Long Zhang

Michel Lecoq
email: mlecoq34@gmail.com

Nosema locustae (Protozoa, Microsporidia), a Biological Agent for Locust and Grasshopper Control

Keywords: locust, grasshopper, biological control, Nosema locustae, application, epizootics

Effective locust and grasshopper control is crucial as locust invasions have seriously threatened crops and food security since ancient times. However, the preponderance of chemical insecticides, effective and widely used today, is increasingly criticized as a result of their adverse effects on human health and the environment. Alternative biological control methods are being actively sought to replace chemical pesticides. Nosema locustae (Synonyms: Paranosema locustae, Antonospora locustae), a protozoan pathogen of locusts and grasshoppers, was developed as a biological control agent as early as the 1980s. Subsequently, numerous studies have focused on its pathogenicity, host spectrum, mass production, epizootiology, applications, genomics, and molecular biology. Aspects of recent advances in N. locustae show that this entomopathogen plays a special role in locust and grasshopper management because it is safer, has a broad host spectrum of 144 orthopteran species, vertical transmission to offspring through eggs, long persistence in locust and grasshopper populations for more than 10 years, and is well adapted to various types of ecosystems in tropical and temperate regions. However, some limitations still need to be overcome for more efficient locust and grasshopper management in the future.

Introduction

Locust and grasshopper (L&G) outbreaks, often resulting in huge plagues, have been a serious threat to global food security since ancient times [START_REF] Zhang | Locust and Grasshopper Management[END_REF]. Traditional control of L&G consists mainly of the application of chemical pesticides, which often results in many side effects. These include toxic chemical residues on food, adverse health effects on humans and nontarget animals, and environmental pollution. One of the most promising alternatives to chemical pesticides is biological control. Although there are many natural enemies of L&G [START_REF] Greathead | Natural Enemies of Tropical Locusts and Grasshoppers: Their Impact and Potential as Biological Control Agents[END_REF][START_REF] Lomer | Biological Control of Locusts and Grasshoppers[END_REF][START_REF] Goettel | Microbial Control of Grasshoppers and Locusts[END_REF], only a few have been developed as biological control agents or potential agents, including the microsporidian Nosema locustae (synonym: Antonospora locustae, Paranosema locustae) and the fungus Metarhizium acridum, both of which have been quite widely used in the control of L&G.

N. locustae, a unicellular eukaryote (Figure 1) with an obligate intracellular lifestyle [START_REF] Solter | Microsporidian Entomopathogens[END_REF][START_REF] Keeling | Microsporidia: Biology and Evolution of Highly Reduced Intracellular Parasites[END_REF][START_REF] Keeling | Origins of Microsporidia[END_REF], was the first to be commercially developed for L&G control since its discovery and characterization of its potential as a biological control agent against these species [START_REF] Canning | The Life Cycle of Nosema locustae Canning in Locusta migratoria migratorioides (R. & F.) and its Infectivity to other Hosts[END_REF][START_REF] Canning | The Pathologenicity of Nosema locustae Canning[END_REF][START_REF] Henry | Importance of Timing, Spore Concentrations, and Levels of Spore Carrier in Applications of Nosema locustae (Microsporida: Nosematidae) for Control of Grasshoppers[END_REF]. Due to its slow action and various other constraints, N. locustae was considered to be of limited application [START_REF] Lockwood | Biological Control of Rangeland Grasshoppers and Locusts[END_REF][START_REF] Lockwood | The History of Biological Control with Nosema locustae: Lessons for Locust Management[END_REF]. However, as the only microsporidian agent for L&G control, N. locustae has been studied extensively [START_REF] Zhang | Locust and Grasshopper Management[END_REF][START_REF] Henry | Pest Control by Nosema locustae, a Pathogen of Grasshoppers and Crickets[END_REF][START_REF] Henry | Natural and Applied Control of Insects by Protozoa[END_REF][START_REF] Brooks | CRC Handbook of Natural Pesticides[END_REF][START_REF] Lomer | Biological control of Locusts and Grasshoppers[END_REF][START_REF] Johnson | Nosematidae and other Protozoa as Agents for Control of Grasshoppers and Locusts: Current Status and Prospects[END_REF]. In recent years, there has been a renewed interest in N. locustae, mainly due to work in China, where it is produced in large quantities and used extensively, and Argentina, where its long-term persistence appears to reduce the frequency and intensity of grasshopper outbreaks [START_REF] Lange | The Development of the Microsporidium Paranosema (Nosema) locustae for Grasshopper Control: John Henry's Innovation with Worldwide Lasting Impacts[END_REF]. Recent advances in areas such as mass production, formulation, application, and epizootiology have further promoted the application of this pathogen. Numerous studies on its host spectra, molecular biology, and evolution have provided a solid fundamental basis for the use of N. locustae for L&G management.

Host Spectrum

N. locustae was first identified in a laboratory population from the migratory locust Locusta migratoria (Linnaeus, 1758) by Elizabeth Canning in 1953 [START_REF] Canning | A New Microsporidian, Nosema locustae n.sp., from the Fat Body of the African Migratory Locust, Locusta migratoria migratorioides R. & F. Parasitol[END_REF]. This pathogen occurs under natural conditions and has been found in various areas of the United States (Montana, Northern Dakota, Minnesota, Oregon, Wyoming, Colorado, Arizona, and Idaho), Canada (Saskatchewan, Ontario) and South America (western Pampas and northwestern Patagonia, Argentina), as well as Asia (Rajasthan and Vidarbha, India; Inner Mongolia, Hainan and Qinghai, China) and Africa (Karoo, South Africa) [START_REF] Lange | The Host and Geographical Range of the Grasshopper Pathogen Paranosema (Nosema) locustae Revisited[END_REF]. The highest level of infection was reported of about 5.5% in Melanoplus sanguinipes (Fabricius, 1798) in Idaho between 1963 and 1967 [START_REF] Henry | Epizootiology of Infections by Nosema locustae Canning (Microsporidia: Nosematidae) in Grasshoppers[END_REF].

N. locustae has a wide host range restricted to orthopteran insects. Henry [START_REF] Henry | Extension of the Host Range of Nosema locustae in Orthoptera[END_REF] in 1969 provided an initial list of 55 North American species that he knew to be susceptible. Brooks [START_REF] Brooks | CRC Handbook of Natural Pesticides[END_REF] published in 1988 a new list of worldwide susceptible orthopterans, expanding the host range to 95 species. The last review was done in 2005 by Lange [START_REF] Lange | The Host and Geographical Range of the Grasshopper Pathogen Paranosema (Nosema) locustae Revisited[END_REF], who arrived at a total of 121 species. Since then, various authors have been added to this list (Table 1), and we now reach a total of 144 susceptible orthopterans. Although N. locustae has a broad host spectrum in Orthoptera, it is incapable of affecting non-orthopteran insects. It has been shown that the honey bee (Apis mellifera) and the lepidoterans Heliothis zea and Agrotis ipsilon were not susceptible [START_REF] Brooks | CRC Handbook of Natural Pesticides[END_REF]. Menapace et al. [START_REF] Menapace | Adult Honey Bees are not Susceptible to Infection by Nosema locustae[END_REF] reported that honey bees were not infected, even when fed high doses of spores. The American cockroach Periplaneta americana and the spider Butalus occidentalis were also found to be non-susceptible [START_REF] Krall | Efficiency and Environmental Impact for Biological Control of Nosema locustae on Grasshoppers in Cape Verde, a Synthesis Report[END_REF]. N. locustae has been demonstrated to be safe for vertebrates. Brooks [START_REF] Brooks | CRC Handbook of Natural Pesticides[END_REF] summarized the assessment tests on primary skin irritation, acute dermal toxicity, acute inhalation toxicity and pathogenicity, subacute oral toxicity, acute oral toxicity, acute pathogenicity, and possible hazards of N. locustae to vertebrates, including rabbits, guinea pigs, rainbow trout, giant toads, mallards, ring-necked pheasant, mice, and rats. No significant effects were observed. The toxicity of N. locustae at 20 million spores/mL to certain non-target organisms, such as Coturnix japonica, Apis mellifera, Bombyx mori, Daphnia magna, and Brachydanio rerio, was examined in China. The results showed that there was no risk to honey bees per os at the maximum exposure dose tested, no deaths in the five animal species tested in the contact toxicity experiments, and N. locustae was relatively safe for nontarget beneficial organisms in the environment [START_REF] Yuan | Toxic Effects of Nosema locustae on Environmental Non-Target Bene-ficial Organisms[END_REF].

Pathogenicity

N. locustae, as an obligate parasite, reproduces in host target cells. Its infection involves the polar tube of the spore to inject its plasma into the target cells [START_REF] Solter | Microsporidian Entomopathogens[END_REF]. It causes high mortality in L&G. The locust's main target organ is the host's adipose tissue (fat body) [START_REF] Canning | The Pathologenicity of Nosema locustae Canning[END_REF]. N. locustae penetrates fat body cells and produces meronts, sporonts, sporoblasts, and spores. In the migratory locust, Locusta migratoria migratorioides (Reiche & Fairmaire, 1849), the younger the nymphs, the more susceptible they are, but even newly emerged adults are still susceptible [START_REF] Canning | The Pathologenicity of Nosema locustae Canning[END_REF]. Studies by Tounou et al. [START_REF] Tounou | Laboratory Assessment of the Potential of Paranosema locustae to Control Immature Stages of Schistocerca gregaria and Oedaleus senegalensis and Vertical Transmission of the Pathogen in Host Populations[END_REF] on the effects of N. locustae on the desert locust, Schistocerca gregaria (Forskål, 1775) and the Senegalese grasshopper, Oedaleus senegalensis (Krauss, 1877), showed that N. locustae has high pathogenicity on the young nymphal instars of these two species. The median survival time for first, second, third, fourth, and fifth nymphal instars was 6, 9, 10, 14, and 15 days, respectively, when locusts were inoculated with 1 × 10 7 spores on 10 g wheat bran in groups. Similar results were obtained with the Senegalese grasshopper using the same treatment method, with median survival times for instars 1, 3, and 5 being 5, 9, and 15 days, respectively. The median survival time therefore increased with the age of the locust and with decreasing inoculation doses. For instance, third-instar desert locust nymphs were inoculated in groups with 5.62 × 10 6 or 3.16 × 10 4 spores with a median survival time of 14 and 16 days, respectively. However, cumulative mortality increases with the increase of spore concentration and decreases with the increasing age of the nymphs. N. locustae caused high mortality in young desert locust nymphs. Mortality was 100% in first and second nymphal instars inoculated in groups with 1 × 10 7 spores of N. locustae, as well as in first nymphal instars with 1 × 10 6 spores. With third and fourth nymphal instars inoculated with 1 × 10 7 spores, cumulative mortality was above 90%. Similar results were obtained with the Senegalese grasshopper, which was inoculated with 1 × 10 7 spores: 100% mortality was observed in the first instar but 88.5% mortality in the third instar. In the fifth instar, mortality was only 66.3% in desert locust and 70% in the Senegalese grasshopper. These results suggest that the appropriate period for the application of N. locustae is mainly between the first and fourth instars, i.e., a possible application period of about 20-28 days, taking into account an average duration of 5-7 days per nymphal instar.

Inoculation of the 1st-5th nymphal instars of Chondracris rosea (De Geer, 1773) with N. locustae resulted in 78.2% to 100% mortality in field caging experiments by Liu and Chen [26]. The LC 50 was approximately 3.88 × 10 5 spores/mL for the 1st-3rd instars and 3.98 × 10 6 spores/mL for the 4th-5th instars. In a caging experiment on a large forest site, the same authors observed that C. rosea mortality was greater than 91.1% when they sprayed N. locustae at a rate of 5 × 10 7 spores/mL. At 1 × 10 8 spores/mL, mortality reached 100% 25 days after treatment [26]. Chen et al. [25] conducted a laboratory experiment with five concentrations of N. locustae spores (1 × 10 4 , 1 × 10 5 , 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 spores/mL) to treat early stages (1st-2nd instar) of the yellow-spined bamboo locust (Ceracris kiangsu). Mortalities were 16.6, 32.9, 29.2, 34.0, and 83.7%, respectively, increasing with spore concentrations. They also found that yellow-spined bamboo locust mortality was 85.1% after the application of a suspension of N. locustae spores at a concentration of 5 × 10 7 spores/mL in field trials in the forest. Zang et al. [START_REF] Zang | Biological Control of Oxya chinensis by using Nosema locustae[END_REF] reported that mortality of Oxya chinensis (Thunberg, 1815) was about 65.4-68.1% 30 days after the field treatment of third-instar nymphs sprayed with 1.5 × 10 10 , 2.25 × 10 10 spores/ha, respectively. The infection rate in survivors was approximately 40%.

Concurrent use of N. locustae and Metarhizium spp. has shown additive effects on locusts and grasshoppers. When fifth-instar nymphs of S. gregaria were inoculated first with N. locustae at doses between 1 × 10 4 and 1 × 10 6 spores on wheat bran in groups, and then 10 days later with M. anisopliae at doses between 1 × 10 2 and 1 × 10 4 spores/nymph, the median survival times ranged from 3 to 9 days, and the shortest duration was only 3 days at doses of 1 × 10 6 spores of Nosema and 1 × 10 4 spores of Metarhizium [START_REF] Tounou | Interaction between Paranosema locustae and Metarhizium anisopliae var. acridum, two Pathogens of the Desert Locust, Schistocerca gregaria under Laboratory Conditions[END_REF]. When the locusts were inoculated in groups with N. locustae at doses of 1 × 10 5 and 1 × 10 6 spores, and Metarhizium at 1 × 10 3 and 1 × 10 4 /nymph, the mortality was at 90% and 97% over 3 days, and both reached 100% 10 days after inoculation. Mortality was only 12.2% in the control, showing a synergistic effect between these two agents. Similar results were obtained with N. locustae directly mixed with Metarhizium spp., with the oriental migratory locust (L. migratoria manilensis Meyen, 1835). Mortality was higher with the mixture compared to treatments with each agent applied separately. The effects of a mixture of M. acridum and N. locustae on third nymphal instars under laboratory conditions showed that at a ratio of 1:1 (M. acridum at 3.90 × 10 6 spores/g locust body weight and N. locustae at 3.99 × 10 6 spores/g locust body weight), locust mortality was about 96.7% 24 days after inoculation, showing an additive effect of these two agents [START_REF] Ding | Virulence of Metarhizium anisopliae and Nosema locustae Against the Nymphs of Locusta migratoria manilensis[END_REF]. When 2nd-3rd-instar nymphs of L. migratoria were inoculated with 1 × 10 6 spores/nymph of N. locustae, and after 3, 6 and 9 days with Metarhizium at 1 × 10 7 conidia/mL, an additive effect was only observed in nymphs inoculated with Nosema and then 9 days later with Metarhizium [START_REF] Tokarev | Interactions of two Insect Pathogens, Paranosema locustae (Protista: Microsporidia) and Metarhizium acridum (Fungi: Hypocreales), during a Mixed Infection of Locusta migratoria (Insecta: Orthoptera) Nymphs[END_REF]. After examining the stage of Nosema development on Days 3, 6, and 9 after inoculation, they found that it was not until the Nosema spore maturation stage that locusts were most susceptible to fungal infection.

Lv et al. [START_REF] Lv | A Family of CSαβ Defensins and Defensin-Like Peptides from the Migratory Locust, Locusta migratoria, and Their Expression Dynamics during Mycosis and Nosemosis[END_REF] identified 4 defensins from migratory locust palp transcriptomes, named LmigDEF1 (78 amino acids), LmigDEF3 (78 amino acids), LmigDEF4 (69 amino acids), and LmigDEF5 (67 amino acids) with theoretical isoelectric points (pI)/molecular weights (Mw, kDa) of 6.48/8.29, 6.56/8.37, 8.23/7.19, and 8.27/6.93, respectively. The expression patterns of LmigDEF1, LmigDEF3, and LmigDEF5 in the fat body and salivary glands were examined by qRT-PCR after the locusts were inoculated with N. locustae. Results indicated that all three defensins varied over time in the fat body and salivary glands after Nosema infection, the transcript level of the LmDEFs being at their lowest in the fat body over the 10 days. This indicates that Nosema infection reduced the locust's immune response via the defensins and may explain why the coordinated use of Nosema and Metarhizium results in higher locust mortality [START_REF] Tounou | Interaction between Paranosema locustae and Metarhizium anisopliae var. acridum, two Pathogens of the Desert Locust, Schistocerca gregaria under Laboratory Conditions[END_REF][START_REF] Ding | Virulence of Metarhizium anisopliae and Nosema locustae Against the Nymphs of Locusta migratoria manilensis[END_REF]. In particular, for about four days when Nosema were sporulating after inoculation, the locusts were more easily infected by the fungus due to the lower level of the three types of defensins in the fat body [START_REF] Tokarev | Interactions of two Insect Pathogens, Paranosema locustae (Protista: Microsporidia) and Metarhizium acridum (Fungi: Hypocreales), during a Mixed Infection of Locusta migratoria (Insecta: Orthoptera) Nymphs[END_REF]. In addition, Chen et al. [START_REF] Chen | A Novel Spore Wall Protein from Antonospora locustae (Microsporidia: Nosematidae) Contributes to Sporulation[END_REF] demonstrated the key role of N. locustae sporulation in locust mortality. They identified a spore wall protein, AlocSWP2 from N. locustae, containing four cysteines. AlocSWP2 has been detected in the wall of mature spores, sporoblasts, and sporonts during sporulation in the host body by immunocytochemistry localization experiments. AlocSWP2 was detected in the fat body of infected locust only on Day 9 after inoculation using RT-PCR. The survival percentage of infected locusts that received a dsRNA injection of AlocSWP2 on Days 15, 16, and 17 after inoculation of Nosema spores was significantly higher than that of infected locusts without dsRNA treatment. Similarly, the number of spores in locusts infected with Nosema and treated with RNAi of AlocSWP2 was significantly lower than that in infected locusts without RNAi of this gene. This indicates that this N. locustae spore wall protein is involved in sporulation, contributing to host mortality.

L. migratoria migratorioides infected with N. locustae demonstrated reduced sustainable flight capacity [START_REF] Canning | The Pathologenicity of Nosema locustae Canning[END_REF]. Zhang et al. [START_REF] Zhang | The Effect of Nosema locustae Infection on Flying Ability of Locust (Locusta migratoria manilensis)[END_REF] confirmed this with the oriental migratory locust (L. migratoria manilensis). Flight capacity of infected and healthy adult locusts, 5 to 15 days after emergence, was determined using a flying mill for 18 h. On average, the flight distance of healthy versus infected locusts was 14,279 vs. 864 m; flight speed, 1.23 vs. 0.51 m/s, flight time, 3 vs. 0.33 h; maximum flight distance, 72,538 vs. 1544 m; and maximum sustained flight time, 6.7 h vs. 0.1 h. The decrease in flight capacity may be due to the fact that N. locustae destroyed the fat bodies reducing the supplement of glyceride and fat as energy resources. In L. migratoria manilensis infested with N. locustae, the glyceride content decreased rapidly, while lipase activity increased in both hemolymph and total fat [START_REF] Chen | Effect of Nosema locustae on the Content of Fat in Locusta migratoria manilensis[END_REF].

Locusts and grasshoppers infected with N. locustae have reduced fertility [START_REF] Henry | Pest Control by Nosema locustae, a Pathogen of Grasshoppers and Crickets[END_REF][START_REF] Zhang | Epizootic and Transmission of Nosema locustae in the Populations of Grasshoppers and Locusts[END_REF]. Reduced vitellogenin was observed in fourth-instar nymphs of L. migratoria manilensis inoculated with N. locustae: vitellogenin levels in the fat body, hemolymph, and ovaries were very low compared to the control [START_REF] Chen | Effect of Nosema locustae on the Content of Vitellogenin of Locusta migratoria manilensis[END_REF]. The maximum vitellogenin level in infected vs. healthy locusts, respectively, were 4.663 vs. 18.655 mg/mL in the fat body, 2.627 vs. 7.603 mg/mL in the hemolymph, and 4.927 vs. 73.367 mg/mL in the ovaries. This explains why the reproductive capacity of infected locusts is low.

The disease caused by N. locustae is transmitted vertically in eggs and egg pods [START_REF] Solter | Microsporidian Entomopathogens[END_REF]. Infected females have been reported to lay eggs containing spores of N. locustae [START_REF] Zhang | Epizootic and Transmission of Nosema locustae in the Populations of Grasshoppers and Locusts[END_REF]. Raina et al. [START_REF] Raina | Transovarial Transmission of Nosema locustae (Microsporida: Nosematidae) in the Migratory Locust Locusta migratoria migratorioides[END_REF] reported vegetative stages of N. locustae in the yolk of the oocyte and spores in the eggs of L. migratoria. When fourth-instar nymphs were inoculated with a dose of 1.5 × 10 6 spores, the infected parents laid eggs and the prevalence of infection was 100% in the next generation. The disease was vertically transmitted up to 14 generations, and mortality due to vertical transmission at times reached more than 90%. Parents of S. gregaria and O. senegalensis infected with N. locustae produced progeny with a 50% infection rate, indicating high vertical transmission in these species [START_REF] Tounou | Laboratory Assessment of the Potential of Paranosema locustae to Control Immature Stages of Schistocerca gregaria and Oedaleus senegalensis and Vertical Transmission of the Pathogen in Host Populations[END_REF].

Genomics and Molecular Biology

Understanding the molecular biology of N. locustae is fundamental in determining its biology and molecular interactions with the host and improving applications in its use for pest control. A complete genomic sequence of N. locustae has recently been revealed by Chen et al. [START_REF] Chen | Complete Genome of a Unicellular Parasite (Antonospora locustae) and Transcriptional Interactions with its Host Locust[END_REF], which is a major achievement. Sequencing of its genome yielded 3,170,203 nucleotides, encoding for 1857 predicted genes, of which 1755 are single-exon genes, and 102 are multiple exon genes. A total of 17 scaffolds, ranging from 88.763 to 388.82 kb, were identified and assigned to 17 chromosomes. Genomic and protein sequences from N. locustae and several other single-celled organisms were used to study evolutionary relationships in genetic synteny and collinearity using the MCScanX method. Results showed that within the microsporidia, most genes exhibit good collinearity [START_REF] Chen | Complete Genome of a Unicellular Parasite (Antonospora locustae) and Transcriptional Interactions with its Host Locust[END_REF].

In addition to genomic sequencing, Chen et al. [START_REF] Chen | Complete Genome of a Unicellular Parasite (Antonospora locustae) and Transcriptional Interactions with its Host Locust[END_REF] performed an analysis of locust midgut transcripts versus locust fat body transcripts from N. locustae infected locusts. They found that the abundant expression of locust antimicrobial peptides and other defense genes, such as peroxiredoxin and amine oxidase in the midgut, may explain the lower number of microsporidian spores in the midgut of the host. In the fat body, however, the large number of N. locustae spores present may be related to the fact that several locust phenol oxidases and peroxisome proliferator-activated receptors have been inhibited to allow it to escape the host immune response. This revealed the interactions of N. locustae and the host at the transcript molecular level, in particular why N. locustae can reproduce massively in the locust fat body and not in the gut.

Identification of the genes and proteins of the polar tube is certainly useful in understanding the interactions between N. locustae and its hosts at the molecular level. Two polar tube protein genes, PTP1 and PTP2, have been identified, and it has been suggested that PTP1 plays a key role in interactions with the host cell surface [START_REF] Polonais | Microsporidian Polar Tube Proteins: Highly Divergent but Closely Linked Genes Encode PTP1 and PTP2 in Members of the Evolutionarily Distant Antonospora and Encephalitozoon Groups[END_REF]. Two other orthologous polar tube proteins, named AlPTP2b and AlPTP2c, exhibit elastomeric characteristics. The AlPTP2b and AlPTP2c genes encode for proteins of 568 and 599 amino acids with deduced molecular weights of 55,399 and 56,664 Da, respectively. These proteins are highly conserved (84.2% identity), larger than the previously reported AlPTP2 (287 amino acids) [START_REF] Polonais | Identification of Two new Polar Tube Proteins Related to Polar Tube Protein 2 in the Microsporidian Antonospora locustae[END_REF].

Fast et al. [START_REF] Fast | Phylogenetic Analysis of the TATA Box Binding Protein (TBP) Gene from Nosema locustae: Evidence for a Microsporidia-Fungi Relationship and Spliceosomal Intron Loss[END_REF] identified a gene for the TATA box binding protein (TBP) of N. locustae. The predicted amino acid sequence of the TBP gene consists of 259 amino acids. In the phylogenetic analysis of TBP, the authors emphasized that TBP from N. locustae is close to that of fungi and supported previous studies on the evolution of microsporidia with tubulins, HSP70, and the greater subunit of RNA polymerase II (RPB1) proteins [START_REF] Edlund | Phylogenetic Analysis of β-Tubulin Sequences from Amitochondrial Protozoa[END_REF][START_REF] Keeling | Alpha-Tubulin from Early-Diverging Eukaryotic Lineages and the Evolution of the Tubulin Family[END_REF][START_REF] Germot | Evidence for Loss of Mitochondria in Microsporidia from a Mitochondrial-Type HSP70 in Nosema locustae[END_REF][START_REF] Hirt | A Mitochondrial Hsp70 Orthologue in Vairimorpha necatrix: Molecular Evidence that Microsporidia Once Contained Mitochondria[END_REF][START_REF] Hirt | Microsporidia are Related to Fungi: Evidence from the Largest Subunit of RNA Polymerase II and other Proteins[END_REF]. However, in a study on the large subunit (LSU) rRNA, Peyretaillade et al. [START_REF] Peyretaillade | Microsporidian Encephalitozoon cuniculi, a Unicellular Eukaryote with an Unusual Chromosomal Dispersion of Ribosomal Genes and a LSU rRNA Reduced to the Universal Core[END_REF] proposed that the origin of microsporidia was not specifically linked to a particular group of eukaryotes. In general, there are only a few studies on proteins and genes of N. locustae [START_REF] Burri | Microsporidian Mitosomes Retain Elements of the General Mitochondrial Targeting System[END_REF], and even rarer studies on their functions, especially pathogen-host interactions at the molecular level.

Mass Production and Products

The production of N. locustae spores is done in vivo. The rearing of infected hosts is the main process for mass production (Figure 2). Grasshoppers Melanoplus bivittatus (Say, 1825) have been used as hosts in the United States. Henry et al. [START_REF] Henry | Importance of Timing, Spore Concentrations, and Levels of Spore Carrier in Applications of Nosema locustae (Microsporida: Nosematidae) for Control of Grasshoppers[END_REF] pointed out that several factors influence production yield. Cage size and the number of individuals in each cage, light in the cage, and time of harvest, as well as factors such as grasshopper species and sex, are important. The general process is as follows: grasshopper nymphs are reared to the 4th-5th instars, inoculated with N. locustae spore suspension, and then reared until they die. The cadavers are collected and crushed, screened, and centrifuged to obtain a high concentration of spores, which are stored between -10 and -20 • C. In the United States, after several improvements in mass production techniques, a yield of 1 × 10 9 to 3 × 10 9 spores per grasshopper has been achieved. However, the use of Melanoplus differentialis (Thomas, 1865) as a host resulted in up to 7.1 × 10 9 spores per individual [START_REF] Henry | Effect of Grasshoppers Species, Cage Density, Light Intensity, and Method of Inoculation on Mass Production of Nosema locustae (Microsporida: Nosematiodae)[END_REF]. The time of harvest is an important factor in influencing the spore yield; the further away from the inoculation date, the more spores are obtained [START_REF] Johnson | Nosematidae and other Protozoa as Agents for Control of Grasshoppers and Locusts: Current Status and Prospects[END_REF]. In China, L. migratoria manilensis has been selected as a host to produce spores of N. locustae. In this species, there is no diapause, and individuals can be used year-round. Several factors influence the efficiency of mass production, including the inoculation concentration, the stage of development for inoculation, and the harvesting time [START_REF] Wang | Mass Production and Application of Nosema locustae Against Grasshoppers[END_REF]. A higher concentration of spores in the inoculation suspension resulted in rapid locust death, and a lower concentration increased the time to harvest and reduced the number of spores. The best inoculation concentration was found to be 1 × 10 6 spores/mL, the stage for inoculation the fourth instar, and the harvest time about 30-40 days after inoculation. The average spores/individual yield was approximately 6 × 10 9 , the highest being about 9.9 × 10 9 . In China, several insectariums have been established for the mass production of N. locustae since the 1990s. Currently, the average spore yield per individual can reach 22 × 10 9 to 34 × 10 9 . There are about 5-6 harvests per year, and the total yield can reach 2 × 10 15 spores for each insectarium [START_REF] Johnson | Nosematidae and other Protozoa as Agents for Control of Grasshoppers and Locusts: Current Status and Prospects[END_REF].

The products are usually formulated either as bran bait, aqueous suspension, or water-based suspension. They are prepared from high concentrations of spores stored at low temperature, diluted directly with water or mixed with wheat bran in an appropriate ratio. In China, a new water-based suspension has been developed. The spores are mixed with xanthan gum, sorbic acid, and other environmentally friendly additives. The suspension is more stable and homogeneous and can be stored for about one year at room temperature [START_REF] Zhang | Research and Application of New Green Control Technology Extension Mode of Locusts in China[END_REF]. It is better adapted to the needs of users and should be widely applied. To date, several commercial products have been registered such as Nolo Bait™, Semaspore™, and Grasshopper Attack™ in the United States since the 1980s [START_REF] Lockwood | The History of Biological Control with Nosema locustae: Lessons for Locust Management[END_REF]. In China, Nosema locustae products for locust control have been developed since the 1990s and are currently distributed by Beijing JiaJing Biotechnology Ltd. (Beijing, China). They are based on a highly pathogenic strain (AL2008L-04) that significantly improves product efficacy, a high-yield production technology, and an aqueous suspension formulation that can be stored at room temperature [START_REF] Jiajing | [END_REF].

Application and Epizootics

The three main components whose interactions can lead to a reduction in locust and grasshopper populations and epizootics are N. locustae, its hosts, and its environments. N. locustae has been used to control L. migratoria manilensis since the 1990s. A field trial was conducted on Hainan Island, China, in areas close to rice fields [START_REF] Zhang | Relationships between Application Rate of Nosema locustae and Infection of Locusta migratoria manilensis in Hainan Province[END_REF]. The locust population consisted of first to third instars with pretreatment densities of 0.9, 1.5, and 2.2 individuals/m 2 in the three treatment plots and 0.6 in the control plot. The plots were treated at 50 × 10 9 , 99 × 10 9 , and 150 × 10 9 spores/ha. At 25 days after treatment, the locust population density was reduced by 70.2%, 74.0%, and 78.9%, respectively, in the treated plots but increased by 58.1% in the control plot. The prevalence of N. locustae infection in survivors was 29.4%, 31.1%, and 31.8% at 30 days and 40.6%, 40%, and 35.7% at 40 days after treatment in the treated plots. The prevalence of infection persisted the following summer (one year) in the three treated plots, respectively, at 23.5%, 13%, and 9.1% levels.

In the United States, in Montana, studies indicated that the prevalence of N. locustae during the treatment season was variable, but mainly around 30%, with the highest being 50% when the spore application rate was 1.4 × 10 10 in 1.12 kg of wheat bran/ha after three applications [START_REF] Henry | Importance of Timing, Spore Concentrations, and Levels of Spore Carrier in Applications of Nosema locustae (Microsporida: Nosematidae) for Control of Grasshoppers[END_REF][START_REF] Henry | Experimental Application of Nosema locustae for Control of Grasshoppers[END_REF]. In Canada, in a grasshopper population consisting mainly of third instars of Melanoplus sanguinipes, M. packardii (Scudder, 1878), and Camnula pellucida (Scudder, 1862) treated with 2.5 × 10 9 or 5.0 × 10 9 spores in 1.68 kg wheat bran per ha, an infection rate of approximately 50% was observed 4-5 weeks after treatment [START_REF] Ewen | Evaluation of Nosema locustae (Microsporida) as a Control Agent of Grasshopper Populations in Saskatchewan[END_REF]. Toward the end of the season, about 12 weeks after treatment, this rate was about 95-100%. Zhang et al. [START_REF] Zhang | A Preliminary Survey on Epizootics of Infection of Nosema locustae Among Grasshoppers in Rangeland[END_REF] reported the prevalence of N. locustae in the years following the application of spore-based baits to mixed grasshopper populations-consisting of Oedaleus asiaticus Bey-Bienko, 1941, Myrmeleotettix palpalis (Zubovski, 1900), Angaracris rhodopa Fischer von Waldheim, 1836, A. barabensis (Pallas, 1773) and Dasyhippus barbipes (Fischer von Waldheim, 1846)-in Inner Mongolia rangelands, a region where temperatures are below -10 • C in winter and 20 to 30 • C in summer and rainfall is about 200 mm per year. In two areas treated in 1991 and 1992 with 7.5 × 10 9 spores/1.5 kg wheat bran bait/ha, locust infection rates of N. locustae were 14.5% and 19.8% in the second year after treatment. In a third area treated in 1988 with N. locustae baits, the observed infection rates were 6.8% and 23% two and six years after treatment. Mortality rates of 60-80% in mixed grasshopper populations were achieved following applications of N. locustae, in Inner Mongolia rangeland, at a rate of 3 × 10 10 spores/ha for two consecutive years [START_REF] Zhang | A Preliminary Study on Strategies of Management of Grasshoppers in crop-Grassland Ecosystem in Inner Mongolia[END_REF]. N. locustae was even used in the highlands (3270-3350 m ASL) in Qinghai province of China, and the disease could still be transmitted and persisted for a long time among grasshoppers [START_REF] Gong | Transmission of Nosema locustae Disease in Grasshopper Populations in Qinghai Grassland[END_REF], up to 10 years with an infection rate exceeding 50% in some years [27]. In three locations in Canada in two consecutive years (1988)(1989), Johnson and Dolinski [START_REF] Johnson | Attempts to Increase the Prevalence and Severity of Infection of Grasshoppers with the Entomopathogen Nosema locustae Canning (Microsporida: Nosematidae) by Repeated Field Application[END_REF] found that N. locustae persisted in grasshopper populations. They suggested that the activity of N. locustae was not inhibited despite severe weather conditions, hot in summer and very cold in winter, with air temperature down to -39 • C.

N. locustae was reportedly applied in a forest to control the yellow-spined bamboo locust (Ceracris kiangsu) [25]. In a 1.5 ha area of bamboo sprayed with 50 kg of N. locustae spore suspension at a concentration of 5 × 10 7 spores/mL, mortality of C. kiangsu was 85% 15 days after treatment. N. locustae was also used in 2018 to control the yellow-spined bamboo locust in bamboo forest ecosystems in Phongsaly province, Lao [START_REF] Phithalsoun | An Effective Biological Control Method of Yellow-Spined Bamboo Locust (Ceracris kiangsu) has been de-Veloped in Lao PDR and Vietnam[END_REF]. Nymphs were in the third and fourth instars at a density of over 100 individuals/m 2 . Treatment was carried out on May 13 at an application rate of 2 × 10 7 spores/mL. After 17 days, many dead locusts were observed in the treated area, where the average density was less than 10 individuals/m 2 , a reduction of more than 90% compared to the untreated control area. N. locustae spores were observed in all dead locusts that were collected from the treated plots and examined individually under a microscope in the laboratory. The average grade of infection was three out of five, corresponding to severe disease. In contrast, the infection rate of locusts collected from the control area was zero. In addition, 46% of surviving locusts in treated plots were infected with N. locustae, and two and a half months after treatment, the density was very low, about 0-2 individuals/m 2 . It was concluded that N. locustae could be an effective agent to control high densities of yellow-spined bamboo locusts in bamboo forests.

In Africa, Tounou et al. [START_REF] Tounou | Combined Field Efficacy of Paranosema locustae and Metarhizium anisopliae var. acridum for the Control of Sahelian Grasshoppers[END_REF] conducted field trials to assess the effects of N. locustae and Metarhizium anisopliae against various grasshopper species: Pyrgomorpha cognata Krauss, 1877, Acrotylus blondeli Saussure, 1884 (both predominant species), and Oedaleus senegalensis. Both agents were mixed with wheat bran as bait with M. anisopliae alone, N. locustae + M. anisopliae, N. locustae spores alone, or N. locustae + sugar. The same treatments were carried out with O. senegalensis in the early stages of development and resulted in mortalities of 64-85%. The population density during the three weeks of monitoring decreased by 44.7 ± 6.9% in the N. locustae plot, 52.8 ± 8.4% in the N. locustae + sugar plot, 73.7 ± 5.5% in the M. anisopliae plot, and 89.1 ± 1.8% in the N. locustae + M. anisopliae plot. The prevalence of N. locustae in adult grasshoppers surviving at 28 days after application was 48.1 ± 2.3, 28.9 ± 4.8, and 27.4 ± 3.7% in the three treatments with N. locustae. The results suggest that these two biological control agents have the potential to control the early stages of grasshoppers in Africa. These conclusions differ from those of Lima et al. [START_REF] Lima | Biological Control of Grasshoppers in the Cape Verde Islands[END_REF], who found no significant difference between treated and untreated plots and concluded that N. locustae could not be used to control O. senegalensis in the Cape Verde archipelago. However, they did not specify the developmental stage of the treated grasshoppers, and it is possible that efficacy was less on the fifth nymphal instar or on adults.

Long-term epizootics of N. locustae have been investigated in Argentina by Lange et al., who have shown that this pathogen can persist for many years after its introduction [START_REF] Lange | Newly-Associated Microsporidium Continues Causing Epizootics and Expanding its Host Range[END_REF][START_REF] Lange | Long-term Patterns of Occurrence of Nosema locustae and Perezia dichroplusae (Microsporidia) in Grasshoppers (Orthoptera: Acrididae) of the Pampas, Argentina[END_REF][START_REF] Lange | New Case of Long-Term Persistence of Paranosema locustae (Microsporidia) in Melanopline Grasshoppers (Orthoptera: Acrididae: Melanoplinae) of Argentina[END_REF]. These authors, in their field survey, found that the prevalence of N. locustae ranged from 1.8 to 41% in 9 of 13 sites between 1995 and 2003. In 1990, in another field survey in an area treated 11 years earlier, Lange and Azzaro [START_REF] Lange | New Case of Long-Term Persistence of Paranosema locustae (Microsporidia) in Melanopline Grasshoppers (Orthoptera: Acrididae: Melanoplinae) of Argentina[END_REF] observed infection rates of 2.9%, 3.5%, and 3.6% by N. locustae spores in three grasshopper species, Dichroplus elongatus Giglio-Tos, 1894, D. maculipennis (Blanchard, 1851), and Scotussa lemniscata (Stål, 1861). Lange et al. [START_REF] Lange | Newly-Associated Microsporidium Continues Causing Epizootics and Expanding its Host Range[END_REF] examined grasshoppers collected from sites treated with N. locustae in Argentina in the late 1970s, early 1980s, and mid-1990s and found that the highest prevalence of N. locustae in grasshopper populations was about 50%.

Conclusions and Prospects

N. locustae is becoming increasingly biologically understood, as well as for its potential in L&G control. Recent studies have shown that it can be applied in different ways to achieve control objectives and plays a more important role than ever in locust and grasshopper management programs. It has a fairly broad host spectrum, and at least 144 orthopteran species are susceptible. However, it is very safe for non-orthopteran insects and other non-target animals [START_REF] Johnson | Nosematidae and other Protozoa as Agents for Control of Grasshoppers and Locusts: Current Status and Prospects[END_REF][START_REF] Yuan | Toxic Effects of Nosema locustae on Environmental Non-Target Bene-ficial Organisms[END_REF]. It is transmitted orally and is therefore probably less sensitive to environmental factors than fungal-type agents. This is certainly the reason why good results have been recorded at a 3000-4000 m altitude on the Tibetan plateau in China and in high-temperature regions such as Hainan province in China or Lao and Vietnam. N. locustae has been demonstrated to be efficient in the control of L&G in rangelands, crop fields, and forest ecosystems. Its disease can persist for many years after application in L&G populations and can be part of their long-term management.

When L&G are at low density, a low dose of N. locustae will slowly kill them as a long-term control method. However, recent field trials have indicated that N. locustae can also be used at high locust densities, as has been demonstrated to control second and third instars of the yellow-spined bamboo locust. N. locustae also exhibits a synergistic effect when used in admixture with the fungus Metarhizium spp. and can potentially weaken the host immune response by reducing its defensins. The simultaneous use of N. locustae and Metarhizium spp. could be encouraged to control high-density locust outbreaks.

L&G mortality caused by N. locustae is dose-dependent; the higher the dose used, the higher the mortality. However, N. locustae spores are produced restrictively in vivo. The mass rearing of locusts to produce spores is expensive; therefore, increasing the spore yield per locust or grasshopper is a bottleneck for mass production and large-scale application. Understanding the molecular mechanisms of N. locustae and its interactions with its host may be valuable to explore ways to mass-produce N. locustae spores in order to improve the traditional in vivo mass production method or to find an alternative, as well as to increase the effects on L&G. Genome sequencing and transcriptomic analysis of N. locustae genes and proteins have provided a valuable basis for future studies to improve the genetics of N. locustae and screen for high-virulence strains [START_REF] Zhou | Selection of Antonospora locustae (Protozoa: Microsporidae) with Higher Virulence Against Locusta migratoria manilensis (Orthoptera:Acrididae)[END_REF]. The development of new formulations and other technologies to maintain high spore vitality at room temperature will also be useful in promoting the wider application of N. locustae.

Figure 1 .

 1 Figure 1. Spores of N. locustae under scanning electron microscopy (photo by Long Zhang).

Figure 2 .

 2 Figure 2. Mass producing process of N. locustae spores.

Table 1 .

 1 List of orthopteran species infected by N. locustae but not included in Lange[START_REF] Lange | The Host and Geographical Range of the Grasshopper Pathogen Paranosema (Nosema) locustae Revisited[END_REF].

	Species Acrotylus patruelis (Herrich-Schäffer, 1838) Aiolopus thalassinus (Fabricius, 1781)	Inoculation Infection (Caging)	X Field Trial Infection [29] X
	Anacridium melanorhodon (Walker, 1870)		X
		America
	Amblytropidia australis Bruner, 1904 Dichroplus vittigerus (Blanchard, 1851)		X X	[30]

Funding: This work is supported by a grant from Shandong Academy of Agricultural Sciences, and a grant from the Ministry of Agriculture of China (15206030).

Acknowledgments: Authors would like to thank anonymous reviewers' comments and suggestions.

Author Contributions: L.Z. conceived the article and drafted the manuscript with substantial input from M.L. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.

Disclaimer: Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the authors.


Ksenia Oguievetskaia
email: ksenia@medit.frwww.medit.fr

Laetitia Martin-Chanas

Artem Vorotyntsev

Olivia Doppelt-Azeroual

Xavier Brotel

Stewart A Adcock

Alexandre G De Brevern

Francois Delfaud

Fabrice Moriaud

Mitotic Kinesin Eg5 Inhibitors Generation By Computational MED-Portion Based Drug Design At PDB Scale

Keywords: FBDD, anti-mitotic, Eg5, KSP, mitotic kinesins Abbreviations SCF Surface Chemical Feature PDB Protein Data Bank KSP Kinesin Spindle Protein HYD Hydrophobic

Eg5, a mitotic kinesin exclusively involved in the formation and function of the mitotic spindle has attracted interest as an anticancer drug target. Eg5 is co-crystallized with several inhibitors bound to its allosteric binding pocket. Each of these occupies a pocket formed by loop 5/helix α 2 (L5/α2). Recently designed inhibitors additionally occupy a hydrophobic pocket of this site. The goal of the present study was to explore this hydrophobic pocket with our MED-SuMo fragment based protocol, and thus discover novel chemical structures that might bind as inhibitors. The MED-SuMo software is able to compare and superimpose similar interaction surfaces upon the whole PDB. In a fragment based protocol, MED-SuMo retrieves MED-Portions that encode protein-fragment binding sites and are derived from cross-mining protein-ligand structures with libraries of small molecules. We have excluded intra-family MED-Portions derived from ligands that occupy the hydrophobic pocket and predicted new potential ligands that would fill simultaneously both pockets. Some of the latter are identified in libraries of synthetically accessible molecules by the MED-Search software.

Introduction

During metaphase, the mitotic spindle maintains a constant shape and size though their principal constituent, the microtubules are continuously being polymerized, depolymerized and transported towards the two spindle poles [START_REF] Mitchison | Mitosis: a history of division[END_REF][START_REF] Mitchison | Dynamic instability of microtubule growth[END_REF][START_REF] Mitchison | Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence[END_REF]. Motor proteins from the kinesin family are involved in the mitotic spindle assembly, as well as an important number of cellular processes such as intracellular vesicles transport, chromosome segregation, cell division and motility in a tight collaboration with proteins of the cytoskeleton tubulin and actin [START_REF] Vale | The design plan of kinesin motors[END_REF][START_REF] Amos | Structure and dynamics of molecular motors[END_REF].

Inhibition of the mitotic spindle formation is an interesting target in cancer chemotherapy. Anti-mitotic agents used to date for cancer treatment, target microtubule stability (e.g., Vinca alkaloids and taxanes). They cause serious sideeffects, such as neurotoxicity (reviewed in [START_REF] Wood | Past and future of the mitotic spindle as an oncology target[END_REF]). Furthermore the development of resistance against these anti-mitotic agents restricts their application.

Another approach to inhibit the mitotic spindle formation has emerged. This alternative acts by inhibiting the mitotic motors that interact with microtubules [START_REF] Mayer | Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen[END_REF]. Eg5 (also known as Kinesin Spindle Protein, KSP, or kinesin-5s) has emerged from recent studies as a leading candidate for targeted antimitotic therapies. This kinesin is exclusively involved in the formation and function of the mitotic spindle, by driving a relative sliding of microtubules in the mitotic spindle (for review see [START_REF] Kwok | Microtubule flux: drivers wanted[END_REF]). The inhibition of Eg5 leads to cells with monopolar spindles (monaster) during mitosis, resulting in a cell cycle arrest and apoptosis, without interfering with other microtubule dependent processes. Several small molecules are now known that inhibit human Eg5 by binding to its catalytic motor domain. These discoveries are attracting a potential interest in this protein as an anticancer drug target.

The first inhibitor discovered by the groups of Stuart Schreiber and Tim Mitchison is a small cell permeable molecule, monastrol [START_REF] Mayer | Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen[END_REF]. One year after the resolution of the structure of the unbound Eg5 [START_REF] Turner | Crystal structure of the mitotic spindle kinesin Eg5 reveals a novel conformation of the necklinker[END_REF], the structure of the Eg5 bound to monastrol was also solved [START_REF] Maliga | Evidence that monastrol is an allosteric inhibitor of the mitotic kinesin Eg5[END_REF]. Monastrol binds specifically to the Eg5 and allosterically inhibits the ATPase activity of the kinesin. Further studies brought several inhibitors into clinical trials and several structures of the Eg5 with diverse types of inhibitors are now solved (Figure 1) [START_REF] Yan | Inhibition of a mitotic motor protein: where, how, and conformational consequences[END_REF][START_REF] Cox | Kinesin spindle protein (KSP) inhibitors. Part 1: The discovery of 3,5-diaryl-4,5dihydropyrazoles as potent and selective inhibitors of the mitotic kinesin KSP[END_REF][START_REF] Cox | Kinesin spindle protein (KSP) inhibitors. Part 4: Structure-based design of 5-alkylamino-3,5-diaryl-4,5-dihydropyrazoles as potent, water-soluble inhibitors of the mitotic kinesin KSP[END_REF][START_REF] Fraley | 3-(Indol-2-yl)indazoles as Chek1 kinase inhibitors: Optimization of potency and selectivity via substitution at C6[END_REF][START_REF] Tarby | Inhibitors of human mitotic kinesin Eg5: characterization of the 4-phenyltetrahydroisoquinoline lead series[END_REF][START_REF] Kim | Synthesis and SAR of pyrrolotriazine-4-one based Eg5 inhibitors[END_REF]. Most of these inhibitors interact with an hydrophobic (HYD) pocket that is inoccupied by the monastrol (Figure 2). This difference in the binding mode results in a ten fold increase of the affinity in the case of the monastrol and its homologue mon-97 (PDB code 2ieh): (S)-monastrol inhibits the basal ATPase activity with an IC 50 value of 1.7 µM, whereas the (R)-mon-97 has an IC 50 value of 110 nM [START_REF] Garcia-Saez | Structure of human Eg5 in complex with a new monastrol-based inhibitor bound in the R configuration[END_REF]. The current knowledge of the available inhibitors for this target enlarges the scope for rational drug design approaches to discover more potent inhibitors for the Eg5.

The SuMo software was originally developed to detect structural similarities between proteins, in particular protein binding sites [START_REF] Jambon | A new bioinformatic approach to detect common 3D sites in protein structures[END_REF]. MED-SuMo, the commercially available version for drug design applications, is able to detect potential binding sites, characterize an unknown protein binding site and probe active-site selectivity within a protein family. In MED-SuMo, all chemical groups that can make interactions with other molecules are represented by Surface Chemical Features (SCFs) such as hydrogen-bond donor and acceptor, aromatic group, hydrophobic group, and so forth. Once detected, all neighboring SCFs are grouped in triplets which represent nodes of a graph encoding the protein interacting surface. The graph is then compared to a precompiled database of similarly constructed graphs (Figure 3, [START_REF] Jambon | A new bioinformatic approach to detect common 3D sites in protein structures[END_REF][START_REF] Jambon | The SuMo server: 3D search for protein functional sites[END_REF]). MED-SuMo is extremely efficient, allowing application to the entire PDB, and therefore maximizing the probability of finding relevant local similarities on the protein binding site [START_REF] Jambon | A new bioinformatic approach to detect common 3D sites in protein structures[END_REF][START_REF] Jambon | The SuMo server: 3D search for protein functional sites[END_REF][START_REF] Doppelt | Functional annotation strategy for protein structures[END_REF][START_REF] Moriaud | Computational Fragment-Based Approach at PDB Scale by Protein Local Similarity[END_REF][START_REF] Doppelt-Azeroual | MED-SuMo Applications[END_REF]. The number of macromolecular structures publicly available from the Protein Data Bank (PDB) is raising quadratically, with 7 065 structures deposited in 2008 and more than 500 new entries currently released every month. In total, more than 55 000 macromolecule structures are accessible as of January 27th, 2009 (http://www.rcsb.org/pdb/statistics).

Recently, we developed a fragment based drug design approach based on MED-SuMo/MED-Fragmentor/MED-Hybridise technologies [START_REF] Moriaud | Computational Fragment-Based Approach at PDB Scale by Protein Local Similarity[END_REF]. For this new approach, the MED-Fragmentor software component was designed to generate a MED-SuMo database of MED-Portions describing local protein-fragment interactions by cross-mining the PDB and a chemical library of small molecules. The MED-Portions are defined by several criteria including chemical moiety represented in 3D and the corresponding SCF environment. Local graph similarities are found using the MED-SuMo algorithm thus retrieving from the database MED-Portions aligned in 3D to the query structure. These MED-Portions can be combined using their 3D coordinates with the MED-Hybridise software.

The goal of this study was to design inhibitors of Eg5 by populating the HYD pocket of the allosteric binding site. Our fragment-based protocol is applied to populate the HYD pocket with chemical moieties of inter-family MED-Portions that MED-SuMo retrieves by local similarity search upon the whole PDB. Intrafamily MED-Portions are discarded to bias towards the generation of new ligands compared to the known kinesin PDB ligands. The retrieved MED-Portions were combined using the MED-Hybridise software and 3D substructure filtering rules to focus on the most promising hybrids having similar binding mode to known inhibitors. This protocol allowed us to generate hybrid molecules that can be new potential ligands, among which, a certain number can be retrieved by MED-Search software in publicly available chemical libraries, ChemBridge, Maybridge, and PubChem database [START_REF]The PubChem Project[END_REF][START_REF] Chembridge | [END_REF][START_REF] Maybridge | [END_REF].

Results and discussion

Definition of the custom SCF dictionary Several water molecules are observed in the allosteric binding site of Eg5, especially inside the HYD pocket. The default MED-SuMo dictionary does not consider these molecules for the graph generation of the binding site. They could however bring important structural information, when they are involved in hydrogen bond bridging the ligand and the protein target. We therefore decided to add H-bond donors and H-bond acceptors on the water molecules defined in the PDB file. As a result these can be superposed with the H-bond donors and acceptors on either the water molecules or the protein SCFs. Since the graph generation of the database is done at 4.5 Å around the ligand, only the water molecules in the near neigbourhood of the ligand are taken into account, those mostly involved in H-bond interactions.

With this modified dictionary each binding site is defined by a larger number of SCFs, since water molecules can be considered as H-bond donors or acceptors. In addition the SCFs that are already engaged in intra-protein H-bond are not eliminated, resulting in formation of graphs containing a greater number of triplets. For example, for the query defined by any protein atom at 10 Å around monastrol (NAT ligand) in the 1x88 structure [START_REF] Maliga | Structural Basis of Eg5 Inhibition by Monastrol[END_REF], 125 SCFs are considered in the default database and form 3 918 triplets, compared to the modified dictionary database where 187 SCFs are considered and form 6 090 triplets. This increasing number of SCF in query signature results generally in hits having larger number of SCFs and by consequence with greater MED-SuMo score [27]. In this work, all H-bond donors and acceptors SCFs are chemical features without any specified orientation.

This new SCF dictionary is able to better detect conserved secondary structures surrounding binding site, and to better populate the binding sites or surfaces where water molecules are present. An inconvenient consequence is that some of the SCFs considered are already engaged in an H-bond and could not interact with MED-Portions chemical moieties. This approximation seems to be better than removing them because backbone carbonyl and NH can be involved in two H-bonds, one intra-protein and one with a water molecule. Nonetheless, we are able to retrieve with this modified dictionary database with additional pertinent MED-Portions (data not shown).

Choice of the Query structure and selection of collected MED-Portions In order to predict new categories of inhibitors that could fill the HYD pocket, we have chosen to use a structure containing an inhibitor that does not fill the HYD pocket. We constructed the query at 10 Å around monastrol of the 1x88 entry in the PDB. The query is then submitted to MED-SuMo to be compared to the MED-Portions database built with the new dictionary described above (Figure 4). During MED-SuMo comparison an automatic calculation of steric clashes between query protein and retrieved MED-Portions chemical moieties has been done (as described in Background and methods). We kept only the MED-Portions chemical moieties that possessed fewer than 10 bumps with the query once superimposed inside its binding pocket. To show that the protocol is capable of exploring the HYD pocket with inter-family MED-Portion chemical moieties, we have eliminated all the MED-Portion chemical moieties that are directly derived from co-crystallized ligands of Eg5 that are targeting this pocket (Figure 2, see Background and Methods for detailed description). The retained MED-Portions (1092 MED-Portions chemical moieties) constitute the starting point for the next steps.

Hybridization and filtering of the MED-Portions into potential ligands

The retained MED-Portions chemical moieties were subjected to five hybridization and filtering steps with MED-Hybridise software [START_REF] Moriaud | Computational Fragment-Based Approach at PDB Scale by Protein Local Similarity[END_REF]. Hybridization of all the MED-portions would produce a very large number of hybrids. Here we focused the hybrid generation by targeting the HYD pocket. At the same time the hybrids have been selected to keep interactions with the L5/α2 pocket to mimic the known inhibitors binding.

To do so a sublist of rings populating one or the other pockets, called 'seeds', has been selected from the MED-Portions (Figure 5, 83 in the HYD pocket, 153 in the L5/α 2 pocket, see Background and methods for detailed description). The first step consisted of the hybridisation of these 'seeds' with the whole list of MED-Portions chemical moieties using our Chain Combine algorithm of MED-Hybridise (described in [START_REF] Moriaud | Computational Fragment-Based Approach at PDB Scale by Protein Local Similarity[END_REF]). At each following step the 'seeds' were hybridised to the original MED-Portions and the hybrids generated during the previous step. During each hybridisation step exact 3D duplicates (same chemical structure and same 3D coordinates) are discarded.

In addition we applied a 3D sub-structure filter keeping only the hybrids that explored either the HYD or the L5/α2 subpocket with at least one ring (as described in Background and methods). This rigorous filtering resulted in a limited number of hybrids (4115) that possessed the required characteristics (Table 1).

Description of generated hybrids derived from exclusively interfamily MED-Portions chemical moieties

The resulting hybrids have then been filtered for bad geometry and then minimized in the query binding site using CHARMm forcefield implemented in Discovery Studio 2.0 [START_REF]10188 Telesis Court, Suite 100[END_REF]. Several hybrids having a low energy after minimization are shown on Figure 6. They all result from the hybridization of inter-family MED-Portions chemical moieties deriving from various ligands (Figure 6). All hybrids have been chosen to have an aromatic ring that binds in the L5/α 2 pocket. Indeed, except for the bulky ligand (pyrrolo-triazine-4-one analogue) in 2gm1 [START_REF] Kim | Synthesis and SAR of pyrrolotriazine-4-one based Eg5 inhibitors[END_REF], all the other Eg5 co-crystallized ligands from the PDB have an aromatic ring of five or six atoms in that pocket. In L5/α 2 pocket several conserved residues are involved in the binding of the inhibitors in the solved crystal structures of Eg5 from the PDB: Glu116, Glu118, Arg119, Pro137 and Leu214 [START_REF] Garcia-Saez | Structure of human Eg5 in complex with a new monastrol-based inhibitor bound in the R configuration[END_REF]. All those residues are also involved in the predicted binding of the selected hybrids (Figure 6). Given the restriction during the hybridisation protocol to keep hybrids targeting the HYD pocket, most of the hybrids are interacting with the hydrophobic residues Arg221, Leu160, Ile136 and several are forming Tstacking interactions with Phe239 (Figure 6(a)). Some hybrids are also predicted to be involved in H-bond interactions. The hybrid represented in Figure 6(a) (Hybrid 1215) has a third group that points toward the solvent like the (S)monastrol ethyl ester group. The nitrogen of the amide is predicted to form a Hbond with the Tyr211. The hybrids represented in Figure 6(b) (Hybrid 969) and (c) (Hybrid 1317) are both predicted to form a hydrogen bond with Glu118 backbone as does the (S)-monastrol.

One of the MED-Portions chemical moieties is present in the three selected hybrids. It derives from a ligand co-crystallized in the 1q0z PDB structure [START_REF] Jansson | Crystal structure of aclacinomycin methylesterase with bound product analogues: implications for anthracycline recognition and mechanism[END_REF] and is very similar to a sub-structure of the Eg5 inhibitor cocrystallized in 2fme [START_REF] Tarby | Inhibitors of human mitotic kinesin Eg5: characterization of the 4-phenyltetrahydroisoquinoline lead series[END_REF]. However it is derived from a very different ligand (10decarboxymethylaclacinomycin A) and is co-crystallized with a protein of the aclacinomycin methylesterase (RdmC) family.

The hybrids selected at that step are hit-like and can be considered as new leads compared to the known PDB ligands that could potentially bind to the allosteric site of Eg5.

Comparison of the obtained hybrids to compound libraries with MED-Search

Having generated new chemistry for potential kinesin inhibitors, we also checked if molecules synthetically accessible were found to be potential ligands targeting the HYD pocket. Hybrids generated at the last step were searched in diverse chemical libraries including known kinesin inhibitors, commercial libraries like the ChemBridge diverset, Maybridge hitfinder and the entire PubChem database [START_REF]The PubChem Project[END_REF][START_REF] Chembridge | [END_REF][START_REF] Maybridge | [END_REF]. A new module called MED-Search has been developed for that purpose.

As hybrids can still contains Du atoms that were considered as wildcard atoms during hybridization, MED-Search needs to deal with that special type of atom.

To accomplish this task we needed an accurate search algorithm capable to treat Du atoms as wildcard atoms that could match any atom type or even nothing. Due to a huge amount of comparisons it was crucial to be able to pre-filter crudely and very quickly the possible matches. For that purpose, the SCINS representation [30,Jenkins, 2004 #27] of the molecules were compared. Only the retained matches are submitted to an exact comparison using the SMART representation [START_REF] Moriaud | Computational Fragment-Based Approach at PDB Scale by Protein Local Similarity[END_REF]. During the search, if a match is found, the Du atoms of the hybrid are replaced by the real atoms of the matched molecule. All the molecules coming out of MED-Search do not have Du atoms, and are known, chemically accessible molecules. Their 3D coordinates are kept and they are thus aligned in 3D in the reference frame of the query binding site.

We have also generated and searched the Murcko scaffolds computed according Bemis and Murcko [START_REF] Bemis | The properties of known drugs. 1. Molecular frameworks[END_REF]. For the generation of the scaffolds exocyclic double bonds were kept. The results of the comparison are listed in Table 1.

Amongst the hybrids found exactly in the PubChem library several are predicted to bind both in the HYD and in the L5/α 2 pockets (Figure 7). Some contain the substructure highlighted in the hybrid description coming from the MED-Portion derived from the 1q0z ligand. One of them has been minimized in the query binding site, while keeping he protein side chains fixed (Figure 8). This molecule does not make hyrdrophobic interaction in the back of the hydrophobic pocket but it interacts with all the important residues involved in the binding of monastrol analogs and has a different chemistry. It is predicted to be involved in two hydrogen bonds with Glu118 and Leu 214.

Summary and conclusion

Development of resistance in cancer therapies to drugs that target directly the microtubules, as well as their serious side-effects such as neurotoxicity have forced the research to chase for other targets that would more specifically inhibit the mitotic spindle function. Eg5, a mitotic kinesin exclusively involved in the formation and function of the mitotic spindle has attracted interest as an anticancer drug target.

Eg5 is co-crystallized with several inhibitors bound to its allosteric binding site. All of them occupy a pocket of this site formed by loop 5 and helix α 2 (L5/α2), some occupy as well the hydrophobic pocket (HYD) (Figure 2). The inhibitors that are binding to both pockets simultaneously have shown sometimes a ten fold higher affinity then the ones occupying only the L5/α2 pocket.

The goal of the present study was to obtain potential new ligands that explore this HYD pocket with our MED-SuMo fragment based protocol. We have excluded intra-family MED-Portions derived from ligands that occupy the HYD pocket and predicted new potential hybrids that would fill simultaneously both pockets. The MED-Portions chemical moieties have then been combined with the MED-Hybridise software. Some of the latter were found in chemical libraries with the MED-Search software and could therefore be tested experimentally. Based on structural information, the obtained hybrids bring new ideas about the possible scaffolds and chemical functions that could be binding the allosteric site of Eg5 and occupy both pockets.

Background and methods

The results presented in this paper were generated using software developed by MEDIT SA, except where stated otherwise.

MED-SuMo technology

The MED-SuMo technology is described briefly hereafter. For further details as well as for the manner of mining the PDB to extract MED-Portions, to populate protein surfaces with MED-Portions and to hybridize the MED-Portions refer to the papers and the thesis work of Jambon et al. [START_REF] Jambon | A new bioinformatic approach to detect common 3D sites in protein structures[END_REF][START_REF] Jambon | The SuMo server: 3D search for protein functional sites[END_REF]27] and a recent paper of Moriaud et al. describing fragment based drug design protocol derived from our core technology [START_REF] Moriaud | Computational Fragment-Based Approach at PDB Scale by Protein Local Similarity[END_REF].

MED-SuMo is derived from the SuMo software [START_REF] Jambon | The SuMo server: 3D search for protein functional sites[END_REF]. MED-SuMo is able to locate similar regions on macromolecular surfaces associated with similar chemical groups. It applies a heuristic based on a 3D representation of macromolecular surfaces using Surface Chemical Features (SCFs) like, for example, H-bond donor, H-bond acceptor, formal positive and negative charges, hydrophobic, aromatic, or more specific features like amide and guanidinium.

Each feature describes a putative physical interaction including its precise geometrical characteristics. The MED-SuMo comparison methodology can be divided into two major steps: [START_REF] Mitchison | Mitosis: a history of division[END_REF] The Graph Formation: SCFs are displayed on the protein structure through a lexicographic analysis of the atoms in the PDB files, i.e., a residue is represented by a set of representative SCFs. The correspondence between the SCFs and the chemical groups is stored in the MED-SuMo dictionary. SCFs are assembled into triplets with specific geometric characteristics. Such geometric characteristics include edge length, perimeter length, and angles. The triplet network is then stored as a graph data structure. The triplets are vertices and the edges are connecting adjacent triplets in the MED-SuMo database. [START_REF] Mitchison | Dynamic instability of microtubule growth[END_REF] The Graph Comparison: To compare two graphs, MED-SuMo looks for compatible triplets; composed of compatible SCF. These triplets are called comparison 'seeds'. When a seed is detected, MED-SuMo extends the comparisons to the vertices of the neighbourhood, until no more similarities are found. These comparisons generate the formation of similar patches (common groups of SCFs) between two graphs, weighted by the MED-SuMo score. The MED-SuMo search heuristics is based also on two criteria that consider the SCFs 3D positions flexibility and so improve the search for compatible graphs. These comparisons are usually performed between a query and a database of precompiled graphs.

Steric clash filtering during the MED-SuMo comparison

During MED-SuMo comparison, a filter of steric clashes is applied. This is particularly important for our fragment based approach where we need to find relevant MED-Portions chemical moieties that could potentially bind the target. This filter is based on the count contacts between the query protein and MED-Portion chemical moieties.

Using SCF superimposition of the query protein and a retrieved hit, atoms of MED-Portions are placed in the query binding site. Distances between each atom of the MED-Portion chemical moiety, except the Du atoms, and the atoms of the query protein are calculated. If the distance between two atoms is shorter than the sum of their Van der Waals radii weighted by a coefficient a contact is registered. This coefficient was set to 0.8. This value was was empirically determined to allow flexibility in the clash detection. The filter can be set on the number of atoms clashing or on the total number of clashes for the given MED-Portions. Finally, if the number of clashes is greater than a user defined value, the MED-Portion is excluded.

In this study, only the MED-Portions chemical moieties that exhibit more than ten steric clashes with the query protein are discarded.

MED-Portion definition MED-Portions are the MED-SuMo representation of protein-fragment patterns

obtained by fragmentation of the protein-ligand structures using the MED-Fragmentor [START_REF] Moriaud | Computational Fragment-Based Approach at PDB Scale by Protein Local Similarity[END_REF]. MED-Portions are defined by several criteria: (1) a chemical moiety where atoms are topologically matching a molecule of less then 250 Da from molecular libraries. Therefore, these molecules are expected to be synthetically accessible molecules or building blocks, [START_REF] Mitchison | Dynamic instability of microtubule growth[END_REF] open valences filled by Du atoms that indicate where it was connected in the original ligand, and (3) the protein interaction surface surrounding that chemical moiety described by the SCFs. The pre-calculated MED-SuMo fragment data base contains 340 888 "binding-sites" and thus 340 888 MED-Portions. MED-Portion chemical moieties are collected in the reference frame of the query and form a pool which is used to generate new 3D hybrid compounds by hybridization.

Custom dictionary database

To use the structural information provided by water molecules crystallized into the binding sites, the MED-SuMo dictionary was modified. Indeed, MED-SuMo software allows the advanced user to modify the dictionary of SCF definitions by editing a text file. Given the potential importance of the water molecules for the considered target, we have taken them into account. This allows them to match with either water molecules, or H-bond donors and acceptors on the hit protein structure. In the classical MED-SuMo dictionary, H-bond donor and acceptor SCFs have a defined orientation. The H-bond donor SCF is also projected to the location of a potential H-bond acceptor. For water molecules it is not possible to derive any orientation or projection for the H-bond donor or acceptor groups. Therefore we added two new ponctual geometrical variant of H-bond acceptor and H-bond donor in the dictionary syntax. Both are neither orientated nor projected. In this new version of the MED-SuMo dictionary, the water molecules are considered as part of the receptor and are described with one H-bond acceptor and H-bond donor.

In MED-SuMo only chemical groups of the same type can be compared and possibly considered as equivalent. One aim of the new dictionary is to be able to match the H-bond donor and/or acceptor of the water molecules with similar groups on the proteins. Therefore former orientated H-bond donors and acceptors on the protein structure are replaced by the new punctual H-bond donor and acceptor. Once the SCFs are positioned on the macromolecular structure their positions and orientations are filtered. In the default MED-SuMo configuration a filtering step discards any SCFs likely to be involved in intra-molecular hydrogen bonds. With the new dictionary this filter is skipped. However the SCFs too buried to interact with a potential ligand are still discarded.

Selection of collected MED-Portions

From the collected MED-Portions we eliminated the ones derived from the Eg5 ligands that are located in the targeted hydrophobic pocket (2gm1, 2g1q, 2q2y, 2uyi, 2uym, 2pg2, 2q2z, 1yrs, 2fl6, 2ieh, 2fky, 2fl2, 3cj0). MED-Portions derived from three structures of Eg5 were kept, since their ligands are not positioned in the HYD pocket: 1x88, 2fme and 1q0b. The selected MED-Portions' atoms were typed with OpenBabel [START_REF]The Open Babel Package[END_REF].

Selection of seeds

To create new ligands that would bind simultaneously the targeted HYD and the L5/α2 pocket, we selected a sub-list of rings derived from the retrieved MED-Portions chemical moieties that explored these pockets as the starting points, the seeds. The seeds were selected as follows. We fragmented the retrieved MED-Portions in rings and linker parts. Then we filtered the rings, to keep only those that had at least one carbon atom inside one of the pockets. The emplacement of this atom was selected at 3 Å around the deepest one buried in the L5/α2 pocket of the 1x88 ligand and deepest one buried in the HYD pocket of the mon-97 ligand from the 2ieh PDB code structure as it was placed inside the 1x88 PDB code structure binding pocket after the superposition of the two structures by MED-SuMo (Figure 9). The selected rings were decorated with Du atoms at every possible position defined by implicit hydrogen atoms.

Hybridization parameters and substructure filtering

We applied 5 hybridization steps with the Chain Combine algorithm of MED-Hybridise [START_REF] Moriaud | Computational Fragment-Based Approach at PDB Scale by Protein Local Similarity[END_REF]. The basic principle of Chain Combine is to look for overlapping bonds between a pair of aligned MED-Portions. The Du atoms of the MED-Portions are considered as wildcard atoms during the hybridization steps.

We have hybridized the seeds to the whole list of selected MED-Portions during the first step. Then we hybridized the seeds to the molecules obtained in the previous step. During steps 2 to 5 we used the seeds, without Du atoms for the substructure 3D filtering, by keeping only the hybrids that were superstructures of the one of those seeds. After each step we filtered the obtained hybrids for 3D duplicates (RMSD 2.0 during the first step, RMSD 4.0 during the 2nd to the 4th steps, and RMSD 6.0 afterwards). These 4841 molecules have been submitted to the MED-Search module as explained later.

In parallel, several filtering steps were applied, molecules with intramolecular clashes and elevated strain energy, the ones containing a phosphate atom or a ribose are discarded. The remaining hybrids were minimized in the binding site of the query structure with the CHARMm forcefield included in Discovery Studio 2.0 [START_REF]10188 Telesis Court, Suite 100[END_REF]. The query structure is 1x88 used for the MED-SuMo run after removing all ligands and water molecules. During the minimization the receptor is considered rigid.

MED-Search module

The aim of the MED-Search software is to search the designed hybrid molecules that contain in many cases dummy atoms, versus a large compound library rapidly and accurately. It uses a two-step algorithm.

The first step rapidly skips molecules that can be trivially seen to not match without discarding false negatives. For reasons of efficiency, this step is performed via comparison of a novel mini-fingerprint that encode molecules' scaffold structures. The design of this mini fingerprint was inspired by the Novartis SCINS classifier [START_REF] Czerminski | Using ROCS, EON, FEPOPS and 2D methods for prospective and retrospective similarity searching: experiences and results[END_REF][START_REF] Jenkins | A 3D similarity method for scaffold hopping from known drugs or natural ligands to new chemotypes[END_REF]. It consists of an array of thirteen integer values, corresponding to the thirteen values in the SCINS classifier. The queries are read from the input file and SCINS fingerprints for these molecules are generated during the first loop of comparison against the first target molecule. Molecules are further compared with an exact comparison only when their Tanimoto similarity metric exceeds a 0.99 threshold value.

The second step is the exact structure search that analyses the connectivity of the compared molecules and then compares corresponding atom pairs. Two atoms are treated as matching if they have the same atom type (Sybyl-style atom type), if they are or not in a ring and if they are or not aromatic. These rules apply only to real atoms. Du atoms are considered as wildcard atoms and can match any atom or even nothing.

At this stage, Du atoms can be replaced with real atom types to complete the match. During the comparison, the algorithm tries first to superimpose the non-Du atoms of the query. If the exact matching is successful, then the mapping of Du atoms is done and they are replaced according to the corresponding atom in the hit. Where there is no possible match, the Du atom can be deleted. The exact structure of 3077 hybrids were compared to the chemical libraries (20 million compounds) with MED-Search. This comparison took forty hours on eight CPUs (2 Xeon Quad Core 5335, 16 GB RAM).

Fig. 1 2D representation of known Eg5 inhibitors. The 3,5-diaryl-4,5-dihydropyrazole (a) is a potent and selective inhibitor of Eg5 that is active in cells at low nanomolar concentrations. It was identified in a high throughput screening by Merck [START_REF] Cox | Kinesin spindle protein (KSP) inhibitors. Part 1: The discovery of 3,5-diaryl-4,5dihydropyrazoles as potent and selective inhibitors of the mitotic kinesin KSP[END_REF]). X-ray crystallographic evidence is presented which demonstrates that this inhibitor bind the same allosteric pocket as the monastrol (b) and mon-97 (c) of Eg5 that is distant from the nucleotide and microtubule binding sites. Further studies showed that by appending a propylamine substituent at the C5 carbon of a dihydropyrazole core, lead to compounds with increased potency and aqueous solubility (d and e) Fig. 2 (a) View of the Eg5 binding site surface from the 1x88 PDB structure showing the cocristallized monastrol (green) and the mon-97 (clear blue) from the 2ieh PDB structure, retrieved from the superposition of the two protein structures with MED-SuMo. Both ligands occupy the sub-pocket formed by loop 5 (L5) and the helix α 2 (α 2). The mon-97 occupies as well the hydrophobic pocket HB shown in (b) Grey: hydrophobic, blue: hydrophilic Fig. 3 MED-SuMo core algorithm: graph creation-comparison. 1. The protein molecular structure is scanned and mapped to a dictionary of physicochemical/geometric constructors that detect the MED-SuMo Surface Chemical Features (SCFs, represented by colored circles). 2. The geometric rules are applied to build a network of triplets (green triangle). 3. This network is transposed into a graph data structure. 4. The resulting graph is compared to the precompiled database of similarly constructed graphs Fig. 4 Query definition. The binding site is defined at 10 Å around every atom of the (S)-monastrol (NAT) of the 1x88 structure, MED-SuMo detects 187 Surface Chemical Futures (SCF, described in Background and methods section) (a). By building the query with these SCFs, the graph contains 6090 triplets (b) which are then submitted to the database Fig. 5 Hybridisation seeds from the HYD pocket (salmon). The seeds used in the hybridization steps have been selected as described in Background and methods. Green: monastrol ligand from the 1x88 structure. Blue: mon-97 ligand from the 2ieh structure Fig. 6 Examples of the selected hybrids and the MED-SuMo chemical moieties that have been used for their formation presented in 3D coordinates, inside the 1x88 allosteric binding site. In L5/α 2 pocket several conserved residues are involved in the binding to the inhibitor in all the crystal structures of Eg5 from the PDB: Glu116, Glu118, Arg119, Pro137 and Leu214. All those residues are also involved in the predicted binding of the selected hybrids. In the HYD pocket, all the hybrids are interacting with the hydrophobic residues Arg221, Leu160, Ile136 and especially are forming T-stacking interacting with Phe239. Hybrid 1215 represented in (a) has a third group that points toward the solvent like the (S)-monastrol ethyl ester group. The nitrogen of the amide is predicted to form a hydrogen bond with the Tyr211. Hybrid 1317 represented in (b) and hybrid 969 (c) are both predicted to form a hydrogen bond with Glu118 background as does the (S)monastrol. For each of the hybrids above MED-Portions were derived from the following PDB structures : (d) 1fo4 in blue, 1n78 in orange, 1q0z in green and 2ox0 in red; (e) 1n78 in dark blue, 1r3w in pink, 1q0z in purple and 1oo5 in clear blue (f) 1r3w in clear blue, 1q0z in orange and 1eqb in green 1fo4: xanthine dehydrogenase, ligand 2-hydroxybenzoic acid 1n78: glutamyl-trna synthetase, ligand glutamol-amp 1q0z: aclacinomycin methylesterase, ligand 10-decarboxymethylaclacinomycin a 2ox0: histone demethylase, ligand n-oxalyolglycine 1r3w: uroporphyrinogen decarboxylase, ligand coproporphyrin iii 1oo5: nitroreductase prodrug-activating system, ligand flavin mononucleotide 1eqb: serine hydroxymethyltransferase, ligand 5-formyl-6-hydrofolic acid Fig. 7 Examples of hybrids found in PubChem with MED-Search. These molecules should be considered as scaffold to be decorated as they are often too hydrophobic to be tested.

Fig. 8 (

 8 Fig.8 (a) Example of a hybrid that matched a PubChem molecule. This hybrid is derived from three MED-Portions represented in their 3D coordinates (b) derived from the following PDB structures: 3pax in orange, 1q0z in green and 1gos in red 3pax: poly (ADP-ribose) polymerase, ligand 3-methoxybenzamide 1q0z: aclacinomycin methylesterase, ligand 10-decarboxymethylaclacinomycin a 1gos: human monoaminne oxidase b, ligand flavin-adenine dinucleotide

Table 1

 1 Statistics on the hybrids. Hybrids were generated as described. They contain Du atoms and are filtered for 3D duplicates and 2D duplicates. The 2D filtered hybrids have been submitted to MED-Search and the listed sources of molecules. The numbers of found molecules represent the number of molecules in a particular library that matched the hybrids. Wildcard can match nothing or any atom type

	Hybrid count (unique 3D)

Acknowledgments

We thank Raphaël Guerois for providing us useful comments and suggestions for the initiation of this study (Commissariat à l'Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay, and Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette, F-91191, France). Olivia Doppelt-Azeroual is a Ph.D. student funded by the ANRT. This work was supported by the Carriocas collaborative project (http://www.carriocas.org/) and funded by the French office "Direction Générale des Entreprises".