Clifford W Fong Eigenenergy
email: cwfong@internode.on.net

Ionophores and inhibition of SARS-CoV-2 ion channels

Keywords: SARS-CoV-2, Vero E6-hTMPRSS2 cells, polyether ionophores, carrier ionophores, E protein ion channel, ion channel inhibitors, voltage controlled ion channels, excitation energy, HOMO-LUMO energy gap, molecular volumes, TD DFT, quantum mechanics

This study uses a previously described predictive quantum mechanical TD DFT method which can describe the time dependent behaviour of inhibitors of voltage controlled ion channels. The method is shown to apply to a widely diverse range of polyether antibiotic ionophores that inhibit SARS-CoV-2 infected Vero E6-hTMPRSS2 cells. The key determinants are (a) the excitation energy of the first excited state of the inhibitor, (b) the ground state HOMO-LUMO gap of the inhibitor that govern the dynamic binding between the inhibitor and the protein target, and (c) the molecular volume of the absorbed inhibitors in the virus envelope or the Vero cell membranes. The possibility that disruption of ion homeostasis caused by the antibiotics acting as carrier ionophores of metal ions such as K + or Ca ++ is also explored.

Introduction

SARS-CoV-2 is an enveloped virus and similar to other coronaviruses, and is comprised of four key structural proteins: S, the spike protein, E, the envelope protein, M, the membrane protein, and N, the nucleocapsid protein. Both SARS-CoV and SARS-CoV-2 spike proteins use the angiotensin-converting enzyme 2 (ACE2) protein as a receptor for cellular entry in humans. Spike protein subunits have been widely reported as targets for vaccines, novel therapeutic antibodies, and small-molecule inhibitors.

Like SARS-CoV, SARS-CoV-2 uses the receptor angiotensin-converting enzyme 2 (ACE2) for entry and the serine protease TMPRSS2 for the virus S spike protein activation. The S protein of SARS-CoV-2 has a higher cell membrane fusion capacity compared to SARS-CoV. TMPRSS2 contains several domains: a LDL-receptor like domain, a scavenger receptor cysteine-rich (SRCR) domain and a serine protease domain. TMPRSS2 is co-expressed in human lung tissue with the ACE2 receptor. The inhibitors camostat and nafamostat have been shown to block the catalytic site of TMPRSS2. [START_REF] Hoffmann | SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor[END_REF] Several studies [START_REF] Mcclenaghan | Coronavirus Proteins as Ion Channels: Current and Potential Research[END_REF][START_REF] Hover | Viral dependence on cellular ion channels -an emerging antiviral target[END_REF][4] have reviewed coronavirus proteins as ion channels and their potential as therapeutic targets. Coronavirus E proteins can also function as ion channels. Different electrophysical ion permeabilities were observed in bilayers for the E channel: the α-CoV HCoV-229 (K + > Na + > Cl -) differed from the mouse hepatitis virus (β -CoV) and infectious bronchitis virus (γ -CoV) (both had Na + > K + > Cl -). The electrophysiology of different ions (Na + , K + , Cl -, Ca ++ ions) on the coronavirus 3A, 4A and 8A proteins has also been discussed. Ion homeostasis in viruses has highlighted the importance of ion channel -virus interactions in viral pathogenesis. [START_REF] Fw Charlton | Ion Channels as Therapeutic Targets for Viral Infections: Further Discoveries and Future Perspectives[END_REF] There are three viroporins encoded by the SARS-CoV-2 genome: the envelope protein E, 3A and ORF8a. Viroporins or ion channels are small and mostly hydrophobic multifunctional viral proteins that can alter cellular membranes. SARS 3A is associated with inflammasome activation as well as apoptotic and necrotic cell death. The 3A viroporin in SARS-CoV-2 was found to be a novel dimeric fold, forming a non-selective calcium (Ca ++ ) permeable cation channel. It is thought that calcium influx through 3A might be an activation trigger for calcium-dependent caspases and apoptosis, and the role of calcium permeability in 3A could be a significant therapeutic target [START_REF] Dm Kern | Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs[END_REF] It has been reported that SARS-CoV-2 exploits changes in metal ion concentrations to disguise itself in the body and evade immune surveillance. Varying concentrations of metal ions (positively charged atoms such as magnesium, manganese and calcium) are observed in hospitalized COVID-19 patients. [START_REF] Viswanathan | A metal ion orients SARS-CoV-2 mRNA to ensure accurate 2′-O methylation of its first nucleotide[END_REF] It is thought that SARS-CoV-2 virion entry, host-membrane fusion and the ensuing virus replication in the host cells are governed by ion currents, ie for example Ca ++ ions are necessary to promote viral fusion peptide insertion into the lipid bilayer and the cellular endocytosis pathway. [START_REF] Ep Navarese | Ion channel inhibition against COVID-19: A novel target for clinical investigation[END_REF] The E protein which is the best characterized protein of SARS-CoV-2 is a small hydrophobic protein of ∼74-109 amino acids comprised of a charged cytoplasmic tail and a hydrophobic domain. This protein consists of three parts: NT (negatively charged), TMD (not recharged), and CT (negatively charged). [START_REF] Mcclenaghan | Coronavirus Proteins as Ion Channels: Current and Potential Research[END_REF][START_REF] Hover | Viral dependence on cellular ion channels -an emerging antiviral target[END_REF][4] E protein contributes to ion channel, viroporin activity, and virus assembling. Envelope protein forms a cation-selective channel and plays a key role in the virus's ability to replicate itself and stimulate the host cell's inflammation response. The structure (determined in the closed state) of the E protein [START_REF] Vs Mandala | Structure and drug binding of the SARS-CoV-2 envelope protein transmembrane domain in lipid bilayers[END_REF] is similar to an influenza protein called the M2 proton channel. Both viral proteins are made of bundles of several helical proteins. The SARS-CoV-2 E protein is very different to the ion channel proteins of influenza and HIV-1 viruses. The structure in the open state is still to be determined. Several amino acids at one end of the channel may attract positively charged ions such as calcium into the channel. It was found that amantadine, used to treat influenza and hexamethylene amiloride, used to treat high blood pressure both weakly block the entrance of the E channel. [START_REF] Vs Mandala | Structure and drug binding of the SARS-CoV-2 envelope protein transmembrane domain in lipid bilayers[END_REF] Another homology study [START_REF] Sarkar | Structural insight into the role of novel SARS-CoV-2 E protein: A potential target for vaccine development and other therapeutic strategies[END_REF] has identified GLU 8 and ASN 15 in the N-terminal region were in close proximity to form H-bonds, and two distinct "core" structures, the hydrophobic core and the central core, can regulate the opening or closing of the channel.

Cao et al [START_REF] Cao | Computational Study of the Ion and Water Permeation and Transport Mechanisms of the SARS-CoV-2 Pentameric E Protein Channel[END_REF] have modeled by molecular dynamics the pentameric SARS-CoV-2 E protein and shown it is a voltage-dependent hydrophobic channel with monovalent cation selectivity. Water molecules and monovalent cations spontaneously penetrate through the channel under a transmembrane voltage. The homology model of the pentameric structure of the SARS E protein (PDB5X29) was used for the SARS-CoV-2 E protein. Cao used a mixed lipid membrane model composed of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine to simulate the lipid environment. Leu10 and Phe19 are the hydrophobic gates that regulate ion permeability. It is thought that channel activity of the pentameric E protein is necessary for inflammasome activation and is the determinant of SARS-CoV-2 cytokine storm virulence.

Gupta et al [START_REF] Gupta | In-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel[END_REF] used molecular dynamics and docking studies to characterize the binding of many inhibitors to the E ion channel of the SARS-CoV-2 homology model (based on the NMR structure PDB5X29) noting that SARS-CoV and SARS-CoV-2 protein share a 94.74% sequence identity. It was found that some phytochemicals which were highly effective inhibitors reduced the random motion of the human SARS-CoV2 E protein. Also Val25 and Phe26 played a key role while interacting with effective inhibitors.

While there have been many recent reports of small molecule inhibitors that are effective against the spike protein of SARS-CoV-2, fewer studies have been made targeting the E protein ion channel. Tomar et al [START_REF] Pps Tomar | Targeted Drug Repurposing Against the SARS-CoV-2 E Channel Identifies Blockers with in vitro Antiviral Activity[END_REF] have reported the screening of 3000 approved drugs using three independent bacteria-based assays. Ten drugs were identified with significant antiviral activity in Vero E6 tissue culture.

Voltage gated ion channels are a class of transmembrane proteins that form ion channels that are activated by changes in the electrical membrane potential near the channel. The membrane potential alters the conformation of the channel proteins, regulating their opening and closing. Cell membranes are generally impermeable to ions, thus they must diffuse through the membrane through transmembrane protein channels. Voltage gated ion channels have a particular ion selectivity and a particular voltage dependence. Many are also time-dependent in that they do not respond immediately to a voltage change but only after a delay.

Efimova 2020 [14] studied changes in the transmembrane distribution of the electrical potential by measuring changes caused by 22 alkaloids to the boundary potential of planar lipid bilayers (POPC, DOPE/DOPS or mixture of 67 mol% POPC and 33 mol% cholesterol). Membranes were also modified by the addition of pore forming anti-microbial agents GrA, SyrE, CecA, and Nys. It was found that the alkaloids had a major effect on the membrane dipole potential. The lifetime and conductance of single pores induced by the pore forming agents were also strongly influenced by the membrane dipole potential. The tested alkaloids did not increase the ion permeability of lipid bilayers in the absence of pore-forming agents.

Broadly there are three kinds of cellular membrane potentials: the surface potentials, (ΔΨ s ), between the membrane surface and bulk solvent, resulting from the accumulation of charges at the membrane surfaces; the transmembrane potential, (ΔΨ m ), across the membrane, from one surface to the other, and determined by imbalance of charge in the aqueous solutions; and the dipole potential, (ΔΨ d ), a membrane-internal potential from the dipolar components of the phospholipids and interface water, which creates a much larger electric field that is highly localized to the interface between the hydrophobic and hydrophilic layers. The dipole potential is much larger than the surface and transmembrane potentials. The absorption of drugs into the lipophilic membrane can alter the ΔΨ d the membrane dipole potential by distorting the membrane and affecting conformations. ΔΨ m is considered to be the driving force of voltage gated ion transitions.

Efimova concluded that the dipole potential made the main contribution to the potential drop at the interface after the adsorption of alkaloids, despite some of the alkaloids having a non-zero charge at pH 7.4 based on their pKa values. The standard measures of lipophilicity, the octanol/water partition coefficient log P o/w (or logD o/w for the ionized species) were strongly correlated with the maximum dipole potential at the 0.75 level. No correlations were observed with the dipole moments of the alkaloids. [14] Efimova 2015 [START_REF] Ss Efimova | Modifiers of the dipole potential of lipid bilayers[END_REF] also similarly studied changes in the dipole potential of lipid bilayer membranes caused by the adsorption of some flavonoids, muscle relaxants, thyroid hormones, and xanthene and styrylpyridinium dyes. A quantitative relationship was found between the ratio of the maximum change in the bilayer dipole potential upon saturation and the absolute value of the unmodified membrane.

Svenningsen [START_REF] Eb Svenningsen | Ionophore antibiotic X-206 is a potent inhibitor of SARS-CoV-2 infection in vitro[END_REF] has determined the in vitro efficacy of a wide range of polyether ionophore antibiotics against the SARS-CoV-2 infection in Vero E6-hTMPRSS2 cells. In particular the antibiotic X-206 showed displayed >500-fold selectivity and was able to inhibit viral replication even at sub-nM levels. The antiviral mechanism of the polyether ionophores is currently not understood. An earlier evaluation of a panel of 290 drugs and drug candidates against MERS-CoV and SARS-CoV, revealed that 9 different ion channel inhibitors including the polyether ionophores salinomycin and monensin could inhibit the pathogenesis effects of MERS but not SARS-CoV. [START_REF] Dyall | Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection[END_REF] Zhang has noted that the ionophore valinomycin is active against coronaviruses such as SARS-CoV, MERS-CoV, human coronavirus OC43 (HCoV-OC43), and has potential to also be active against SARS-CoV-2 [START_REF] Zhang | Valinomycin as a potential antiviral agent against coronaviruses: A review[END_REF].

Study objectives:

Identify predictive methods which can describe the time dependent behaviour of polyether antibiotics possibly acting as inhibitors of voltage controlled ion channels or influencing ion homeostasis in SARS-CoV-2 infected cells

Results

Time-dependent density-functional theory (TDDFT) is a quantum mechanical theory which can describe the properties and dynamics of molecular systems in the presence of time-dependent potentials, such as electric or magnetic fields. The excitation energies of molecular systems such as the influence of chemical inhibitors on the electrical fields in voltage gated ion channels offer potential to describe such behaviour as a function of varying structural properties of such inhibitors. The time dependencies involved are (a) the dynamic inhibition equilibrium of ion channels by chemical species, (b) the electrical field imposed on the particular inhibitor-ion channel interaction.

We have previously shown [START_REF] Fong | Inhibition of SARS-CoV-2 envelope ion channel and the relationship with the membrane dipole potential[END_REF] that the excitation energy and the ground state (HOMO-LUMO) energy gap can predicatively describe linear changes in the electrical dipole transmembrane potential of planar lipid bilayers for a wide and diverse range of flavanoids, xanthenes dyes and alkaloids. In alkaloids the lifetime and conductance of single pores induced by the pore forming agents were also strongly influenced by the membrane dipole potential. The tested alkaloids did not increase the ion permeability of lipid bilayers in the absence of pore-forming agents. We also showed that drugs which have antiviral activity by inhibiting the E protein ion channel of SARS-CoV-2 can be predicatively described by a linear dependence on the excitation energy of the drugs as well as their dynamic ground state binding to the E protein as measured by the (HOMO-LUMO) gap. [START_REF] Fong | Inhibition of SARS-CoV-2 envelope ion channel and the relationship with the membrane dipole potential[END_REF] We have used our general LFER method [START_REF] Fong | Predicting inhibition of repurposed drugs for SARS-CoV-2 viral activity and cellular entry[END_REF] which seeks unconstrained linear free energy relationships amongst the vertical excitation energy of the first excited state, the ground state (HOMO-LUMO) energy gap, the lipophilicity (ΔG CDS,Octanol ) and the molecular volume in n-octanol (as a surrogate for the lipophilic virus coating or host cell membrane). In a study of the changes in the transmembrane distribution of the electrical potential caused by the absorption of 22 alkaloids in planar lipid bilayers, it was found that the lipophilicity, as measured by the octanol/water partition coefficient log P o/w (or logD o/w for the ionized species) was strongly correlated with the maximum dipole potential at the 0.75 level. [14] Table 1 shows the EC 50 values for eleven ionophore antibiotics from Svenningsen [START_REF] Eb Svenningsen | Ionophore antibiotic X-206 is a potent inhibitor of SARS-CoV-2 infection in vitro[END_REF] and the calculated excitation energies, ground state HOMO-LUMO gaps, lipophilicities and molecular volumes in n-octanol.

Svenningsen [START_REF] Eb Svenningsen | Ionophore antibiotic X-206 is a potent inhibitor of SARS-CoV-2 infection in vitro[END_REF] has determined the in vitro efficacy of a wide range of polyether ionophore antibiotics against the SARS-CoV-2 infection in Vero E6-hTMPRSS2 cells. For the free acid antibiotics the best correlation for EC 50 was found for eq 1 and 2. Calculated molecular volumes have been normalized by 100 times to allow a direct comparison of the coefficients for the excitation energy and molecular volumes. Ionomycin was excluded as a demonstrated outlier from all correlations. It can be seen that eq 1 and eq 2 are very similar, suggesting that the EC 50 values for the SARS-CoV-2 are about equally dependent on the molecular volumes of the absorbed antibiotics in the lipophilic viral envelope and the excitation energies and (HOMO-LUMO) gaps. Because of the small experimental data set for EC 50 values, it is noted that separate linear correlations between EC 50 and molecular volume {F 12.5, Signif 0.007}, EC 50 and excitation energies { F 2.1, Signif 0.18}, and EC 50 and (HOMO-LUMO) { F 3.2, Signif 0.10} were also reasonably strong correlations.

Since the antibiotics examined by Svenningsen are known ionophores it is pertinent to investigate whether the EC 50 experimental values may be dependent on these antibiotics acting partly or substantially as carrier ionophores which can influence ion homeostasis within the virus and or Vero cells. Under the in vitro experimental conditions (Dulbecco's Modified Eagle's Medium (DMEM) and fetal calf serum, pH ~ 7.5) may provide sources of ions such as K + , Na + and Ca ++ . All of the ionophores are known to be active carriers of K + particularly, with A23187 calcimycin and ionmycin being active carriers of Ca ++ ions. Examination of the antibiotics acting as ion carriers shows that the most stable forms of the {ionophore-metal ion} complexes are electrogenically neutral species where the anionic form of the antibiotic complexes with the K + ion in such a fashion that the antibiotic component wraps around the metal ion partially shielding the metal ion, and increasing the lipophilicity of the complexes. However {indanomycin-K + } 0 is an exception, since it is a much smaller molecule. A23187 calcimycin requires two anionic antibiotic moieties to complex with the Ca ++ to form a neutral complex, whereas in ionomycin the di-anionic antibiotic moiety complexes with the Ca ++ ion to form a neutral complex.

Table 2 shows the molecular specifiers for these {antibiotic-metal} complexes in n-octanol. Figures 1 and2 show the molecular complexes {X-206-K + } 0 (formed from the X-206 anion and K + ) and {X-206-K + } + (formed from the neutral X-206 and K + ), with lipophilicities (ΔG CDS,Octanol ) of -2.74 and -3.20 kcal/mol respectively. {X-206-K + } 0 is more stable than {X-206-K + } + by 0.57 kcal/mol. Figure 3 shows {Monensin-K + } 0 (formed from the anionic Monensin and K + ) with the intramolecular H bond, and Figure 4 showing {Monensin-K + } + (formed from the neutral Monensin and K + ) without the intramolecular H bond and carrying a formal positive charge. It is noteworthy that {Monensin-K} 0 has a lipophilicity of -0.66 kcal/mol, while {Monensin-K} + has virtually the same lipophilicity of -0.71 kcal/mol. The formal positive charges on {X-206-K + } + and {Monensin-K} + are highly delocalized. {Monensin-K + } 0 is more stable than {Monensin-K + } + by 0.82 kcal/mol. Figure 5 shows {Ionomycin-Ca ++ } 0 with the dianionic form of ionomycin complexing with Ca ++ , and Figure 6 shows {dicalcimycin-Ca ++ } 0 formed by two calcimycin anions complexing with Ca ++ . Analysis of the EC 50 values from Svenningsen assuming the active inhibitory species are the neutral {ionophore-metal ion} complexes (as per Table 2 shown as the K + complexes or otherwise as the Ca ++ complexes for dicalcimycin and narasin) gives eq 3 and 4. It can be seen that eqs 3 and 4 are quite similar to the corresponding eqs 1 and 2, with similar precision. The strong similarities infers that the active species that inhibits the SARS-Cov-2 in Vero E6-hTMPRSS2 cells could be either the free acid form of the antibiotic ionophores or possibly the neutral {ionophore-M n+ } 0 complexes.

Discussion

The absorption of drugs into the lipophilic envelope of viruses or the lipophilic membrane of cells can be dependent on the molecular size of the drugs as they distort the viral envelope affecting electrical fields in voltage gated ion channels, and perturb ΔΨ d of the cell membrane.

[14] [START_REF] Mares | Identification of Electric-Field-Dependent Steps in the Na + , K + -Pump Cycle[END_REF] The (HOMO-LUMO) gap is a ground state property that determines the inherent chemical reactivity of the drugs and governs the dynamic equilibrium between various drugs and the lipid membrane. The excitation energy of the first excited state is a measure of how a drug will be excited under a dynamic electrical potential. Eqs 1 and 2 suggest that EC 50 results for the ionophore antibiotics studied by Svenningsen indicate that ion channel inhibition of the SARS-CoV-2 virus is likely occurring, since the dependency on the excited state of the antibiotics indicates that time-dependent potentials are involved in the chemical inhibition of the electrical fields in voltage controlled ion channels. The strong dependency on the molecular volume for the adsorbed antibiotics and how they may influence the ion channel potential of the viral envelope, or the dipole potential of the cell membrane also supports this notion. Dependency on the (HOMO-LUMO) gap is consistent with the innate reactivity of the antibiotics. It is not known which ion channels or viroporins are involved in Svenningsen's study, but the envelope protein E, 3A and ORF8a are likely present. We have previously shown that the inhibition of the SARS-CoV-2 envelope protein E is determined by equations similar to eqs 1 and 2 being primarily dependent on excitation energy and the (HOMO-LUMO) gap. [START_REF] Fong | Inhibition of SARS-CoV-2 envelope ion channel and the relationship with the membrane dipole potential[END_REF] Eqs 3 and 4 are suggestive that since the antibiotics examined by Svenningsen are known ionophores, the EC 50 experimental values could be dependent on these antibiotics acting partly or substantially as carrier ionophores which can influence ion homeostasis within the virus and or Vero cells. There is evidence that metal ions and accompanying disruption of ion homeostasis may be involved in the pathogenesis of Covid-19. [START_REF] Mcclenaghan | Coronavirus Proteins as Ion Channels: Current and Potential Research[END_REF][START_REF] Fw Charlton | Ion Channels as Therapeutic Targets for Viral Infections: Further Discoveries and Future Perspectives[END_REF][START_REF] Viswanathan | A metal ion orients SARS-CoV-2 mRNA to ensure accurate 2′-O methylation of its first nucleotide[END_REF][START_REF] Ep Navarese | Ion channel inhibition against COVID-19: A novel target for clinical investigation[END_REF] The neutral {ionophore-M n+ } 0 complexes (formed from the anionic ionophore and metal ion) are shown to have similar lipophilicity and greater stability to the charged {ionophore-M n+ } n+ (formed from the neutral ionophore and metal ion) species where the formal charge is delocalized, and accordingly are similarly likely to penetrate the lipophilic virus envelope or Vero cell membrane. The pH of the experimental DMEM medium is ~7.5 which allows some anionic ionophore to be present which can then complex with K + or Ca ++ to form the neutral {ionophore-M n+ } 0 complexes, which are stabilized by an intramolecular hydrogen bond between the anionic acid group and a hydroxyl group (see Figures 123456)

Conclusions

This study uses a previously described predictive quantum mechanical TD DFT method which can describe the time dependent behaviour of inhibitors of voltage controlled ion channels. The method is shown to apply to a widely diverse range of polyether antibiotic ionophores that inhibit SARS-CoV-2 infected Vero E6-hTMPRSS2 cells. The key determinants are (a) the excitation energy of the first excited state of the inhibitor, (b) the ground state HOMO-LUMO gap of the inhibitor that govern the dynamic binding between the inhibitor and the protein target, and (c) the molecular volume of the absorbed inhibitors in the virus envelope or the Vero cell membranes. The possibility that disruption of ion homeostasis caused by the antibiotics acting as carrier ionophores of metal ions such as K + or Ca ++ is also explored.

Materials and methods

All calculations were carried out using the Gaussian 09 package. Energy optimizations were at the DFT/B3LYP/6-31G(d) (6d, 7f) level of theory for all atoms in water. TD DFT vertical calculations were conducted at the TD DFT/B3LYP/6-31G(d,p) (6d, 7f) with the empirical dispersion GD3BJ correction for neutral and charged compounds with optimized geometries in n-octanol, using the IEFPCM/SMD solvent model. Ground state HOMO and LUMO were calculated at the DFT/B3LYP/6-31G(d,p) (6d, 7f) with empirical dispersion GD3BJ in n-octanol.

Eq 1

 1 10 antibiotics (free acid form) from Svenningsen EC 50 = -0.09Excitation Energy -0.11MolVol + 1.15 Where R 2 = 0.730, SEE = 0.112, SE(ExcitEn)= 0.05, SE(MolVol) = 0.03, F=9.47, Significance= 0.010. P-values: ExcitEn= 0.012, MolVol=0.018; Eq 2 examines the 10 antibiotics (free acid form) from Svenningsen when tested against the HOMO-LUMO gap and the molecular volume Eq 2 10 antibiotics (free acid form) from Svenningsen EC 50 = -0.095(HOMO-LUMO) -0.11MolVol + 1.215 Where R 2 = 0.720, SEE = 0.114, SE(HOMO-LUMO)= 0.057, SE(MolVol) = 0.03, F=8.98, Significance= 0.0116. P-values: (HOMO-LUMO)= 0.14, MolVol=0.014;

Eq 3 10

 10 antibiotics as neutral {ionophore-M n+ } 0 complexes EC 50 = -0.089Excitation Energy -0.15MolVol + 1.411 Where R 2 = 0.612, SEE = 0.134, SE(ExcitEn)= 0.049, SE(MolVol) = 0.05, F=5.54, Significance= 0.036. P-values: ExcitEn= 0.114, MolVol=0.018; Eq 4 10 antibiotics as neutral {ionophore-M n+ } 0 complexes EC 50 = -0.102(HOMO-LUMO) -0.156MolVol + 1.560 Where R 2 = 0.667, SEE = 0.125, SE(HOMO-LUMO)= 0.044, SE(MolVol) = 0.051, F=7.03, Significance= 0.021. P-values: (HOMO-LUMO)= 0.061, MolVol=0.012;

Fig 1 (Fig 3 (Fig 5 (

 135 Fig 1(left) {X-206-K + } 0 showing intramolecular H bond left edge & Fig 2(right) {X-206-K + } + without the intramolecular H bond

Table 1 . Molecular specifiers for free acid form of ionophores: EC 50 inhibition of SARS- CoV-2 infected Vero E6-hTMPRSS2 cells from ref 16 Free acid Ionophores

 1

		Excitation Energy eV	(HOMO-LUMO) eV	ΔG CDS,Octanol kcal/mol	Molecular cm 3 /mol Volume	EC 50 µM
	Nigericin	4.641	4.984	0.54	472	0.09
	Monensin	5.154	5.703	-1.26	465	0.1
	Salinomycin	4.132	5.737	-1.98	518	0.1
	Indanomycin	3.45	4.179	-1.78	328	0.63
	Lasalocid	4.251	5.34	-1.7	439	0.36
	A23187	3.491	4.02	0.96	440	0.26
	Calcimycin
	Narasin	3.698	5.039	-1.76	577	0.07
	Nanchangmycin	3.659	4.861	0.09	673	0.07
	Maduramycin	3.854	4.647	2.28	724	0.06
	X-206	5.638	6.195	-1.81	643	0.014
	Ionomycin	3.849	4.898	-0.81	633	3

Table 2 . Molecular specifiers for {ionophore-M n+ } 0 complexes: EC 50 inhibition of SARS- CoV-2 infected Vero E6-hTMPRSS2 cells from ref 16 {Ionophore-M n+ } 0 Complexes

 2

		Excitation Energy eV	(HOMO-LUMO) eV	ΔG CDS,Octanol kcal/mol	Molecular cm 3 /mol Volume	EC 50 µM
	{Nigericin-K}(0)	4.65	5.052	-0.85	609	0.09
	{Monensin-K}(0)	5.429	6.4	-0.66	528	0.1
	{Salinomycin-K}(0)	3.974	4.577	-1.32	525	0.1
	{Indanomycin-K}(0)	3.488	4.185	-2.12	380	0.63
	{Lasalocid-K}(0)	4.105	4.609	-1.25	559	0.36
	{Dicalcimycin-Ca}(0)	2.803	3.226	1.6	705	0.26
	{Narasin-K}(0)	3.974	4.550	-1.41	592	0.07
	{Nanchangmycin-K}(0)	3.858	4.775	-0.18	673	0.07
	{Maduramycin-K}(0)	2.575	3.605	1.54	634	0.06
	{X-206-K}(0)	5.202	5.696	-2.74	634	0.014
	{Ionomycin-Ca}(0)	4.035	4.989	-2.47	533	3


Clifford W Fong Eigenenergy
email: cwfong@internode.on.net

Inhibition of SARS-CoV-2 envelope ion channel and the relationship with the membrane dipole potential

Keywords: SARS-CoV-2, E protein ion channel, ion channel inhibitors, transmembrane electrical dipole potential, voltage gated ion channels, TD DFT, excitation energy, HOMO-LUMO energy gap, docking binding energy, quantum mechanics

This study has identified a predictive quantum mechanical TD DFT method which can describe the time dependent behaviour of inhibitors of voltage gated ion channels. The key determinants are the excitation energy of the first excited state of the inhibitor and the ground state HOMO-LUMO gap of the inhibitor that govern the dynamic binding between the inhibitor and the protein target. The method applies to the inhibition of the E protein ion channel of SARS-CoV-2.

Introduction

SARS-CoV-2 is similar to other coronaviruses, and is comprised of four key structural proteins: S, the spike protein, E, the envelope protein, M, the membrane protein, and N, the nucleocapsid protein. Both SARS-CoV and SARS-CoV-2 spike proteins use the angiotensin-converting enzyme 2 (ACE2) protein as a receptor for cellular entry in humans. Spike protein subunits have been widely reported as targets for vaccines, novel therapeutic antibodies, and small-molecule inhibitors.

The E protein is a small hydrophobic protein of ∼74-109 amino acids comprised of a charged cytoplasmic tail and a hydrophobic domain. This protein consists of three parts: NT (negatively charged), TMD (not recharged), and CT (negatively charged). [START_REF] Mcclenaghan | Coronavirus Proteins as Ion Channels: Current and Potential Research[END_REF][START_REF] Hover | Viral dependence on cellular ion channelsan emerging antiviral target[END_REF][START_REF] Jl Nieto-Torres | Subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein[END_REF] E protein contributes to ion channel, viroporin activity, and virus assembling. Envelope protein form a cation-selective channel and plays a key role in the virus's ability to replicate itself and stimulate the host cell's inflammation response. The structure (determined in the closed state) of the E protein [START_REF] Vs Mandala | Structure and drug binding of the SARS-CoV-2 envelope protein transmembrane domain in lipid bilayers[END_REF] is similar to an influenza protein called the M2 proton channel. Both viral proteins are made of bundles of several helical proteins. The SARS-CoV-2 E protein is very different to the ion channel proteins of influenza and HIV-1 viruses. The structure in the open state is still to be determined. Several amino acids at one end of the channel may attract positively charged ions such as calcium into the channel. It was found that amantadine, used to treat influenza and hexamethylene amiloride, used to treat high blood pressure both weakly block the entrance of the E channel. [START_REF] Vs Mandala | Structure and drug binding of the SARS-CoV-2 envelope protein transmembrane domain in lipid bilayers[END_REF] Another homology study [START_REF] Sarkar | Structural insight into the role of novel SARS-CoV-2 E protein: A potential target for vaccine development and other therapeutic strategies[END_REF] has identified GLU 8 and ASN 15 in the N-terminal region were in close proximity to form H-bonds, and two distinct "core" structures, the hydrophobic core and the central core, can regulate the opening or closing of the channel.

Cao et al [START_REF] Cao | Computational Study of the Ion and Water Permeation and Transport Mechanisms of the SARS-CoV-2 Pentameric E Protein Channel[END_REF] have modeled by molecular dynamics the pentameric SARS-CoV-2 E protein and shown it is a voltage-dependent hydrophobic channel with monovalent cation selectivity. Water molecules and monovalent cations spontaneously penetrate through the channel under a transmembrane voltage. The homology model of the pentameric structure of the SARS E protein (PDB5X29) was used for the SARS-CoV-2 E protein. Cao used a mixed lipid membrane model composed of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine to simulate the lipid environment. Leu10 and Phe19 are the hydrophobic gates that regulate ion permeability. It is thought that channel activity of the pentameric E protein is necessary for inflammasome activation and is the determinant of SARS-CoV-2 cytokine storm virulence.

Gupta et al [START_REF] Gupta | In-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel[END_REF] used molecular dynamics and docking studies to characterize the binding of many inhibitors to the E ion channel of the SARS-CoV-2 homology model (based on the NMR structure PDB5X29) noting that SARS-CoV and SARS-CoV-2 protein share a 84.74% sequence identity. It was found that some phytochemicals which were highly effective inhibitors reduced the random motion of the human SARS-CoV2 E protein. Also Val25 and Phe26 played a key role while interacting with effective inhibitors.

While there have been many recent reports of small molecule inhibitors that are effective against the spike protein of SARS-CoV-2, fewer studies have been made targeting the E protein ion channel. Tomar et al [START_REF] Pps Tomar | Targeted Drug Repurposing Against the SARS-CoV-2 E Channel Identifies Blockers with in vitro Antiviral Activity[END_REF] have reported the screening of 3000 approved drugs using three independent bacteria-based assays. Ten drugs were identified with significant antiviral activity in Vero E6 tissue culture.

Voltage gated ion channels are a class of transmembrane proteins that form ion channels that are activated by changes in the electrical membrane potential near the channel. The membrane potential alters the conformation of the channel proteins, regulating their opening and closing. Cell membranes are generally impermeable to ions, thus they must diffuse through the membrane through transmembrane protein channels. Voltage gated ion channels have a particular ion selectivity and a particular voltage dependence. Many are also time-dependent in that they do not respond immediately to a voltage change but only after a delay.

Efimova 2020 [START_REF] Ss Efimova | Alkaloids Modulate the Functioning of Ion Channels Produced by Antimicrobial Agents via an Influence on the Lipid Host[END_REF] studied changes in the transmembrane distribution of the electrical potential by measuring changes caused by 22 alkaloids to the boundary potential of planar lipid bilayers (POPC, DOPE/DOPS or mixture of 67 mol% POPC and 33 mol% Cholesterol). Membranes were also modified by the addition of pore forming anti-microbial agents GrA, SyrE, CecA, and Nys. It was found that the alkaloids had a major effect on the membrane dipole potential, The lifetime and conductance of single pores induced by the pore forming agents were also strongly influenced by the membrane dipole potential. The tested alkaloids did not increase the ion permeability of lipid bilayers in the absence of pore-forming agents.

Broadly there are three kinds of cellular membrane potentials: the surface potentials, between the membrane surface and bulk solvent, resulting from the accumulation of charges at the membrane surfaces; the transmembrane potential, across the membrane, from one surface to the other, and determined by imbalance of charge in the aqueous solutions; and the dipole potential, a membrane-internal potential from the dipolar components of the phospholipids and interface water, which creates a much larger electric field that is highly localized to the interface between the hydrophobic and hydrophilic layers.

Efimova concluded that the dipole potential made the main contribution to the potential drop at the interface after the adsorption of alkaloids, despite some of the alkaloids having a non-zero charge at pH 7.4 basing on their pKa values. The standard measures of lipophilicity, the octanol/water partition coefficient log P o/w (or logD o/w for the ionized species) were strongly correlated with the maximum dipole potential at the 0.75 level. No correlations were observed with the dipole moments of the alkaloids. [START_REF] Ss Efimova | Alkaloids Modulate the Functioning of Ion Channels Produced by Antimicrobial Agents via an Influence on the Lipid Host[END_REF] Efimova 2015 [START_REF] Ss Efimova | Modifiers of the dipole potential of lipid bilayers[END_REF] also similarly studied changes in the dipole potential of lipid bilayer membranes caused by the adsorption of some flavonoids, muscle relaxants, thyroid hormones, and xanthene and styrylpyridinium dyes. A quantitative relationship was found between the ratio of the maximum change in the bilayer dipole potential upon saturation and the absolute value of the unmodified membrane.

Study objectives: (a) Identify a predictive method which can describe the time dependent behaviour of inhibitors of voltage gated ion channels (b) Identify predictive criteria that can apply to the inhibition of the E ion channel of SARS-CoV-2

Results

Time-dependent density-functional theory (TDDFT) is a quantum mechanical theory which can describe the properties and dynamics of molecular systems in the presence of time-dependent potentials, such as electric or magnetic fields. The excitation energies of molecular systems such as the influence of chemical inhibitors on the electrical fields in voltage gated ion channels offer potential to describe such behaviour as a function of varying structural properties of such inhibitors. The time dependencies involved are (a) the dynamic inhibition equilibrium of ion channels by chemical species, (b) the electrical field imposed on the particular inhibitor-ion channel interaction.

The data from Efimova 2015, 2020 [START_REF] Ss Efimova | Alkaloids Modulate the Functioning of Ion Channels Produced by Antimicrobial Agents via an Influence on the Lipid Host[END_REF][START_REF] Ss Efimova | Modifiers of the dipole potential of lipid bilayers[END_REF] can be used to test if TDDFT methods can usefully describe the relationship between changes in the dipole potential of lipid bilayer membranes caused by the adsorption of some flavonoids, xanthenes dyes, and alkaloids. Table 1 shows the excitation energies and ground state HOMO-LUMO gaps of a number of drugs in n-octanol along with their changes in membrane dipole potentials. Octanol was chosen as it is known to be a good surrogate for lipophilic behaviour of cell membranes, with mainly hydrophobic properties balanced with some hydrophilic behaviour. We have previously shown that octanol better represents a cell membrane environment where some water interaction can occur, whereas octane is a better representative of a more hydrophobic environment found in deep pockets where drug-protein environments exists with very little water interaction. [START_REF] Fong | Predicting inhibition of repurposed drugs for SARS-CoV-2 viral activity and cellular entry[END_REF] Also Efimova found the octanol/water partition coefficient log P o/w (or logD o/w for the ionized species) was strongly correlated with the maximum dipole potential at the 0.75 level. [START_REF] Ss Efimova | Alkaloids Modulate the Functioning of Ion Channels Produced by Antimicrobial Agents via an Influence on the Lipid Host[END_REF] For 13 flavanoids the best correlation was found for eq 1: It is noted that the outliers capsaicin and dihydrocapsaicin had the strongest effect of all the tested alkaloids. Capsaicin binds to the ion channel-type TRPV1receptor. Capsaicin invokes long-onset current in comparison to other inhibitors, possibly due to a significant rate-limiting step. [START_REF] Geppetti | The concept of neurogenic inflammation[END_REF] It appears that eqs 1-3 demonstrate that the change in lipid membrane dipole potential (including where ion channels are active) for a wide and diverse range of drugs can be described by a dependence on the vertical excitation energy of the first excited state and the ground state HOMO-LUMO reactivity gap of the drugs.

Analysis of Tomar's data [START_REF] Pps Tomar | Targeted Drug Repurposing Against the SARS-CoV-2 E Channel Identifies Blockers with in vitro Antiviral Activity[END_REF] for inhibitors of the SARS-CoV-2 E ion channel gives eq 4(a): Eq 4(a) includes the neutral and positively charged species for Kasugamycin and Mavorixafor as both species may be active antivirals under the experimental conditions. Similarly the neutral Saroglitazar magnesium complex and the negatively charged Saroglitazar ion from dissociation of the complex may also be active species. The SaroglitazarMg has been modeled with the Mg-O bond lengths set at 1.92 Å and 2.02Å to simulate a more dissociative structure at the longer bond length. The P-values show that the excitation energy and the HOMO-LUMO gap are significant at the 0.035 and 0.041 levels, despite the limited available data set. Eq 4(b) includes ΔG CDS,Octanol which represent the lipophilic free energy of solvation in n-octanol, [START_REF] Ss Efimova | Modifiers of the dipole potential of lipid bilayers[END_REF] as well as the excitation energy and HOMO-LUMO gap. This result is only roughly indicative given the small data set, and the poor P-value for ΔG CDS,Octanol at 0.193 significacnce. Eq 4(b) suggests that the lipophilicity of n-octanol may have some small influence on the antiviral activity of the tested inhibitors of the E ion channel. The magnitude of the coefficients for the excitation energy and HOMO-LUMO gap show the lipophilicity effect is very small if the influence is indeed real.

Analysis of Gupta's docking study for 18 highly effective inhibitors (see Table 3) of the SARS-CoV-2 E ion channel gives eq 5. Eq 5 18 drugs from Gupta 2021 Binding Energy = 1.44Excitation Energy -0.80HOMO-LUMO -12.13

Where R 2 = 0.385, SEE = 0.652, SE(ExcitEn)= 0.58, SE(HOMO-LUMO) = 0.46, F=4.70, Significance= 0.026, P-values : ExcitEn=0.026, HOMO-LUMO=0.110

Eq 5 illustrates that the docking binding energy is strongly correlated to the excitation energy of the first excited state of the inhibitor, with the correlation with the HOMO-LUMO gap being much weaker. No dependency on ΔG CDS,Octanol was found. These computational results in eq 5 are consistent with the biological experimental antiviral activity results in eq 4(a).

Discussion

The results from eq 1-3 clearly demonstrate that the excitation energy and the HOMO-LUMO energy gap can adequately describe changes in the electrical dipole transmembrane potential of planar lipid bilayers for a wide and diverse range of flavanoids, xanthenes dyes and alkaloids. In alkaloids the lifetime and conductance of single pores induced by the pore forming agents were also strongly influenced by the membrane dipole potential. The tested alkaloids did not increase the ion permeability of lipid bilayers in the absence of pore-forming agents.

The HOMO-LUMO gap is a ground state property that determines the inherent chemical reactivity of the drugs and governs the dynamic equilibrium between various drugs and the lipid membrane. The excitation energy of the first excited state is a measure of how a drug will be excited under a dynamic electrical potential.

The individual eqs 1-3 each have a moderately high statistical significance for the limited data sets, but the overall pattern clearly suggests both the excitation energy and HOMO-LUMO gap are fundamental descriptors of how the transmembrane dipole potential interacts with adsorbed drugs under an applied electrical voltage.

Eq 4(a) indicates that drugs shown to have antiviral activity by inhibiting the E protein ion channel of SARS-CoV-2 can be described by a dependence on the excitation energy of the drugs as well as their dynamic ground state binding to the E protein as measured by the HOMO-LUMO gap. Here the voltage gated ion channel provides a dynamic electrical field to the adsorbed inhibitors. Eq 5 representing the docking binding behaviour of various drugs to the E ion channel is in strong agreement with the results from eq 4(a): ie that the excitation energy and HOMO-LUMO gap are fundamental descriptors of inhibitors of the SARS-CoV-2 E ion channel.

Cao [START_REF] Cao | Computational Study of the Ion and Water Permeation and Transport Mechanisms of the SARS-CoV-2 Pentameric E Protein Channel[END_REF] and Delemotte [START_REF] Delemotte | Exploring the Complex Dynamics of an Ion Channel Voltage Sensor Domain via Computation[END_REF] have used MD simulations to determine the free energies of the voltage sensor domain activation and predict the time dependence of the resulting gating currents. Such efforts are very intensive, and the SARS-CoV-2 E ion channel may be computationally accessible. The addition of time dependent inhibitor-ion channel interaction would add considerable complexity to the voltage gating description. Docking studies between the E ion channel and potential inhibitors would be static not dynamic representations. The approach described in this study is easily accessed, but requires some actual experimental inhibition data of the SARS-CoV-2 E ion channel to build a predictive data base.

Tomar [START_REF] Pps Tomar | Targeted Drug Repurposing Against the SARS-CoV-2 E Channel Identifies Blockers with in vitro Antiviral Activity[END_REF] noted that the antiviral results for 5-azacytidine are anomalously high (possibly due to hypomethylation of DNA), which is reflected in eq 4, with 5-azacytidine being a high outlier. It has been shown that the anomaly is not due to possible tautomers such as the imino tautomer or other tautomers derived from the analogous KP1212 [START_REF] Li | Tautomerism provides a molecular explanation for the mutagenic properties of the anti-HIV nucleoside 5-aza-5,6-dihydro-2-deoxycytidine[END_REF]. However, 5-azacytidine is very unstable in aqueous medium, a temperature dependent process. A rapid and reversible hydrolysis occurs, which leads to N-formylribosylguanylurea, followed by N-formylribosylguanylurea being irreversibly hydrolysed into ribosylguanylurea. [15] It is possible that the experimental viral activity results for 5-azacytidine [START_REF] Pps Tomar | Targeted Drug Repurposing Against the SARS-CoV-2 E Channel Identifies Blockers with in vitro Antiviral Activity[END_REF] may be masked by stability and concentration problems with the drug itself, leading to unusually high results.)

It is noted that Tomar's conclusion [START_REF] Pps Tomar | Targeted Drug Repurposing Against the SARS-CoV-2 E Channel Identifies Blockers with in vitro Antiviral Activity[END_REF] that Memantine is a weak inhibitor of the SARS-CoV-2 E ion channel is in agreement with Mandala's conclusion for Amantidine [START_REF] Vs Mandala | Structure and drug binding of the SARS-CoV-2 envelope protein transmembrane domain in lipid bilayers[END_REF], which is structurally almost the same as Memantine but has two methyl groups at the 3,5 position instead of H on the adamantane scaffold. The excitation energies and the HL gaps are almost identical. Hexamethylene amiloride (HMA) is predicted from eq4(a) to have a antiviral activity of -33 for the neutral species, or -36 for the di-ion species. (see Table 2)

Conclusions

This study has identified a predictive quantum mechanical TD DFT method which can describe the time dependent behaviour of inhibitors of voltage gated ion channels. The key determinants are the excitation energy of the first excited state of the inhibitor and the ground state HOMO-LUMO gap of the inhibitor that govern the dynamic binding between the inhibitor and the protein target. The method applies to the inhibition of the E protein ion channel of SARS-CoV-2.

Materials and methods

All calculations were carried out using the Gaussian 09 package. Energy optimizations were at the DFT/B3LYP/6-31G(d) (6d, 7f) level of theory for all atoms in water. TD DFT vertical calculations were conducted at the TD DFT/B3LYP/6-31G(d,p) (6d, 7f) with the empirical dispersion GD3BJ correction for neutral and charged compounds with optimized geometries in n-octanol, using the IEFPCM/SMD solvent model. Ground state HOMO and LUMO were calculated at the DFT/B3LYP/6-31G(d,p) (6d, 7f) with empirical dispersion GD3BJ in n-octanol.

[

Eq 1 13 flavanoids from Efimova 2015 ΔΔ

 2015 Dipole Potential = 168.98Excitation Energy -125.67HOMO-LUMO -112.80 Where R 2 = 0.490, SEE = 14.96, SE(ExcitEn)= 61.02, SE(HOMO-LUMO) = 50.98, F=4.81, Significance= 0.034. P-values: ExcitEn= 0.019, HOMO-LUMO=0.033; For 7 xanthene dyes, separate linear correlations were found with the excitation energies and the ground state HOMO-LUMO, but it was not possible to test if a correlation similar to eq 1 existed (insufficient data): Eq 2(a) 7 xanthene dyes from Efimova 2015 Δ Dipole Potential = 8.36Excitation Energy -35.87 Where R 2 = 0.589, SEE = 12.74, SE(ExcitEn)= 3.12, F=7.175, Significance= 0.043 = P-value Eq 2(b) 7 xanthene dyes from Efimova 2015 Dipole Potential = 8.98HOMO-LUMO -41.21 Where R 2 = 0.619, SEE = 12.28, SE(HOMO-LUMO) = 3.15, F=8.118, Significance= 0.035= P-value For 14 alkaloids the best correlations were found for eq 3(a) and (b): Eq 3(a) 14 alkaloids from Efimova 2020 (excluding capsaicin and dihydrocapsaicin outliers) Δ Dipole Potential = 4.41Excitation Energy -43.12 Where R 2 = 0.220, SEE = 8.18, SE(ExcitEn)= 2.40, F=3.38, Significance= 0.091 = P-value Eq 3(b) 14 alkaloids from Efimova 2020 (excluding capsaicin and dihydrocapsaicin outliers) Δ Dipole Potential = 4.37HOMO-LUMO -45.30 Where R 2 = 0.228, SEE = 8.14, SE(HOMO-LUMO) = 2.32, F=3.53, Significance= 0.084= P-value

Eq 4 (

 4 a) 13 drugs from Tomar 2020 (excluding outlier 5-azacytidine) Δ Antiviral Activity = 62.62Excitation Energy -52.42HOMO-LUMO -43.59 Where R 2 = 0.386, SEE = 12.21, SE(ExcitEn)= 25.76, SE(HOMO-LUMO) = 22.42, F=3.14, Significance= 0.087, P-values : ExcitEn=0.035, HOMO-LUMO=0.041

Eq 4 (Δ

 4 b) 13 drugs from Tomar 2020 (excluding outlier 5-azacytidine) Antiviral Activity = 1.43ΔG CDS + 58.13Excitation Energy -48.00HOMO-LUMO -44.31 Where R 2 = 0.496, SEE = 11.66, SE(ExcitEn)= 24.78, SE(HOMO-LUMO) = 21.63, F=2.96, Significance= 0.080, P-values : ΔG CDS = 0.193, ExcitEn=0.035, HOMO-LUMO=0.041

Table 1 Membrane dipole potentials [9,10] and calculated excitation energies and HOMO- LUMO energy gaps

 1 15] C Balouzet, C Chanat, M Jobard, ML Brandely-Piat, F Chast, Stability of 25 mg/mL Azacitidine Suspensions Kept in Fridge after Freezing, Pharm. Technol. Hosp. Pharm. 2017, 2, 11-16

	Alkaloids	Dipole	Excitation	HOMO-
		Potential	Energy	LUMO
		mV	eV	eV
	1,7-Dimethylxanthine	-21	4.676	5.13
	Theophylline	-40	4.63	5.09
	3-Isobutyl-1-	-20	4.602	5.07
	methylxanthine
	Quinine	-16	3.536	4.15
	Quinine Ion	-16	3.984	4.53
	Piperine	-40	3.343	3.61
	Melatonin	-15	4.396	4.96
	Colchicine	-29	3.387	3.80
	Hordenine	-23	5.062	5.79
	Hordenine Ion	-23	5.094	5.89
	Synephrine	-24	5.06	5.83
	Synephrine Ion	-24	5.082	5.88
	Conessine	-12	5.946	6.37
	Conessine DiIon	-12	6.696	6.96
	Capsaicin	-92	5.066	5.85
	Dihydrocapsaicin	-92	5.098	5.94
	Flavanoids	Changes	Excitation	HOMO-
		in Dipole	Energy	LUMO
		Potential	eV	eV
	Phloretin	-59	3.941	-5.79
	Phlorizin	-37	3.913	-5.75
	Quercetin	-42	3.414	-5.64
	Myricetin	-44	3.423	-5.71
	Rutin	-17	3.815	-5.84
	Biochanin A	-37	3.619	-5.64
	Genistein	-28	3.639	-5.70
	Genistin	-2	3.802	-5.66
	Catechin	-1	4.957	-5.64
	Taxifolin	-3	3.655	-5.78
	DHAP	-20	3.94	-6.02
	Daidzein	-8	3.758	-5.72
	THAP	-6	3.978	-6.01

Table 2 Antiviral activity in SARS-CoV-2 E ion channel [8] and calculated excitation energies and HOMO-LUMO gaps of inhibitors

 2

	Antiviral	ΔG CDSoct	Excitation	HOMO-
	Activity	kcal/mol	Energy	LUMO
	%			eV	eV
	5-Azacytidine	-98	0.24	4.771	5.91
	Plerixifor	-53	-7.7	4.442	5.19
	Plerixifor OctaIon	-40	-5.01	5.206	6.19
	Mebofenin	-45	2.12	5.038	5.93
	Gliclazide	-41	-0.38	4.64	5.22
	Cyclen	-38	-3.2	6.43	7.27
	Kasugamycin	-27	3.77	4.311	4.90
	Kasugamycin Ion	-27	3.82	4.399	5.21
	Mavorixafor	-41	-3.89	4.149	4.97
	Mavorixafor TriIon	-41	-3.46	3.202	3.61
	Memantine	-3	-2.3	7.247	8.16
	SarogMg1.92A	-18	-0.92	3.777	4.24
	SaroglitazarMg2.02A	-18	-0.32	3.94	4.24
	Saroglitazar Ion	-18	-0.24	3.953	4.43
	5-azacytidine Imino Tautomer	0.45	4.877	5.55
	Amantidine		-2.6	7.258	8.20
	Hexamethylene amiloride		-0.43	5.094	5.89
	(HMA)
	HMA DiIon		-2.43	3.336	3.83

Table 3 Binding energies of inhibitors to SARS-CoV-2 E ion channel [7] and calculated excitation energies and HOMO-LUMO gaps of inhibitors

 3

		Binding	ΔG CDSoct	Excitation	HOMO-
		Energy	kcal/mol	Energy	LUMO
		kcal/mol		eV	eV
	Belachinal	-11.46	-1.55	3.365	3.84
	Macaflavanone E	-11.07	-0.35	3.529	4.02
	Vibsanol B	-11.07	2.06	3.597	4.82
	14R*,15-Epoxyvibsanin C	-10.56	2.99	3.654	4.65
	Macaflavanone C	-10.49	-0.14	3.463	3.93
	Luzonoid D	-10.47	2.18	3.429	3.91
	Grossamide K	-10.5	-10.61	3.829	4.26
	(-)-Blestriarene C	-10.4	-11.27	3.597	4.19
	Macaflavanone F	-10.36	-0.79	3.537	4.02
	Dolichosterone	-10.31	-0.66	4.247	5.95
	Luzanoid A	-9.71	3.26	3.867	4.17
	Luzanoid B	-9.06	2.41	3.717	4.30
	Luzanoid C	-10.12	1.75	3.592	4.03
	Toddaliamide	-8.96	1.19	3.913	4.32
	Methyltoddaliamide	-9.11	1.44	3.913	4.32
	NN Dicyclohexylurea	-9.3	-3.69	6.702	7.84
	Pinoresinol di-3,3-	-9.87	1.09	4.783	5.41
	dimethylallylether
	Pinoresinol-3,3-	-8.84	0.67	4.921	5.62
	dimethylallylether