Structure Infections[END_REF]. In both LEAP 1 and LEAP 2 trials, most AEs were mild or moderate in severity for both arm groups [START_REF] File | Efficacy and Safety of Intravenous-to-oral Lefamulin, a Pleuromutilin Antibiotic, for the Treatment of Community-acquired Bacterial Pneumonia: The Phase III Lefamulin Evaluation Against Pneumonia (LEAP 1) Trial[END_REF][START_REF] Alexander | Oral Lefamulin vs Moxifloxacin for Early Clinical Response Among Adults With Community-Acquired Bacterial Pneumonia: The LEAP 2 Randomized Clinical Trial[END_REF]. Lefamulin remains contraindicated in patients with known hypersensitivity to pleuromutilins [START_REF]XENLETA® (lefamulin) Safety and Tolerability, XENLETA TM Lefamulin Website Healthc. Prof[END_REF]. Interestingly, the concomitant IV or oral administration of lefamulin with strong and/or moderate CYP3A or P-glycoprotein inducers can reduce the efficacy of the drug and should be then avoided. On the other hand, IV or oral coadministration of lefamulin with drugs that strongly or moderately inhibit CYP3A or P-glycoprotein inhibitors might increase the risk of AEs.

Current advances

Lefamulin is currently marketed as treatment for CABP caused by susceptible microorganisms.

Moreover, lefamulin could be an option for the treatment of other serious bacterial infections caused by MRSA and vancomycin-resistant E. faecium, but also by MDR organisms associated with STI (N. gonorrhoeae and M. genitalium). The use of lefamulin could be also considered for patients with high-risk/recurrent CDI [START_REF] Mercuro | Clinical Utility of Lefamulin: If Not Now, When?[END_REF].

FabI enzyme as a drug target

Introduction

FabI is an essential NADH-dependent enzyme which catalyzes the reduction of enoyl-acyl carrier protein (enoyl-ACP) in the last step of the bacterial fatty acid biosynthesis pathway (FAS-II), involved in the synthesis of phospholipids. Contrary to the multifunctional mammalian type I fatty acid synthase (FAS-I), each reaction of FAS-II is catalyzed by a different enzyme [START_REF] Lu | Inhibitors of FabI, an Enzyme Drug Target in the Bacterial Fatty Acid Biosynthesis Pathway[END_REF]. This structural difference between the two pathways is the reason why inhibitors of the FAS-II pathway are selective and safe for use in humans. On the other hand, the complexity in the bacterial fatty acid metabolism can narrow the spectrum of activity of FabI inhibitors [START_REF] Lu | Inhibitors of FabI, an Enzyme Drug Target in the Bacterial Fatty Acid Biosynthesis Pathway[END_REF]. However, their development has continued since FabI represents an essential and validated target in multidrug-resistant S. aureus.

FabI inhibitors can be divided in two groups, depending on their ability to form a covalent or non-covalent adduct with the NAD + cofactor. The covalent inhibitors include the diazaborines and the first-line tuberculosis drug isoniazid whereas inhibitors that do not form a covalent adduct include for instance AFN-1252, the preclinical drug candidate fabimycine [START_REF] Parker | An Iterative Approach Guides Discovery of the FabI Inhibitor Fabimycin, a Late-Stage Antibiotic Candidate with In Vivo Efficacy against Drug-Resistant Gram-Negative Infections[END_REF], and the preservation agent triclosan, forming a much broader and heterogeneous group [START_REF] Lu | Inhibitors of FabI, an Enzyme Drug Target in the Bacterial Fatty Acid Biosynthesis Pathway[END_REF].

AFN-1252

AFN-1252 is a selective inhibitor of S. aureus the previously obtained ester with HBr in glacial acetic acid. The coupling between the two moieties by using HOBt and EDCI leads to the formation of AFN-1252 (Figure 16). Afabicin, the corresponding prodrug of AFN-1252, is obtained in three more steps. Firstly, AFN-1252 is reacted with chloromethyl bis(2-trimethylsilylethyl)phosphate, then the two protecting groups are removed in acidic conditions.

Finally, the addition of the desired base (ethanolamine, NH 4 OH, NaOH) leads to the corresponding salt of afabicin [START_REF] Partridge | Prodrug Derivatives of (e)-N-Methyl-N-((3-Methylbenzofuran-2-Yl)methyl)-3-(7-Oxo-5,6,7,8-Tetrahydro-1,8-Naphthyridin-3-Yl)acryla mide[END_REF]. FabI was confirmed to be the target of AFN-1252 by using biochemistry, macromolecular synthesis, genetics and co-crystallization of AFN-1252-FabI complex (Figure 17). The 3-methylbenzofuran moiety of AFN-1252 occupies a hydrophobic pocket of the enzyme created by residues F204, Y157 and Y147. The oxotetrahydronaphtyridine moiety of the molecule occupies a more solvent exposed region and is involved in two hydrogen bond interactions with A97. The carbonyl of the linking cis-amide form two hydrogen bond interactions with the 2'-hydroxyl of the cofactor and the hydroxyl group of Y157 [START_REF] Kaplan | Mode of Action, In Vitro Activity, and In Vivo Efficacy of AFN-1252, a Selective Antistaphylococcal FabI Inhibitor[END_REF].

Structure-activity relationship studies

High-throughput screening of 305,189 compounds from the GSK compound collection identified the benzodiazepine derivative as a low micromolar inhibitor of S. aureus FabI [START_REF] Payne | Discovery of a Novel and Potent Class of FabI-Directed Antibacterial Agents[END_REF][START_REF] Miller | Discovery of Aminopyridine-Based Inhibitors of Bacterial Enoyl-ACP Reductase (FabI)[END_REF]. In order to improve potency, SAR studies were performed (Figure 18):

• Removal of the ester chain: the synthesis of compounds without the ester chain showed that the chain is not important for the inhibition of FabI.

• Ring-opened variants: ring-opened variants of the benzodiazepine derivative maintained the activity against S. aureus, indicating that the seven membered ring is not necessary for the antibacterial activity.

• Importance of the para amino group: removal of the para amino group caused a substantial decrease in activity.

• Replacement of the aniline moiety with aminopyridine: aminopyridine is an essential moiety engaged in two hydrogen-bonding interactions with Ala 97 from the cofactor.

• Introduction of the unsaturated two-carbon linker: acrylamide moiety was introduced to maximize the overlapping with previously synthesized promising compounds. The synthesis of the corresponding saturated derivatives led to inactive compounds.

In 2020, Parker et al. were able to re-design AFN-1252 into a derivative, AFN-1252-NH3, that accumulates in Escherichia coli and possesses an antibacterial activity against Gram-negative bacteria [START_REF] Parker | An Iterative Approach Guides Discovery of the FabI Inhibitor Fabimycin, a Late-Stage Antibiotic Candidate with In Vivo Efficacy against Drug-Resistant Gram-Negative Infections[END_REF].

6.2.5

In vivo experiments AFN-1252 was orally available in mouse pharmacokinetic studies, where a linear relationship was observed between the administered dose (0.3, 0.6, and 1.0 mg/kg), the observed C max (154, 196, and 304 ng/ml) and AUC 0-t (367, 806, and 1.030 ng-h/ml). The long elimination half-life measured (5-7 h) indicated the potential for once-or twice-daily administration in human. Kaplan et al. also showed that a single oral dose of 1 mg/kg was efficacious in a mouse model of septicemia, with 100% survival from an otherwise lethal peritoneal infection of S. aureus. The group also compared the in vivo efficacy of a single oral administration of AFN-1252 to linezolid, and an ED 50 of 0.15 mg/kg, suggesting that AFN-1252 was 12 to 24 times more potent than linezolid in this model [START_REF] Kaplan | Mode of Action, In Vitro Activity, and In Vivo Efficacy of AFN-1252, a Selective Antistaphylococcal FabI Inhibitor[END_REF].

Phase 1

A double-blind, randomized, placebo-controlled, two-part study was initiated to assess the safety, tolerability and pharmacokinetic properties of an immediate release tablet formulation of AFN-1252 in healthy subjects, following oral administration in an ascending dose trial [START_REF] Hafkin | Safety, tolerability and pharmacokinetics of AFN-1252 administered as immediate release tablets in healthy subjects[END_REF]. In the first part, single doses of 100, 200, 300, or 400 mg of AFN-1252 were administered. In the second part, subjects received 100, 200, 300 and 400 mg twice a day. This study showed oral doses of AFN-1252 to be safe and well tolerated and highlighted the potential of the drug for once or twice-a-day dosing to treat staphylococcal infections. AFN-1252 was well-absorbed with C max at 3-4 h when given once per day and 2.5-9 h when administered twice daily. to the active metabolite AFN-1252 and its antibacterial activity was better than AFN-1252 administered by itself orally [START_REF] Zheng | Comparative study of resazurin reduction and MTT assays for cytocompatibility evaluation of nanofibrous materials[END_REF]. The prodrug was studied in a phase 2 clinical trial to compare its efficacy, safety, and tolerability with those of vancomycin/linezolid in the treatment of ABSSSIs [START_REF]A Phase 2, Randomized, Double-Blind, Multicenter Study of Safety, Tolerability, and Efficacy of Debio 1450 vs Vancomycin (IV)/Linezolid (Oral) in the Treatment of Acute Bacterial Skin and Skin Structure Infections (ABSSSI) Due to Staphylococcus Sensitive or Resistant to Methicillin, clinicaltrials[END_REF]. In a phase 1 clinical trial, the penetration of afabicin into bones in patients undergoing elective hip replacement surgery was evaluated [START_REF]A Phase 1, Open-Label Pharmacokinetic Study to Investigate Drug Penetration in the Bone After Repeated Oral Administration of Debio 1450 to Patients Undergoing Hip Replacement Surgery, clinicaltrials[END_REF]. Moreover, a phase 2 clinical trial is underway to study the safety and efficacy of afabicin in human subjects with bone and joint infections caused S. aureus.

[156].

FtsZ protein as a drug target

Introduction

FtsZ is a bacterial protein involved in cell division forming single-stranded filaments by uanosine -triphosphate (GTP) dependent polymerization. These FtsZ filaments form the cytokinetic ring (called Z-ring) at midcell, able to recruit other critical elements for peptidoglycan synthesis and septum formation, which leads to the induction of cell division. Because FtsZ is essential for the germ and is widely conserved throughout bacteria, it displays all the attractive features of a new target for antibiotic compounds (Figure 19) [START_REF] Haydon | An Inhibitor of FtsZ with Potent and Selective Anti-Staphylococcal Activity[END_REF].

TXA707/TXA709

TXA707 is the first antibacterial compound targeting FtsZ to reach clinical development. It is being developed as an oral prodrug (TXA709) by Taxis Pharmaceuticals to treat MRSA infections.

TXA707 is derived from PC190723, which is coming from a fragment-based medicinal chemistry program starting from 3-methoxybenzamide (3-MBA), a known FtsZ inhibitor in Bacillus subtilis [START_REF] Ohashi | The lethal effect of a benzamide derivative, 3-methoxybenzamide, can be suppressed by mutations within a cell division gene, ftsZ, in Bacillus subtilis[END_REF].

To improve the pharmacokinetic properties of PC190723, the chlorine atom on the pyridyl ring was replaced with a trifluoromethyl group, resistant to metabolization by cytochrome P450 enzymes. Transmission electron micrography studies showed that within 1 hour of treatment with TXA707, S.

aureus cells became enlarged enough, resulting to cell lysis. Furthermore, TXA707 was co-crystallized in complex with FtsZ (Figure 21) [START_REF] Fujita | Structural Flexibility of an Inhibitor Overcomes Drug Resistance Mutations in Staphylococcus aureus FtsZ[END_REF]. Eventually, the target was validated with the selection of TXA707-resistant MRSA mutants (frequencies of resistance: 3.10 8 in MRSA ATCC 43300; 1.10 8 in an MRSA clinical isolate). Twenty resistant clones were isolated and the ftsZ gene was sequenced. Data showed that all TXA707-resistant strains carried a mutation within this gene [START_REF] Kaul | TXA709, an FtsZ-Targeting Benzamide Prodrug with Improved Pharmacokinetics and Enhanced In Vivo Efficacy against Methicillin-Resistant Staphylococcus aureus[END_REF].

It is worthwhile noting that the mechanism of action of TXA707 on FtsZ echoes that of paclitaxel (tradename TAXOL) on tubulin, the eukaryotic structural homologue of FtsZ. Indeed, both compounds prevent polymers disassembly, eventually leading to cell division halt.

Structure-activity relationship studies

The starting point for the development of TXA707 was 3-methoxybenzamide, proven to be an FtsZ inhibitor in B. subtilis [START_REF] Ohashi | The lethal effect of a benzamide derivative, 3-methoxybenzamide, can be suppressed by mutations within a cell division gene, ftsZ, in Bacillus subtilis[END_REF]. The methoxy and amide moieties in their respective positions were shown to be essential for the activity on FtsZ (Figure 22). Authors then investigated modifications around the 3-methoxybenzamide core:

 Substitution of the phenyl ring: substitution was detrimental, except for the addition of halogen atoms in ortho position to the amide.

 Substitution of the methoxy moiety: substitution with other alkoxy chains was possible. It was shown that increasing the length of the alkyl chain up to nine carbon atoms allowed an 8000-fold improvement in potency on B. subtilis compared to the methoxy moiety [START_REF] Czaplewski | Antibacterial alkoxybenzamide inhibitors of the essential bacterial cell division protein FtsZ[END_REF]. However, this also led to an increase in PPB. Subsequent replacement of the alkyl chain with various heterocycles was investigated, where the benzothiazole gave interesting results.

 Substitution of the benzothiazole ring: substitution in position 4 was detrimental. In position 6, substitution was tolerated with small moieties (methyl, chlorine) but larger groups led to inactive compounds (phenyl, methoxy). Lastly, substitution in position 5 was favorable, particularly with a chlorine or bromine atom, phenyl ring, propyl or ethoxy chain.

 Modification of the benzothiazole ring: to further decrease the plasma protein binding, thiazolopyridine analogues were synthesized. This allowed the identification of PC190723, which displayed a good potency on S. aureus (MIC = 1 µg/mL) and a relatively low PPB (85.4% bound) [START_REF] Haydon | Creating an Antibacterial with in Vivo Efficacy: Synthesis and Characterization of Potent Inhibitors of the Bacterial Cell Division Protein FtsZ with Improved Pharmaceutical Properties[END_REF]. Because PC190723 displayed a low solubility, the maximum dose that could be administered orally was not sufficient to display any efficacy. To circumvent this issue, an imide prodrug with a basic piperidine, TXY541, was synthesized, allowing a great improvement in solubility [START_REF] Kaul | Pharmacokinetics and in vivo antistaphylococcal efficacy of TXY541, a 1-methylpiperidine-4-carboxamide prodrug of PC190723[END_REF].

Administration of TXY541 proved to be efficacious in vivo but required high doses (192 mpk against a MRSA strain). To reduce administered doses, metabolization was decreased by replacement of the chlorine atom on the pyridyl ring with a trifluoromethyl group, yielding TXA707 and the corresponding imide prodrug TXA709 [START_REF] Kaul | TXA709, an FtsZ-Targeting Benzamide Prodrug with Improved Pharmacokinetics and Enhanced In Vivo Efficacy against Methicillin-Resistant Staphylococcus aureus[END_REF]. 7.2.4. In vitro activity TXA707 exhibits potent bactericidal activity against a panel of 84 S. aureus clinical strains with various levels of resistance against current treatment options. The most prevalent MIC for TXA707 was 1 µg/mL against the five types of clinical isolates tested: MRSA, VISA, VRSA, DNSSA (Daptomycin Non-Susceptible S. aureus), and LNSSA (Linezolid Non-Susceptible S. aureus). The prodrug TXA709 also showed antibacterial activity against all S. aureus strains tested so far, however its modal MICs were 2-to 4-fold higher than those of TXA707. This is explained by a slower conversion from TXA709 to TXA707 in the culture medium [START_REF] Kaul | TXA709, an FtsZ-Targeting Benzamide Prodrug with Improved Pharmacokinetics and Enhanced In Vivo Efficacy against Methicillin-Resistant Staphylococcus aureus[END_REF]. Overall, these results make TXA707 and its prodrug TXA709 a promising alternative for the treatment of multidrug resistant S. aureus.

Regarding selectivity, it was shown in vitro that PC190723, the parent analogue of TXA707, did not impact positively or negatively the polymerization of tubulin, the eukaryote counterpart of FtsZ [START_REF] Haydon | An Inhibitor of FtsZ with Potent and Selective Anti-Staphylococcal Activity[END_REF]. Moreover, TXA709 demonstrated minimal toxicity on mammalian cells (HeLa and MDCK, IC 50 > 120 µg/mL) [START_REF] Kaul | TXA709, an FtsZ-Targeting Benzamide Prodrug with Improved Pharmacokinetics and Enhanced In Vivo Efficacy against Methicillin-Resistant Staphylococcus aureus[END_REF].

In vivo experiments

Firstly, the pharmacokinetic properties of TXA709 were measured in male BALB/c mice following oral (32 mpk) or IV (24 mpk) administration. TXA709 was not detectable in plasma, indicating a rapid conversion into the active compound TXA707. Maximum plasmatic concentrations were attained after 1h following oral administration, and the bioavailability was calculated to be 95% [START_REF] Kaul | TXA709, an FtsZ-Targeting Benzamide Prodrug with Improved Pharmacokinetics and Enhanced In Vivo Efficacy against Methicillin-Resistant Staphylococcus aureus[END_REF].

The efficacy of TXA709 was demonstrated in a mouse peritonitis model, both with MSSA and MRSA strains. It was demonstrated that a dose of 36 mpk by intravenous administration and a dose of 48 mpk by oral administration were sufficient for the survival of all mice infected by the MRSA strain. TXA709 and TXA707 were further evaluated in a mouse thigh model of MRSA infection. After intravenous administration of TXA709, volume of distribution at steady state for TXA707 was measured at 2.02 L/kg. This value is three times higher than the total water volume of the mouse (0.7 L/kg), indicating that TXA707 is well distributed in the tissues. Moreover, an oral dose of 120 mg/kg of TXA709 in the same mouse thigh infection model resulted in a 2-log reduction in bacterial CFU [START_REF] Kaul | TXA709, an FtsZ-Targeting Benzamide Prodrug with Improved Pharmacokinetics and Enhanced In Vivo Efficacy against Methicillin-Resistant Staphylococcus aureus[END_REF].

Lastly, PK/PD parameters were evaluated in a murine neutropenic thigh infection model and the ratio AUC/MIC was deemed to be the PK/PD index most closely linked to efficacy [START_REF] Lepak | In Vivo Pharmacodynamic Evaluation of an FtsZ Inhibitor, TXA-709, and Its Active Metabolite, TXA-707, in a Murine Neutropenic Thigh Infection Model[END_REF]. 7.2.6. Current advances TXA709 has been evaluated in a non-registered phase 1 clinical trial and no serious AE was reported [START_REF]Our Pipeline -TAXIS Pharmaceutical[END_REF].

Conclusion and perspectives

For the first time in decades, pharmaceutical companies are refuelling the pipeline of antibiotics.

To date, at least eight compounds with new targets and/or MOA are in clinical trials, including two in phase 3, or have been recently approved. They represent a new hope to fight the bad bugs that are becoming more and more resistant to current antibiotics.

Beside these clinical candidates, other promising antibiotics and/or new targets have been reported in the literature. This is the case for a series of novel tetrazole ligands targeting LpxA, the first enzyme in the lipid A pathway [START_REF] Sacco | Structure-Based Ligand Design Targeting Pseudomonas aeruginosa LpxA in Lipid A Biosynthesis[END_REF]. Moreover, over twenty potential candidates targeting the bacterial RNA polymerase enzyme have been designed [START_REF] Sartini | New Antimicrobials Targeting Bacterial RNA Polymerase Holoenzyme Assembly Identified with an in Vivo BRET-Based Discovery Platform[END_REF][START_REF] Kirsch | Beyond the approved: target sites and inhibitors of bacterial RNA polymerase from bacteria and fungi[END_REF].

In addition, other strategies are being explored such as pathoblockers to disarm bacteria or adjuvants like efflux pump inhibitors (EPI) to potentialize current antibiotics.

Unlike antibiotics, which kill bacteria or inhibit growth and so exert a selection pressure, pathoblockers inhibit virulence factors. Virulence is the ability of bacteria to cause disease and is mediated by factors such as toxins, proteases or cytolysins [START_REF] Clatworthy | Targeting virulence: a new paradigm for antimicrobial therapy[END_REF]. Therefore, antivirulence drugs disrupt the relation between the host and the pathogen, causing the disarmament of bacteria which can then be eliminated by the immune system. Because the bacterium viability is not impacted, pathoblockers are believed to reduce resistance development. To date, only one small molecule acting as a pathoblocker has been evaluated in clinical trials: fluorothiazinon (formerly CL-55), developed by the Gamaleya Research Institute of Epidemiology and Microbiology in Russia.

Fluorothiazinon inhibits the type III secretion system, a well conserved system among Gram-negative bacteria allowing the secretion of several virulence factors [START_REF] Zigangirova | Fluorothiazinon, a small-molecular inhibitor of T3SS, suppresses salmonella oral infection in mice[END_REF][START_REF] Nesterenko | A small-molecule compound belonging to a class of 2,4-disubstituted 1,3,4-thiadiazine-5-ones suppresses Salmonella infection in vivo[END_REF][START_REF] Sheremet | Small Molecule Inhibitor of Type Three Secretion System Belonging to a Class 2,4-disubstituted-4H-[1,3,4]-thiadiazine-5-ones Improves Survival and Decreases Bacterial Loads in an Airway Pseudomonas aeruginosa Infection in Mice[END_REF][START_REF] Bondareva | Preventative treatment with Fluorothiazinon suppressed Acinetobacter baumannii-associated septicemia in mice[END_REF]. After completing a phase 1 study [START_REF]An Open Study to Assess the Safety and Pharmacokinetics of Fluorothyazinone Used as a Single-Dose Administration or a Treatment Course in Healthy Volunteers, clinicaltrials[END_REF], fluorothiazinon is currently evaluated in a phase 2 clinical study for the treatment of complicated urinary tract infections caused by P. aeruginosa [START_REF]Randomized, Blind, Placebo-controlled Study to Evaluate Safety and Efficacy of Ftortiazinon in Combination With Maxipime®[END_REF]. Moreover, recent studies reported quorum sensing inhibitors to reduce virulence in A. baumannii [START_REF] Seleem | Drugs with new lease of life as quorum sensing inhibitors: for combating MDR Acinetobacter baumannii infections[END_REF][START_REF] Zhong | Quorum Sensing Inhibition or Quenching in Acinetobacter baumannii: The Novel Therapeutic Strategies for New Drug Development[END_REF].

EPIs are compounds able to block bacterial multidrug efflux transporters. These pumps are able to extrude several classes of antibiotics from the bacteria such as tetracyclines, macrolides, -lactams, fluoroquinolones, therefore decreasing their intra-bacterial concentration and eventually rendering them ineffective [START_REF] Li | The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria[END_REF]. (Over)expression of multidrug efflux transporters, notably from the resistance-nodulation-cell division family, is one of the most worrying resistance mechanisms regarding multidrug antibacterial resistance. One strategy to override this defense mechanism is the conception of EPIs [START_REF] Compagne | Update on the Discovery of Efflux Pump Inhibitors against Critical Priority Gram-Negative Bacteria[END_REF]. Administrating an EPI in combination with an antibiotic effluxed by the targeted bacterial transporters could allow to restore the antibiotic activity and decrease the necessary antibiotic dose, thus reducing the selection pressure [START_REF] Blanco | The development of efflux pump inhibitors to treat Gram-negative infections[END_REF].

Figure 1 .

 1 Figure 1. New drugs in the clinical pipeline or recently approved for the treatment of infections caused by resistant bacteria. For prodrugs, the name of the active pharmaceutical ingredient is written into brackets.

Figure 2 .

 2 Figure2. Description of the synthetic pathway leading to gepotidacin[START_REF] Davies | Tricyclic nitrogen containing compounds as antibacterial agents[END_REF][START_REF] Widdowson | Tricyclic nitrogen containing compounds for treating neisseria gonorrhoea infection[END_REF].

Figure 3 .Figure 4 .

 34 Figure 3. Views of the gepotidacin complex formed with S. aureus DNA gyrase and uncleaved DNA (PDB code: 6QTP). Due to a relative movement of the two GyrA subunits, the two existing positions of gepotidacin are represented.

3 Figure 5 .

 35 Figure 5. Description of the synthetic pathway leading to zoliflodacin [47].

Figure 6 .

 6 Figure 6. Summary of the SARs of zoliflodacin.

Figure 7 .

 7 Figure 7. Description of the synthetic pathway leading to ibezapolstat [70].

Figure 8 .

 8 Figure 8. Binding mode of ibezapolstat (IBZ) to dCTP and DNA pol IIIC. Adapted from Acurx Pharmaceuticals [74].

Figure 9 . 3 . 2 . 4 .

 9324 Figure 9. Summary of the SARs of ibezapolstat from H 2 -HPUra.3.2.4. In vitro activity

  al. at the University of Strathclyde. In the end, MGB-BP-3 was found to be the most active against Gram-positive bacteria. Collaboration with MGB Biopharma enabled the preclinical and clinical development of MGB-BP-3 for the treatment of gastrointestinal infections caused by C. difficile [87-89]. 4.2.1. Chemical Synthesis MGB-BP-3 was synthesized by Anthony et al. by combining two moieties (synthon A and synthon B) [90]. To obtain the synthon A, 1-methyl-4-nitro-pyrrole-2-carboxylic acid is first converted into an acyl chloride which reacts with aminoethyl morpholine to give the corresponding amide. After reduction of the nitro group the intermediate is reacted with the previous acyl chloride to deliver the synthon A. The synthon B is synthesized in three steps starting from methyl 4-(bromomethyl)benzoate. At first, an Arbuzov reaction using triethylphosphite gives the corresponding phosphonate which reacts with quinoline-3-aldehyde via a Horner Wadsworth Emmons reaction. Finally, the saponification of the methyl ester delivers the synthon B. Eventually, the reduction of the nitro group of the synthon A and the subsequent coupling with the synthon B lead to MGB-BP-3 (Figure 10).

Figure 10 .

 10 Figure 10. Description of the synthetic pathway leading to MGB-BP-3 [90].4.2.2. Target and mechanism of action

Figure 11 . 4 . 2 . 4 .

 11424 Figure 11. Summary of the SARs of MGB-BP-3 from distamycin.

4. 2 . 5 .

 25 In vivo experiments MGB-BP-3 was able to protect against death and prolonged post-treatment survival in a hamster model of CDI[START_REF] Kullar | Investigational Treatment Agents for Recurrent Clostridioides difficile Infection (rCDI)[END_REF]. In the study, the efficacy of MGB-BP-3 (free base suspension and colonic delivery microparticles) was compared to that of vancomycin in the treatment of clindamycin-induced C. difficile infection in a hamster CDAD model[START_REF] Ridlon | Bile salt biotransformations by human intestinal bacteria[END_REF].The animals received 30 mg/kg oral clindamycin, followed[START_REF] Glaxosmithkline | A Single-Center, Three-Part, Open Label Study to Evaluate the Relative Bioavailability of Two Formulations, Food Effect, and Interaction With Itraconazole Following Single Dose of GSK2140944[END_REF] hours later by C. difficile (strain BI1) by gavage. The treatment started 20 hours post C. difficile infection and was administered orally for 5 days. The hamsters received MGB-BP-3 as a free base suspension or as colonic delivery microparticles (10 mg bid), vancomycin (25 mg/kg bid) or a vehicle. The animals were observed for up to 13 days post C. difficile administration and scarified when severe infection developed. All untreated and vehicle treated hamster died within 36 hours post infection. On the contrary, MGB-BP-3 free base suspension, MGB-BP-3 microparticle formulation and vancomycin treated hamsters survived throughout the treatment period. 3 days after the end of the treatment, hamsters that received MGB-BP-3 free base deteriorated clinically (wet tail and hypothermia). Conversely, MGB-BP-3 microparticle formulation and vancomycin treated hamsters remained healthy and survived to the end of the study. Moreover, the rates of regrowth of C. difficile isolates in hamster following the end of vancomycin and MGB-BP-3 treatment were compared. C. difficile burden in the colon and caecum was much higher in animals treated with vancomycin compared to MGB-BP-3 microparticles. Finally, MIC values of MGB-BP-3 against C. difficile before and after infection were measured and no change was observed in their values, indicating that resistance did not develop during therapy.In another study, the same hamster model of severe CDAD was used to assess the disposition of two MGB-BP-3 formulations (free base suspension and microparticles) in plasma and intestines after oral administration[START_REF] Allegretti | Recurrent Clostridium difficile Infection Associates with Distinct Bile Acid and Microbiome Profiles[END_REF]. The results indicated that MGB-BP-3 has a favorable PK profile for the treatment of C. difficile infections.4.2.6. Phase 1A group of 40 healthy volunteers took part in a phase 1 study in which MGB-BP-3 exhibited an excellent safety and tolerability profile with no serious AEs. Only one subject in the 125 mg dose cohort experienced transient dizziness[START_REF] Mgb Biopharma | A Single-centre, Double-blind, Placebo-controlled, Study in Healthy Men to Assess the Safety and Tolerability of Single and Repeated Ascending Doses of MGB-BP-3, clinicaltrials[END_REF].4.2.7. Phase 2A phase 2 study was conducted to assess the safety, tolerability, and efficacy of incremental doses of MGB-BP-3 in patients with CDAD. Three sequential ascending doses of 125 mg, 250 mg, and 500 mg given twice daily were investigated. The study showed that a 10 day-administration of 250 mg of MGB-BP-3 given twice daily resulted in an initial cure and 100% durable cure, with no disease recurrence measured four weeks after treatment. This regimen has been selected for further clinical trials[START_REF] Mgb Biopharma | An Exploratory, Open Labelled, Phase IIa Study to Assess Safety, Tolerability and Efficacy of Incremental Doses of MGB-BP-3 in Patients With Clostridium Difficile-associated Diarrhea (CDAD), clinicaltrials[END_REF]. 4.2.8. Current Advances MGB-BP-3 has been granted "qualified infectious disease product" status (QIDP) by the FDA, allowing it to receive fast track designation and priority review. The molecule will also be eligible to participate in the US program DISARM (Developing an Innovative Strategy for Antimicrobial Resistant

5. 2 . 1 .

 21 Chemical synthesis CRS-3123 was initially synthesized by Replidyne Inc,[START_REF] Critchley | Spectrum of activity and mode of action of REP3123, a new antibiotic to treat Clostridium difficile infections[END_REF] following a convergent strategy described in Figure12[101]. Synthon A is prepared in 6 steps, by first condensing 3-thienyl isocyanate and Meldrum acid through a Knoevenagel-type reaction. The thienolate intermediate is then alkylated with iodomethane. Substitution with 3,3-diethoxypropylamine allows the introduction of the alkyl chain. The obtained intermediate is then heated in presence of HMDS (hexamethyldisilazane) to perform an intramolecular cyclization and obtain thieno[3,2-b]pyridine bicycle, bearing a carboxylic acid. Decarboxylation is achieved with sodium methoxide at reflux to give the pyridinolate intermediate, which is next acylated with benzoic anhydride. Lastly, the acetal group is cleaved in acidic conditions to give the aldehyde A. In parallel, synthon B is obtained in 3 steps. 6,8-dibromochroman-4-one is stereoselectively reduced using (1S,2S)-(+)-N-p-tosyl-1,2-diphenylethylene diamine and dichloro(p-cymene)-ruthenium (II) dimer to give the S-alcohol intermediate. S N 2 with DPPA (diphenylphosphoryl azide) allows the replacement of the hydroxy group with an azido group, while achieving inversion of configuration of the chiral carbon. The R-azido derivative is then reduced via a Staudinger reaction to give the synthon B.Reductive amination between synthons A and B with sodium triacetoxyborohydride gives the corresponding intermediate, which is then deprotected using ammonia in methanol to cleave the benzoyl group. Finally, salification with HCl in ethanol affords CRS-3123 as a dihydrochloride salt.

Figure 12 . 5 . 2 . 2 .

 12522 Figure 12. Description of the synthetic pathway leading to CRS-3123 [101]. Where known, yields are indicated.

Figure 13 .

 13 Figure13. Description of the synthetic pathway leading to Lefamulin[START_REF] Mang | Pleuromutilin derivatives for the treatment of diseases mediated by microbes[END_REF].

Figure 14 .

 14 Figure 14. Views of lefamulin complex formed with S. aureus DNA gyrase and uncleaved DNA (PDB code: 5HL7).

  FabI. This molecule belongs to the benzofuran naphthyridines, a novel class of antibiotics. The effort to identify potent and selective FabI inhibitors began with the screening of the GSK proprietary compound collection. From a first high-throughput screening on the S. aureus FabI enzyme, Miller et al. identified a 1,4-benzodiazepine derivative as a low micromolar inhibitor. A structure-guided optimization process led to aminopyridine and oxotetrahydronaphthyridine derivatives and to the selection of AFN-1252 as a lead development candidate [144,145].6.2.1. Chemical SynthesisThe synthesis of AFN-1252 was patented in 2008 by Affinium Pharmaceuticals[START_REF] Pauls | Salts, Prodrugs and Polymorphs of Fab I Inhibitors[END_REF]. The strategy consists of combining two different moieties. The first 3-methylbenzofuran moiety (synthon A) is synthesized starting from 3-methylbenzofuran-2-carboxylic acid. The addition of aqueous methylamine to the acyl chloride intermediate leads to the formation of the corresponding amide, which is then reduced using LiAlH 4 . The second oxotetrahydronaphthyridine moiety (synthon B) is synthesized starting from 6-bromo-3,4-dihydro-1,8-naphthyridin-2(1H)-one bromide. A Heck reaction is performed with tert-butylacrylate. The desired carboxylic acid is then obtained as a HBr salt by treating

Figure 16 .

 16 Figure 16. Description of the synthetic pathway leading to AFN-1252 and the diethanol ammonium salt of afabicin [145,146].

Figure 17 .

 17 Figure 17. Views of AFN-1252 complex formed with S. aureus FabI and the NADPH cofactor (PDB code: 4FS3) .

Figure 18 .

 18 Figure 18. Summary of the SARs of AFN-1252 from the benzodiazepine series. 6.2.4 In vitro activity

Figure 19 .

 19 Figure 19. Involvement of the FtsZ protein towards the formation of the Z-ring.

Figure 20 .

 20 Figure 20. Description of the synthetic pathway leading to TXA709 [159].

Figure 21 .

 21 Figure 21. Views of TXA707 complex formed with S. aureus FtsZ 12-316 (PDB code: 5XDT).

Figure 22 .

 22 Figure 22. Summary of the SARs of TXA709.

Table 1 .

 1 Main information and calculated chemical properties of the eight drug candidates with their progression toward clinical development. Concerning bacteria, there are four types of topoisomerases: DNA gyrases, topoisomerases I, topoisomerases IV and reverse gyrases.

	Name	Gepotidacin	Zoliflodacin	Ibezapolstat	MGB-BP-3
	Family	Triazaacenaphtylene/NBTI Spiropyrimidinetrione	DCBG ([dichlorobenzyl] guanine)	Distamycin
	Mode of action	Inhibition of DNA gyrase and topoisomerase IV	Inhibition of topoisomerase II	Inhibition of DNA polymerase IIIC	Binding to the minor groove of DNA

  6.2.7 Phase 2A phase 2 trial was designed to evaluate the efficacy and safety of 200 mg of AFN-1252, given orally twice-a-day in the treatment of ABSSSI. This study showed that AFN-1252 is well tolerated and highly effective in the treatment of ABSSSI caused by S. aureus, including MRSA. The overall early re-sponse rate at day 3 was 97.3%, where 82.9% of patients had a >20% decrease in the area of erythema, and 77.9% of patients had a >20% decrease in the area of induration. Microbiologic eradication rates for MRSA and MSSA were around 90% at short-and long-term follow-ups. The main drug-related AEs reported, which were mostly mild or moderate, were headache (26.2%) and nausea

	(21.4%) [151,152].
	6.2.8 Prodrug
	AFN-1252 belongs to class II biopharmaceutics classification system (BCS), presenting a limited
	aqueous solubility and oral bioavailability. Therefore, Debiopharm invested in the development of its
	prodrug Afabicin desphosphono (formerly Debio1450, AFN-1720). As expected, the prodrug showed
	little to no activity against S. aureus, E. coli and other species. Afabicin was rapidly converted in vivo

Funding: This work was supported by Institut National de la Santé et de la Recherche Médicale, Université de Lille, Institut Pasteur de Lille, Région Hauts-de-France.

Author Contributions: writing-original draft preparation, F.R., N.C., K.A., M.E; writing-review and editing, F.R., N.C., K.A., M.E, M.F., N.W.; supervision, M.F., N.W. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.