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Introduction

Japanese encephalitis (JE) is a flaviviral brain infection caused by an arbovirus, the Japanese encephalitis virus (JEV). Anthropophilic mosquitoes of the Culex species (mainly the Culex tritaeniorhynchus group) that breed in rice fields are mainly known to transmit JEV. JEV was first reported in Japan in the 1870s. It spread to the south, east, and south-east of Asia and now to the Western Pacific, threatening large populations [1-3]. It can cause severe viral-encephalitis in 0.1–2% of people infected, with a death rate of 20–30%, and of those that survive, suffer from severe neurologic injuries, including persistent motor defects and severe cognitive and language impairments. Acute encephalitis develops in about 0.1–2% of cases, producing serious neurological lesions in 30-50% of the survivors [2-6].

Infections with JEV most often produce no symptoms (asymptomatic), which is why only 0.3% of cases produce clinical features. The first signs of disease appear after an incubation period of between 6 and 14 days, usually begins with a high fever, chills, muscle pain, and meningitis-type headaches accompanied by vomiting. The initial clinical features in children usually involve gastrointestinal symptoms (nausea, vomiting, and abdominal pains). These nonspecific symptoms can continue for 2–4 days. After this period, the patient's condition declines rapidly. About 85% of the infected suffer from seizures. The meningeal syndrome prevails, causing painful neck stiffness. Additionally, motor paralyzes, including hemiplegia and tetraplegia, may also occur. In about 30% of patients, tremors, rigidity, abnormal movements, and other signs of extrapyramidal involvement are present. Recovery usually leaves serious behavioral and neurological injuries such as persistently altered sensorium, extrapyramidal syndrome, epileptic seizures, and severe mental retardation in children [7, 8]. Vaccines for the prevention of JEV are available and have reduced the occurrence of JE in some countries. However, they are not effective against all the clinical subjects causing 10,000 – 15,000 human deaths and 709,000 disability-adjusted life years annually. Regardless of the vaccine development, there is a lack of an absolutely protective or preventive vaccine or antiviral drugs to treat JE. Hence, there is an urgent need to identify lead compounds with antiviral properties against JEV [9] so that a drug could be developed.

JEV belongs in the genus Flavivirus of the Flaviviridae family, which also includes the important human pathogens Zika virus (ZIKV) and the Dengue virus (DENV). Flaviviruses replicate their RNA genome using virally encoded replication proteins. Hindering the flaviviral replication is widely studied and considered to be an effective antiviral drug discovery approach. Recent studies have led to the identification of specific domains in the flaviviral proteins whose inhibition could block viral replication. Antiviral agents for flaviviruses hepatitis C virus, Dengue virus, and West Nile have been reported [10]. Similar inhibitors with antiviral properties for JEV were reported that targets the NS3 - Indirubin, Dehydroepiandrosterone (DHEA), N-nonyl-deoxynojirimycin, and SCH 16 [11]. Nevertheless, only a few JEV inhibitors have been discovered and are undergoing clinical trials at present. It is also important to note that despite the severe consequences of the infection, efforts for drug discovery against JEV have been relatively very limited.

Viruses contain and produce structural, nonstructural proteins, regulatory and accessory proteins for different functions. The nonstructural proteins, coded for by the viral genome, are expressed in infected host cells and not assembled in the virion. These proteins play an important role in the flaviviral RNA genome replication and assembly processes. Specifically, nonstructural proteins NS3 and NS5 are reported as the main components of the viral RNA replication complex associated with the 3′ noncoding region of genomic RNA in the initiation of viral replication. NS5 is the largest and most conserved flavivirus protein encoded in the open reading frame. NS5 harbors two domains that directly affect viral replication - methyltransferase (MTase) in its N-terminal (≈265 residues) responsible for RNA capping (methylation of the 5′ RNA cap structure); and RNA-dependent RNA polymerase (RdRp) within the C-terminal (≈640 residues) for viral replication, and hence was considered a potential drug target in this study [12,13].

The goal of this work was to identify potential antagonists to hinder viral replication via silencing NS5 without causing toxicity to the infected by analyzing the ligand-receptor interactions between the NS5 receptor and the pharmacologically active ligands screened from the ZINC database [14] using a combination of molecular docking and molecular dynamics (MD) simulations. Docking was used to identify the ligand hotspot on the receptor, as well as, to analyze the screened compounds. Molecular dynamics was used for druggability assessment and to further verify binding free energies between the ligand(s) and receptor in a simulated cellular environment while the protein was dynamically flexing.

Computational methods

Protein structure analysis

The crystal structure of JEV NS5 was available in the Protein Data Bank (PDB ID:4K6M) [15, 16]. NS5 performs the main activities pertinent to viral replication with the help of its enzymatic domains – MTase and RdRp. The MTase activity protects viral mRNA from degradation by 5′-exoribonucleases and ensures their recognition by the eukaryotic translation initiation factor. The N-terminal MTase domain (residues 5–266) is connected through a ten-residue linker to the C-terminal RdRp domain formed by residues 276–895. In turn, the RdRp domain is formed by three subdomains called fingers, palm, and thumb. The RdRp domain contains the core polymerase that is essential for the viral RNA synthesis and, thus, is of major interest as a potential drug target. The protein crystal has two chains, A and B, comprising three NS5 hexamers, of which chain A was computationally isolated using VMD [17] for all the studies in this work. The RdRp active site of JEV NS5 protein – chain A was visualized using VMD and used for docking and MD simulations.

NTP interactions with JEV NS5 RdRp

The conserved RdRp domain of JEV NS5 protein was examined using AutoDock Vina [18]. Nucleotide triphosphates (NTPs): adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP) which are building blocks of nucleic acids were used as ligands. The JEV NS5 (PDB ID:4K6M) crystal structure was retrieved from PDB [16]. The 3D structures of the ligands were obtained from the ZINC15 database [14], ATP (ZINC4261765), GTP (ZINC60094177), CTP (ZINC3861746), and UTP (ZINC3861755). The receptor and ligands were prepared using AutoDock Tools [19] and converted to the PDBQT formats. A grid box of size 104 × 102 × 116 Å3 located at the RdRp domain was generated for docking. The default docking protocol was used to dock the native ligands at the RdRp active site of JEV NS5 protein via AutoDock Vina, a fast and accurate tool for screening out small molecules that are less effective for target sites.

Druggability Assessment of JEV NS5 protein

Probe-based mixed-solvent MD simulations were performed to explore the binding site of the JEV NS5 protein [20-22]. The NAMD simulation [23] configuration files were built using the VMD plugin DruGUI [24]. Five water-soluble organic probes, 60% isopropanol and 10% each of isobutene, acetamide, acetate, and isopropylamine were used for druggability analyses to reveal any clusters of hotspots that specify the presence of druggable sites on the receptor. Chemistry at HARvard Macromolecular Mechanics (CHARMM) force fields for larger proteins and the CHARMM General Force Fields (CGenFF) for smaller ligands were used for the simulations [25, 26]. The solvent model for MD was TIP3P. The protein was immersed in a 6 Å width water solvent. The protocol of the simulation had three steps, system minimization and equilibration, and unrestrained MD simulation. First, the system was minimized with 1.0 scale of constraints under 0K for 4ps. The equilibration step then typically raised the system temperature from 100K to 600K, eventually stabilizing at 300K. The whole equilibration step took 1.4ns to complete. Finally, unconstrained MD simulations were carried out for 40ns at 1.01325atm and 300K under isothermal-isobaric (NPT) conditions. The simulation output files were analyzed by the standard protocol described in the plugin documentation for all the probe molecules [24]. All probe molecules were shown with binding free energies (druggability score) given and ranked, with some of the probe molecules grouped into different clusters. These active probe molecules were considered as potential pharmacophores based on their functional groups and probe-protein interactions.

Pharmacophore Identification

Enhanced Ligand Exploration and Interaction Recognition Algorithm (ELIXIR-A) [27-30] (ELIXIR-A) was used for deciphering pharmacophores via protein-ligand interactions analyses done using the NAMD and molecular docking studies. ELIXIR-A is a pharmacophore screening algorithm that is under development in our laboratory. It consists of a computer-guided routine that recognizes pharmacophore points i.e., the ensemble of steric, electrostatic, and hydrophobic properties which are essential for optimal supramolecular interactions with the receptor to inhibit its biological effect. The probe molecules were converted to pharmacophores using the ELIXIR-A VMD plugin. The pharmacophores with good druggability scores were used for further ligand screening.

Ligand Screening and Verification

Using the pharmacophore information obtained from ELIXIR-A, potential compounds were screened from the ZINC15 database [14] using the ZINCPharmer software [31]. The ZINC15 database includes 122 million conformations for approximately 13 million molecules. Structure-based screening focuses on matching small molecule conformations with suitable pharmacophores based on the functional groups present at the binding site. The molecules were screened based on their structural stability and having a minimum of three pharmacophore points. The screened molecules were validated In Silico via AutoDock Vina using the molecular docking method previously described in the NTP interaction section [32]. Vina evaluated the docking of each small molecule using a scoring function and retained the nine most stable conformations with the best binding score (i.e., the lowest binding affinity). The compound with the highest affinity amongst the screened molecules was selected for MD simulations.

MD simulations

To analyze the conformational and interaction stability of the JEV NS5 protein complexed with ZINC 9367, an MD simulation of 100 ns was performed by using the Schrödinger-Desmond platform [33]. JEV NS5 complexed with ATP was also simulated similarly and was considered as a control. The protein was prepared by Maestro’s Protein Preparation Wizard [34, 35]. The missing side chains and loops were added by the Prime module. Ligands were prepared by the LigPrep [36] module that generated 32 stereoisomers per ligand under the OPLS3e force field [37]. These ligands were also ionized using the Epik [38] module at pH 7.0 ± 2.0. AutoDock Vina removed all charge or non-polar hydrogens from the ligands, which were necessary for MD simulations. Here, Schrödinger's Glide [30] XP (extra-precision) module was used to reproduce the docking pose of the complete ligand structure based on the Vina docking results. The reproduced binding pose with the highest glide score (most negative) for each ligand was used as the initial frame for MD. The system was immersed using the TIP3P solvent model under orthorhombic boundary conditions with a buffer distance of 10 Å. The salt concentration of 0.1M was added, and the system charge was neutralized using sodium and chloride ions. Each system was initially minimized under the OPLS3e force field using Desmond’s default relax protocol. After relaxation, the systems were simulated under the NPT ensemble at 300 K and 1.01325 bar pressure for 100 ns. Total 500 frames were recorded at an interval of 200 ps excluding the initial frame. Post-simulation analysis included complex root mean square deviations (RMSD), and ligand/protein root mean square fluctuations (RMSF), and complex interactions given by the Simulation Interaction Diagram (SID) module.

Calculation of the binding free energy

The binding free energy of each protein-ligand complex was computed using the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method [39]. The python script “thermal_mmgbsa.py” from Schrodinger that utilizes the VSGB 2.0 solvation model with the OPLS3e force field was used to calculate the Prime MM/GBSA free energies [40]. For the entire 100 ns simulation, a total of 500 frames were generated, and 50 frames were sampled uniformly from the entire trajectory for calculation. The free energy of binding (ΔG) for each protein-ligand complex is calculated as follows:

Results and discussion

NTP interactions with JEV NS5 RdRp

The docking results showed that the NTPs bind in the same pocket which was previously identified as the active site of the RdRp domain of JEV NS5 protein. The protein-ligand interaction analyses (Figure 1) revealed that THR609, TYR610, SER666, and SER801 were common residues that interacted with the NTPs at the RdRp active site. Table 1 gives the information of the type of bonds formed and the maximum binding affinities of NTPs with the JEV NS5 RdRp domain. Amongst all NTPs, GTP (-7.3 kcal/mol) recorded the highest docking score at the JEV RdRp active site followed by ATP (-6.9 kcal/mol), UTP ( -6.6 kcal/mol), and CTP (-6.5 kcal/mol). Small molecules that can bind to the same region with a much higher affinity (i.e., a more negative docking score) could potentially be promising candidates as potent drugs inhibiting the replication cycle of the virus. Hence, the results generated from molecular docking studies were used to filter the pharmacophores which were later used for compound screening. 

Druggability Assessment of JEV NS5 protein

MD simulations of biological targets in the presence of drug-like probe molecules help characterize the ability of a target protein to bind small molecule drugs with high affinity, also known as protein druggability. The druggability of JEV NS5 protein was assessed via NAMD simulations. Figure 2 shows the system setup and analysis of the NAMD simulations. Small organic molecular probes were used to reveal any druggable sites on the receptor. After equilibration, the system was found to contain 16800 water molecules and 504 probe molecules (i.e., 306 isopropanol, 84 isobutene, 84 acetamide, 84 acetate, and 84 iso-propylamine) shown in Figure 2A. The druggability analysis revealed 330 probe binding hotspots ranging from a minimum ΔG of -2.67 kcal/mol and a maximum of -1.00 kcal/mol (Table S1). The protein surface was supplemented with 153 binding hotspots of isopropanol with the lowest binding free energy of -2.43 kcal/mol. However, isobutene (27 hotspots, -2.11 kcal/mol), isopropylamine (32 hotspots; -2.57 kcal/mol), acetamide (14 hotspots, -2.06 kcal/mol), and acetate (104 hotspots, -2.67 kcal/mol) supplementation were remote. The analysis predicted nine potential sites for drug attachment on the JEV NS5 protein (Table S2) by clustering a maximum of 7 and a minimum of 6 probes. One druggable site (Table S2: Site 2 - Solution 1) formed by a cluster of nine probe binding hotspots overlapped with the RdRp domain (Figure 2B) with an achievable binding affinity of -11.33 kcal/mol and highest drug-like affinity of 5.504 nM occupying an approximate volume of 434.83 Å3 on the receptor.

Pharmacophore Identification

The hotspot information from the druggability simulations combined with the protein-NTP interaction analyses via molecular docking was used by ELIXIR-A to isolate pharmacophores for compound screening. The pharmacophoric features included proton donors or acceptors, aromatic rings, hydrophobic centroids, cations, and anions. Figure 3 illustrates the pharmacophore distribution on the JEV NS5 receptor. From the five probes tested isopropanol, followed by acetate and isobutene have the maximum affinity at the active site of the RdRp domain. Detailed information on the hotspot analysis is given under supplementary data (Table S1).

Ligand Screening and Verification

With the pharmacophore information from ELIXIR-A, potential compounds were screened using the ZINCPharmer software. The screening resulted in four potential ligands: ZINC9972155, ZINC 67912950, ZINC95910070, and ZINC196939367, molecular structures of these are shown in Figure 4. Of the four identified potential drugs for JEV, ZINC 0070/chebulanin, and ZINC 9367/chebulinic acid are medicinally important phytochemicals derived from the fruit of Terminalia chebula and are active constituents of Triphala, an ancient Indian Ayurvedic medicine [41, 42]. Recent studies in arthritic mouse models revealed the anti-inflammatory and anti-arthritic effects of chebulanin [43] and antiangiogenic effects of chebulinic acid [44]. Chebulinic acid has been identified as a promising anti-tumor agent in human colorectal carcinoma and acute myeloid leukemia cell lines due to its potent anti-proliferative, pro-apoptotic, and anti-migratory properties [45, 46]. Also, chebulinic acid has been recognized for its potent direct antiviral activity against HSV-2 and influenza A virus [47, 48].

The binding affinities of ZINC 2155 (-8.1 kcal/mol) and ZINC 2950 (-8 kcal/mol) were lower (i.e., with comparatively higher binding free energies) among the four compounds. ZINC 9367 showed the highest affinity, i.e., lowest binding free energy (-11.96 kcal/mol) for the JEV NS5 protein at the RdRp domain, followed by ZINC 0070 (-9.13 kcal/mol) (Figure 5A). Clearly, ZINC 9367 and ZINC 0070 bind with a greater affinity, thus being the more promising of the four studied inhibitors. All four compounds bind at the active site of the RdRp domain where the NTP native molecules attach, as shown in Figure 5B. ZINC 9367 was able to bind into the active pocket of JEV NS5 with an affinity much higher than any of the NTPs, thus showing a high possibility of inhibiting the replication of JEV via competitive inhibition. We also compared the conformations of ATP and ZINC 9367 from our docking studies to the conformation of ATP bound at the JEV NS5 RdRp active site (PDB ID: 4HDH). The superposition of the two structures (RdRp domain from 4K6M and 4HDH) revealed an RMSD of 6.5540 Å indicative of a structural change at the RdRp domain upon ATP binding. The ligand RMSDs for docked ATP and ZINC 9367 were 4.1093 Å and 11.9828 Å respectively compared to the reference ATP from the 4HDH structure (Figure S1). ZINC 9367 binds to the native structure of RdRp in JEV with a higher affinity than the native substrates, thus capable of impeding substrate availability required for the viral replication process. Further, MD simulations were performed to verify the binding stability of ZINC 9367 into the NTP binding pocket of the RdRp domain of the JEV NS5 protein.

MD simulations

All-atom MD simulations using explicit solvent models were employed to evaluate the stability of the JEV NS5 protein and ligand-protein complexes. The RMSD of the Cα backbone of the JEV NS5 protein complexed with ATP and ZINC 9367 are shown in Figure 6 which reveals that the ZINC 9367 complex is highly stable when compared to the native ligand ATP. The RMSD values for the protein (without ligand) were around 2.5 Å throughout the simulation. The protein backbone deviations for both the complexes were found around 3 Å over the trajectory of the simulation. Towards the end of the simulation, the ZINC 9367 complex converges to a lower RMSD value compared to the apo structure and NS5 complexed with ATP. The inset in Figure 6 illustrates the initial frame (0 ns) and the last frame (100 ns) of the whole simulation. This indicates the interaction stability of both complexes throughout the simulation.

The RMSF plot shown in Figure 7 supports the RMSD results. It shows that major fluctuations were observed towards the tails and in the loop regions of the protein which is typical [49,50]. The residues 255 to 280 of the NS5-ATP complex fluctuate more compared to the apo structure and NS5-ZINC 9367 complex which is due to the presence of a loop (Figure S2). Also, the NS5 protein (without ligand) and complexed with ATP show larger fluctuations around residues 850 which are towards the C-terminal of the protein compared to the NS5-ZINC 9367 complex.

The protein-ligand interaction analysis was done to explore the significant type of interactions and key protein residues involved in ligand binding for both the complexes. Figure 8 shows the histogram and schematic of protein interactions with ATP that were monitored throughout the simulation. Figure 8A reveals that ATP has strong interactions with LYS459, mainly through hydrogen bonding, hydrophobic interactions, water bridges, and some ionic interactions. It interacts with ASP669 only via water bridges and ionic interactions, and through hydrogen bonding and water bridges with SER715. These interactions of ATP with NS5 were retained from docking. While some major interactions with GLU 461, CYS 714, ARG 734, ARG 742, SER 799, TRP 800, and SER 801 were gained over a time period during the simulation.

Figure 8B shows a detailed schematic of the ATP atoms that interact with the protein residues for over 20% of the simulation time. ATP interacts with TRP 800 through hydrophobic contacts (pi-pi stacking) and hydrogen bonding. The phosphate groups of the ATP molecule form major water bridges with CYS 714, ARG 734, ARG 742, and SER 801. The ribose structure contributes to hydrogen bonding with GLU 461 and LYS 459. The aromatic rings of adenine are involved in hydrophobic contacts (pi-pi stacking) with TRP 800 for 44% of the simulation time and pi-cation interaction with LYS 459 for about 23% of the simulation time. Figure 9 gives the protein-ligand interactions for the JEV NS5- ZINC 9367 complex. It shows that ZINC-9367 forms strong interactions with LYS 459, ARG 460, ASP 541, ASP 668, ASP 669, TRP 800, ILE 802, and HIS 803. The stacked bar charts are normalized throughout the trajectory, indicating the percentage of the simulation time a specific contact is maintained. The interaction fraction values in the histograms in Figures 8A and 9A are over 1.0, which is because some protein residues are involved in multiple interactions of the same subtype with the ligands. Comparing the two histograms, clearly, ZINC 9367 is involved in a greater number of contacts with the JEV NS5 protein than ATP, which is the native ligand. Figure 9B shows that the atoms of ZINC 9367 that interact with the JEV NS5 protein residues. ZINC 9367 forms strong hydrogen bonds with ASP 541, ARG 460, ASP 668, ASP 669, and ALA 475 for more than 65% of the simulation time. Like ATP, the aromatic ring in ZINC 9367 also interacts with TRP 800 via pi-pi stacking (41%). An additional ring in ZINC 9367 is involved in pi-cation interactions with ARG 474 (43%) and LYS 459 (29%). Water-bridges are significant even in the NS5-ZINC 9367 complex.

Binding free energy calculations

Accurate prediction of receptor-ligand binding affinities is an important step in the drug discovery process. The binding free energies for protein-ligand complexes were computed using the MM/GBSA method. The distribution of MM/GBSA free energies for the two complexes over the entire trajectory during a 100 ns simulation is shown in Figure 10. The results indicate that ZINC 9367 (ΔGBind = -102.57 kcal/mol) has a higher order of binding strength compared to the native ligand ATP (ΔGBind = -43.88 kcal/mol) at the JEV NS5 RdRp active site.

Conclusion

NTPs were docked on the JEV NS5 protein to determine the high binding affinity locations on the RdRp domain. Followed by a pharmacophore-based druggability analysis, four potential molecules were identified that had a high affinity to the RdRp domain. The affinities of the four lead compounds were orders of magnitude higher than that of the NTPs, the native substrates for the polymerase. Further, to decipher the binding mechanism of ZINC 9367 to the JEV NS5 receptor, a 100 ns MD simulation was performed. Protein-ligand interactions and simulation trajectory analysis revealed that ZINC 9367 forms a stable complex with JEV NS5 protein throughout the entire simulation. MM/GBSA binding free energy calculations support the docking results that ZINC 9367 has a higher binding affinity to the JEV NS5 protein than ATP. The computational results obtained in this study suggest that these compounds have a high potential to inhibit the virus by blocking RNA replication and thus are prime candidates for experimental validation via in vitro studies.

Data Availability

The data that support the findings of this study are available on request from the corresponding author. ELIXIR-A, the algorithm created for pharmacophore mapping has been deposited in GitHub https://github.com/sfernando-BAEN/ELIXIR-A].   

Acknowledgment

We gratefully acknowledge the support from Texas A&M High Performance Research Computing (HPRC) and Laboratory for Molecular Simulation (LMS).

Declaration of competing interest

All the authors declare no conflict of interest.

Supplementary Data

Table S1: Hotspot analysis of JEV NS5
Parameter: temperature 300.00 K
Parameter: delta_g -1.000 kcal/mol
Parameter: n_probes 7
Parameter: min_n_probes 6
Parameter: merge_radius 5.5 A
Parameter: low_affinity 10.00 uM
Parameter: n_solutions 3
Parameter: max_charge 2.0 e
Parameter: n_charged 3
Parameter: n_frames 1
probe binding hotspots with deltaG less than -1.00 kcal/mol (~5 folds enrichment).
330 all-probes binding spots were identified in 3.89s.
Minimum binding free energy is -2.67 kcal/mol.
Hotspot   1 -2.67 kcal/mol 100.0% ACET
Hotspot   2 -2.57 kcal/mol  99.9% IPAM   0.1% IPRO
Hotspot   3 -2.45 kcal/mol 100.0% ACET
Hotspot   4 -2.43 kcal/mol  99.5% IPRO   0.5% ACAM
Hotspot   5 -2.37 kcal/mol 100.0% ACET
Hotspot   6 -2.34 kcal/mol  99.6% ACET   0.4% ACAM   0.0% IPAM
Hotspot   7 -2.29 kcal/mol 100.0% IPRO
Hotspot   8 -2.27 kcal/mol  99.3% ACET   0.7% IPRO
Hotspot   9 -2.24 kcal/mol  99.3% IPRO   0.7% IBUT
Hotspot  10 -2.22 kcal/mol  97.2% IPAM   2.8% IPRO
Hotspot  11 -2.16 kcal/mol  99.8% IPRO   0.2% ACET
Hotspot  12 -2.16 kcal/mol  99.5% ACET   0.5% IPRO
Hotspot  13 -2.15 kcal/mol  97.5% IPRO   2.5% IBUT
Hotspot  14 -2.13 kcal/mol  97.7% ACET   1.3% ACAM   1.1% IPRO
Hotspot  15 -2.12 kcal/mol 100.0% IPRO
Hotspot  16 -2.11 kcal/mol  61.8% IBUT  28.7% IPRO   9.5% ACAM
Hotspot  17 -2.10 kcal/mol  81.6% IPRO  18.4% ACAM
Hotspot  18 -2.09 kcal/mol  99.5% ACET   0.5% ACAM
Hotspot  19 -2.08 kcal/mol  98.9% ACET   1.1% IPRO
Hotspot  20 -2.07 kcal/mol  72.5% IPRO  26.3% IBUT   1.2% IPAM   0.1% ACAM
Hotspot  21 -2.06 kcal/mol  94.8% ACAM   5.2% IPRO
Hotspot  22 -2.06 kcal/mol  61.6% IBUT  38.4% IPRO
Hotspot  23 -2.04 kcal/mol  99.7% IPRO   0.3% IBUT
Hotspot  24 -2.03 kcal/mol  99.1% ACET   0.8% IPRO   0.1% ACAM
Hotspot  25 -1.99 kcal/mol  99.6% ACET   0.3% IPRO   0.1% ACAM
Hotspot  26 -1.97 kcal/mol 100.0% ACET
Hotspot  27 -1.96 kcal/mol 100.0% IPAM
Hotspot  28 -1.95 kcal/mol  62.9% IPRO  35.9% IBUT   1.2% ACAM
Hotspot  29 -1.92 kcal/mol  81.8% IPRO  18.2% IPAM
Hotspot  30 -1.90 kcal/mol  94.9% ACET   4.5% ACAM   0.5% IPRO
Hotspot  31 -1.90 kcal/mol  97.6% ACET   2.4% IPRO
Hotspot  32 -1.89 kcal/mol 100.0% ACET
Hotspot  33 -1.88 kcal/mol  82.4% IPRO  17.6% ACAM
Hotspot  34 -1.87 kcal/mol  75.5% IPRO  24.5% ACET
Hotspot  35 -1.87 kcal/mol  99.5% ACET   0.5% ACAM   0.1% IPRO
Hotspot  36 -1.86 kcal/mol  65.6% IBUT  21.9% ACAM  12.5% IPRO
Hotspot  37 -1.86 kcal/mol  91.5% IPRO   3.8% ACET   3.1% IPAM   1.6% ACAM
Hotspot  38 -1.86 kcal/mol  99.7% IPRO   0.3% IBUT
Hotspot  39 -1.86 kcal/mol  99.8% IPRO   0.2% IBUT
Hotspot  40 -1.85 kcal/mol  98.7% ACET   1.3% IPRO
Hotspot  41 -1.83 kcal/mol  65.3% IPRO  32.5% IBUT   2.2% ACAM
Hotspot  42 -1.83 kcal/mol  49.5% IPRO  26.8% IPAM  23.6% ACET
Hotspot  43 -1.83 kcal/mol  67.0% IBUT  32.0% IPRO   0.8% ACAM   0.2% IPAM
Hotspot  44 -1.82 kcal/mol 100.0% ACET
Hotspot  45 -1.81 kcal/mol  99.8% IPRO   0.2% ACAM
Hotspot  46 -1.81 kcal/mol 100.0% IPRO
Hotspot  47 -1.79 kcal/mol 100.0% ACET
Hotspot  48 -1.79 kcal/mol  62.5% IPAM  29.1% IPRO   4.9% IBUT   2.5% ACAM   1.0% ACET
Hotspot  49 -1.78 kcal/mol  92.3% ACET   7.7% IPRO
Hotspot  50 -1.78 kcal/mol  97.3% ACET   1.3% ACAM   1.2% IPRO   0.3% IBUT
Hotspot  51 -1.76 kcal/mol  98.7% ACET   1.3% IBUT
Hotspot  52 -1.76 kcal/mol  97.5% ACET   2.5% IPRO
Hotspot  53 -1.76 kcal/mol 100.0% IPRO
Hotspot  54 -1.75 kcal/mol  95.0% ACET   4.5% IPRO   0.3% IBUT   0.2% ACAM
Hotspot  55 -1.75 kcal/mol  60.4% IPRO  38.9% IBUT   0.6% ACAM   0.1% IPAM
Hotspot  56 -1.74 kcal/mol  90.7% IPRO   6.2% IBUT   3.0% IPAM   0.1% ACET
Hotspot  57 -1.74 kcal/mol 100.0% IPAM
Hotspot  58 -1.73 kcal/mol  99.9% ACET   0.1% IPRO
Hotspot  59 -1.72 kcal/mol  99.8% ACET   0.2% IPRO
Hotspot  60 -1.71 kcal/mol  99.3% ACET   0.7% IPRO
Hotspot  61 -1.71 kcal/mol  84.2% IPRO  14.3% IBUT   1.5% ACAM
Hotspot  62 -1.71 kcal/mol  96.1% ACET   3.9% IPRO
Hotspot  63 -1.71 kcal/mol  97.6% ACET   2.3% IPRO   0.1% ACAM
Hotspot  64 -1.70 kcal/mol  76.8% IPRO  22.8% IBUT   0.4% IPAM
Hotspot  65 -1.70 kcal/mol  73.0% IBUT  20.6% IPRO   4.3% ACAM   2.1% IPAM
Hotspot  66 -1.69 kcal/mol  42.6% IBUT  35.8% ACET  21.5% IPRO
Hotspot  67 -1.68 kcal/mol  99.1% ACET   0.6% IPRO   0.2% ACAM
Hotspot  68 -1.68 kcal/mol  99.8% IPAM   0.2% IPRO
Hotspot  69 -1.68 kcal/mol  88.4% IPRO  11.6% IPAM
Hotspot  70 -1.68 kcal/mol  97.9% IBUT   2.1% IPRO
Hotspot  71 -1.68 kcal/mol 100.0% IPRO
Hotspot  72 -1.67 kcal/mol  98.5% IPRO   1.5% ACAM
Hotspot  73 -1.66 kcal/mol  94.8% IPRO   5.2% IBUT
Hotspot  74 -1.66 kcal/mol 100.0% IPRO
Hotspot  75 -1.66 kcal/mol 100.0% ACAM
Hotspot  76 -1.65 kcal/mol  65.0% IBUT  33.8% IPRO   1.2% ACAM
Hotspot  77 -1.65 kcal/mol  90.3% IPRO   9.7% IBUT
Hotspot  78 -1.64 kcal/mol  83.0% IBUT  15.7% IPRO   1.2% IPAM   0.1% ACAM
Hotspot  79 -1.63 kcal/mol  93.3% ACET   3.4% IPAM   3.0% ACAM   0.4% IPRO
Hotspot  80 -1.63 kcal/mol  89.4% IPRO  10.5% ACET   0.1% ACAM
Hotspot  81 -1.63 kcal/mol  86.4% IPRO   6.9% IBUT   6.1% IPAM   0.5% ACAM
Hotspot  82 -1.62 kcal/mol  91.4% ACET   8.6% IPRO
Hotspot  83 -1.62 kcal/mol  99.0% ACET   1.0% IPRO
Hotspot  84 -1.61 kcal/mol  96.9% IPAM   3.1% IPRO
Hotspot  85 -1.61 kcal/mol  93.4% IPAM   5.2% IPRO   1.4% ACAM
Hotspot  86 -1.60 kcal/mol  98.4% IPRO   1.6% IBUT
Hotspot  87 -1.59 kcal/mol  95.2% ACET   3.0% ACAM   1.7% IPRO
Hotspot  88 -1.58 kcal/mol  87.6% ACET  12.2% IPRO   0.1% ACAM
Hotspot  89 -1.58 kcal/mol  80.9% IPRO   8.3% ACAM   6.3% IBUT   4.5% IPAM
Hotspot  90 -1.58 kcal/mol  95.0% IPRO   4.2% IBUT   0.7% ACAM
Hotspot  91 -1.57 kcal/mol  75.5% ACET  24.5% ACAM
Hotspot  92 -1.56 kcal/mol  98.8% ACET   1.1% IPRO   0.2% ACAM
Hotspot  93 -1.56 kcal/mol 100.0% IPRO
Hotspot  94 -1.56 kcal/mol  67.2% IPRO  27.6% ACET   5.1% ACAM
Hotspot  95 -1.56 kcal/mol  80.6% IPRO  11.4% ACAM   8.0% IPAM
Hotspot  96 -1.56 kcal/mol  86.4% IPRO   9.9% ACET   3.7% ACAM
Hotspot  97 -1.55 kcal/mol  94.8% IPRO   4.3% IBUT   0.9% ACAM
Hotspot  98 -1.54 kcal/mol 100.0% IPAM
Hotspot  99 -1.54 kcal/mol  68.1% IBUT  31.2% IPRO   0.8% ACAM
Hotspot 100 -1.54 kcal/mol  72.6% IPRO  25.2% IBUT   2.2% ACAM
Hotspot 101 -1.54 kcal/mol 100.0% IPRO
Hotspot 102 -1.54 kcal/mol  93.6% IPAM   5.7% IPRO   0.5% ACAM   0.3% IBUT
Hotspot 103 -1.54 kcal/mol  95.9% ACET   3.3% IPRO   0.5% IPAM   0.3% ACAM
Hotspot 104 -1.53 kcal/mol  79.2% IPRO   9.8% ACET   6.2% IPAM   4.4% ACAM   0.3% IBUT
Hotspot 105 -1.51 kcal/mol 100.0% ACET
Hotspot 106 -1.50 kcal/mol  99.0% ACET   1.0% ACAM
Hotspot 107 -1.50 kcal/mol  80.9% IPRO  13.5% IBUT   3.4% ACET   2.2% ACAM
Hotspot 108 -1.49 kcal/mol  83.7% ACAM  16.3% IPRO
Hotspot 109 -1.48 kcal/mol  93.9% ACET   5.9% IPRO   0.2% ACAM
Hotspot 110 -1.47 kcal/mol 100.0% IPRO
Hotspot 111 -1.46 kcal/mol  75.8% IBUT  22.8% IPRO   1.3% ACAM   0.2% ACET
Hotspot 112 -1.45 kcal/mol 100.0% IPRO

Hotspot 113 -1.44 kcal/mol  95.0% IPRO   5.0% IBUT
Hotspot 114 -1.44 kcal/mol 100.0% ACET
Hotspot 115 -1.44 kcal/mol  70.6% ACAM  27.4% IPRO   2.0% IBUT
Hotspot 116 -1.44 kcal/mol  92.6% IPRO   4.6% ACAM   2.8% IBUT
Hotspot 117 -1.44 kcal/mol  99.4% IPAM   0.6% ACAM
Hotspot 118 -1.43 kcal/mol  52.0% IPRO  40.7% IBUT   7.3% ACAM
Hotspot 119 -1.43 kcal/mol  74.4% IPRO  17.3% IBUT   6.8% ACET   0.8% ACAM   0.8% IPAM
Hotspot 120 -1.43 kcal/mol  84.1% IPRO   7.6% ACAM   4.2% IBUT   4.2% ACET
Hotspot 121 -1.43 kcal/mol 100.0% ACET
Hotspot 122 -1.42 kcal/mol 100.0% IPRO
Hotspot 123 -1.42 kcal/mol  98.1% ACET   1.1% ACAM   0.8% IPRO
Hotspot 124 -1.42 kcal/mol  86.9% IBUT   7.1% IPRO   6.0% IPAM
Hotspot 125 -1.42 kcal/mol  99.6% ACAM   0.4% IPRO
Hotspot 126 -1.41 kcal/mol  67.6% IPAM  26.9% IPRO   5.4% ACAM
Hotspot 127 -1.41 kcal/mol  76.1% IPRO  20.2% IBUT   2.5% ACAM   1.2% IPAM
Hotspot 128 -1.41 kcal/mol  87.8% IPAM  10.2% IPRO   2.0% ACAM
Hotspot 129 -1.39 kcal/mol  87.6% IPRO  10.0% ACAM   2.4% IPAM
Hotspot 130 -1.39 kcal/mol 100.0% ACET
Hotspot 131 -1.39 kcal/mol  97.2% ACET   1.8% IPRO   1.0% ACAM
Hotspot 132 -1.39 kcal/mol  83.6% IBUT  16.4% IPRO
Hotspot 133 -1.38 kcal/mol  84.5% IPRO  15.5% ACAM
Hotspot 134 -1.38 kcal/mol 100.0% ACAM
Hotspot 135 -1.38 kcal/mol  54.8% IPAM  28.2% IPRO  14.0% IBUT   2.9% ACAM
Hotspot 136 -1.37 kcal/mol  92.9% IPRO   7.1% ACAM
Hotspot 137 -1.37 kcal/mol  99.8% ACET   0.2% IPRO
Hotspot 138 -1.36 kcal/mol  96.6% ACET   2.7% IPRO   0.6% IPAM
Hotspot 139 -1.36 kcal/mol  99.6% ACET   0.4% IPRO
Hotspot 140 -1.36 kcal/mol  74.9% IPRO  14.6% IBUT   5.9% ACAM   4.5% IPAM
Hotspot 141 -1.35 kcal/mol  99.6% ACET   0.4% IPRO
Hotspot 142 -1.35 kcal/mol  52.0% ACAM  37.0% IPRO  11.0% IPAM
Hotspot 143 -1.34 kcal/mol  50.1% IPRO  49.9% IBUT
Hotspot 144 -1.34 kcal/mol  83.6% IPRO  16.2% IBUT   0.2% ACET
Hotspot 145 -1.34 kcal/mol  76.1% ACET  20.6% IPRO   2.4% IBUT   0.9% ACAM
Hotspot 146 -1.34 kcal/mol  61.9% IBUT  37.8% IPRO   0.2% IPAM
Hotspot 147 -1.34 kcal/mol  59.5% ACET  25.0% IPRO  10.4% IBUT   5.1% IPAM
Hotspot 148 -1.34 kcal/mol  89.6% IPRO   5.1% IBUT   3.1% ACAM   2.2% IPAM
Hotspot 149 -1.33 kcal/mol  91.1% IPRO   8.3% ACAM   0.4% IPAM   0.2% IBUT
Hotspot 150 -1.32 kcal/mol  75.2% IPRO  13.9% IPAM  10.7% ACAM   0.2% IBUT
Hotspot 151 -1.32 kcal/mol  82.0% ACET  13.9% IPRO   4.1% IBUT
Hotspot 152 -1.32 kcal/mol 100.0% ACAM
Hotspot 153 -1.31 kcal/mol  90.6% IPAM   9.2% IPRO   0.2% ACAM
Hotspot 154 -1.31 kcal/mol  86.1% IPRO  13.9% IBUT
Hotspot 155 -1.30 kcal/mol  75.5% IPAM  19.4% IPRO   2.6% IBUT   2.6% ACET
Hotspot 156 -1.30 kcal/mol  88.1% IPRO   7.7% ACAM   3.7% IBUT   0.5% ACET
Hotspot 157 -1.30 kcal/mol  96.7% ACET   3.1% IPRO   0.2% IPAM
Hotspot 158 -1.30 kcal/mol  66.2% IBUT  33.3% IPRO   0.2% ACAM   0.2% IPAM
Hotspot 159 -1.30 kcal/mol  61.2% IBUT  38.8% IPRO
Hotspot 160 -1.29 kcal/mol  97.4% ACET   2.6% IPRO
Hotspot 161 -1.29 kcal/mol  84.1% IPRO  13.7% ACAM   2.1% IBUT
Hotspot 162 -1.29 kcal/mol 100.0% IPRO
Hotspot 163 -1.29 kcal/mol  74.4% IPRO  22.2% IBUT   1.9% ACAM   1.4% IPAM
Hotspot 164 -1.28 kcal/mol  78.7% IPRO  13.3% IBUT   6.5% ACAM   1.4% IPAM
Hotspot 165 -1.28 kcal/mol  91.0% IPAM   7.8% ACAM   1.2% IPRO
Hotspot 166 -1.28 kcal/mol  91.2% IPRO   7.1% ACAM   1.7% IPAM
Hotspot 167 -1.28 kcal/mol  79.2% ACET  15.4% IPRO   4.2% IBUT   1.2% ACAM
Hotspot 168 -1.27 kcal/mol  91.9% IPAM   7.8% IPRO   0.2% ACAM
Hotspot 169 -1.27 kcal/mol  93.6% IPRO   5.9% ACAM   0.5% ACET
Hotspot 170 -1.27 kcal/mol  77.1% IPRO  11.9% ACET   6.2% IBUT   4.7% ACAM
Hotspot 171 -1.27 kcal/mol  99.5% IPAM   0.5% IPRO
Hotspot 172 -1.26 kcal/mol  67.3% IPRO  29.2% ACET   3.5% ACAM
Hotspot 173 -1.26 kcal/mol  80.8% ACET   9.2% IBUT   8.5% IPRO   1.5% ACAM
Hotspot 174 -1.26 kcal/mol  99.8% ACET   0.2% IPRO
Hotspot 175 -1.26 kcal/mol  99.5% ACET   0.5% IPRO
Hotspot 176 -1.25 kcal/mol  80.4% IPRO  17.3% IBUT   1.3% ACET   1.0% ACAM
Hotspot 177 -1.25 kcal/mol 100.0% ACET
Hotspot 178 -1.25 kcal/mol  71.2% IPRO  28.2% IPAM   0.5% ACAM
Hotspot 179 -1.25 kcal/mol  55.9% IBUT  40.3% IPRO   3.8% ACAM
Hotspot 180 -1.25 kcal/mol  81.7% ACET  18.0% IPRO   0.3% ACAM
Hotspot 181 -1.25 kcal/mol  92.5% IPRO   7.5% ACET
Hotspot 182 -1.25 kcal/mol  95.9% IPRO   4.1% ACAM
Hotspot 183 -1.25 kcal/mol  95.4% IPRO   3.6% IBUT   1.0% ACAM
Hotspot 184 -1.24 kcal/mol  91.8% ACET   8.2% IPRO
Hotspot 185 -1.24 kcal/mol  47.9% IPRO  32.7% ACET  18.3% IBUT   0.8% ACAM   0.3% IPAM
Hotspot 186 -1.24 kcal/mol  97.9% ACET   2.1% IPRO
Hotspot 187 -1.24 kcal/mol  86.2% IPRO   9.1% ACAM   4.7% IBUT
Hotspot 188 -1.24 kcal/mol  49.9% IBUT  49.1% IPRO   1.0% ACAM
Hotspot 189 -1.24 kcal/mol 100.0% IPRO
Hotspot 190 -1.24 kcal/mol 100.0% IBUT
Hotspot 191 -1.23 kcal/mol  94.8% ACET   3.7% IPRO   0.8% IPAM   0.8% IBUT
Hotspot 192 -1.23 kcal/mol  96.3% IPAM   3.7% ACET
Hotspot 193 -1.23 kcal/mol  99.7% ACET   0.3% IBUT
Hotspot 194 -1.23 kcal/mol  70.4% IPRO  19.8% ACAM   5.5% ACET   3.4% IPAM   0.8% IBUT
Hotspot 195 -1.23 kcal/mol  60.2% IPRO  36.6% IBUT   3.2% ACAM
Hotspot 196 -1.23 kcal/mol  66.0% IPRO  34.0% ACET
Hotspot 197 -1.22 kcal/mol  85.9% IPRO  14.1% IBUT
Hotspot 198 -1.22 kcal/mol  66.3% ACAM  32.3% ACET   1.3% IPRO
Hotspot 199 -1.22 kcal/mol  87.8% IPRO  11.6% IBUT   0.5% ACAM
Hotspot 200 -1.21 kcal/mol 100.0% IPRO
Hotspot 201 -1.21 kcal/mol  95.9% IPRO   4.1% ACAM
Hotspot 202 -1.21 kcal/mol  49.9% IPRO  47.7% IBUT   2.2% IPAM   0.3% ACAM
Hotspot 203 -1.21 kcal/mol 100.0% ACET
Hotspot 204 -1.20 kcal/mol  78.5% IPRO  21.5% IBUT
Hotspot 205 -1.20 kcal/mol  93.9% IPRO   3.6% ACAM   1.9% IPAM   0.3% ACET   0.3% IBUT
Hotspot 206 -1.20 kcal/mol  68.4% IPRO  19.4% ACAM  12.2% IBUT
Hotspot 207 -1.20 kcal/mol  99.4% ACAM   0.6% IPRO
Hotspot 208 -1.20 kcal/mol 100.0% ACET
Hotspot 209 -1.19 kcal/mol  72.8% IPRO  25.8% IBUT   1.1% ACAM   0.3% IPAM
Hotspot 210 -1.19 kcal/mol  64.8% IPRO  24.5% IBUT   6.2% ACAM   4.5% ACET
Hotspot 211 -1.18 kcal/mol  51.0% IBUT  47.6% IPRO   1.4% ACAM
Hotspot 212 -1.18 kcal/mol  90.3% ACAM   9.7% IPRO
Hotspot 213 -1.18 kcal/mol  71.2% ACET  28.8% IPRO
Hotspot 214 -1.18 kcal/mol  99.4% ACET   0.6% IPRO
Hotspot 215 -1.18 kcal/mol  54.9% IPAM  45.1% IPRO
Hotspot 216 -1.17 kcal/mol  63.2% IPRO  33.9% IBUT   2.9% ACAM
Hotspot 217 -1.17 kcal/mol  58.0% IPRO  23.5% IBUT  14.2% ACAM   4.3% IPAM
Hotspot 218 -1.17 kcal/mol  79.4% ACAM  20.0% IPRO   0.6% IBUT
Hotspot 219 -1.17 kcal/mol 100.0% IPAM
Hotspot 220 -1.17 kcal/mol 100.0% IPAM
Hotspot 221 -1.17 kcal/mol  83.7% IPRO  15.4% IBUT   0.9% ACAM
Hotspot 222 -1.17 kcal/mol  79.9% IPRO  12.5% IBUT   7.6% IPAM
Hotspot 223 -1.17 kcal/mol  99.1% IPAM   0.6% IPRO   0.3% ACAM
Hotspot 224 -1.17 kcal/mol  59.6% IBUT  40.1% IPRO   0.3% ACAM
Hotspot 225 -1.17 kcal/mol  57.3% IPRO  36.0% ACAM   6.7% IBUT
Hotspot 226 -1.17 kcal/mol  76.8% IBUT  23.2% IPRO
Hotspot 227 -1.17 kcal/mol  71.5% IPRO  15.9% IBUT  11.5% ACET   1.2% ACAM
Hotspot 228 -1.16 kcal/mol  77.0% IPRO  16.2% IBUT   4.7% ACAM   2.1% IPAM
Hotspot 229 -1.16 kcal/mol  94.7% ACET   2.4% IPRO   1.5% ACAM   1.2% IBUT   0.3% IPAM
Hotspot 230 -1.16 kcal/mol  62.5% IPRO  29.8% IBUT   4.8% IPAM   3.0% ACAM
Hotspot 231 -1.15 kcal/mol  98.8% ACET   0.6% ACAM   0.6% IPRO
Hotspot 232 -1.15 kcal/mol  85.0% ACET  14.7% IPRO   0.3% ACAM
Hotspot 233 -1.15 kcal/mol  57.1% IPRO  22.8% IPAM  16.5% ACAM   2.7% IBUT   0.9% ACET
Hotspot 234 -1.15 kcal/mol  99.7% ACET   0.3% IPRO
Hotspot 235 -1.15 kcal/mol  97.3% ACET   2.7% IPRO
Hotspot 236 -1.15 kcal/mol  90.3% IPRO   8.8% ACAM   0.9% ACET
Hotspot 237 -1.15 kcal/mol 100.0% IPRO
Hotspot 238 -1.14 kcal/mol  37.2% IBUT  32.3% IPRO  30.5% IPAM
Hotspot 239 -1.14 kcal/mol  98.8% ACET   0.9% IPRO   0.3% ACAM
Hotspot 240 -1.14 kcal/mol  89.3% IPRO   6.1% IBUT   4.6% ACAM
Hotspot 241 -1.14 kcal/mol 100.0% IPRO
Hotspot 242 -1.14 kcal/mol  82.5% IPRO   6.8% ACET   5.8% IBUT   4.0% IPAM   0.9% ACAM
Hotspot 243 -1.14 kcal/mol  98.8% ACET   0.9% IPRO   0.3% ACAM
Hotspot 244 -1.13 kcal/mol  98.1% ACET   1.6% IPRO   0.3% ACAM
Hotspot 245 -1.13 kcal/mol  56.2% ACET  36.6% IPRO   3.8% ACAM   3.4% IBUT
Hotspot 246 -1.13 kcal/mol  52.4% IPRO  23.8% IPAM  16.9% ACAM   6.9% ACET
Hotspot 247 -1.12 kcal/mol  98.1% ACET   0.6% ACAM   0.6% IPAM   0.6% IPRO
Hotspot 248 -1.12 kcal/mol  98.4% ACET   0.9% IBUT   0.6% IPRO
Hotspot 249 -1.12 kcal/mol  93.1% IPRO   6.9% IBUT
Hotspot 250 -1.12 kcal/mol 100.0% ACAM
Hotspot 251 -1.11 kcal/mol  98.4% IPRO   1.6% ACAM
Hotspot 252 -1.11 kcal/mol  98.7% IPRO   1.3% IBUT
Hotspot 253 -1.11 kcal/mol  78.8% IPRO  11.3% ACAM  10.0% IPAM
Hotspot 254 -1.10 kcal/mol  56.0% ACET  24.8% IPRO  19.2% IPAM
Hotspot 255 -1.10 kcal/mol  81.3% IPRO  13.4% ACAM   5.2% IPAM
Hotspot 256 -1.10 kcal/mol  70.5% ACET  29.5% IPRO
Hotspot 257 -1.10 kcal/mol  94.1% IPRO   3.9% IBUT   2.0% IPAM
Hotspot 258 -1.10 kcal/mol 100.0% ACET
Hotspot 259 -1.10 kcal/mol  80.5% IPRO  19.1% ACAM   0.3% IBUT
Hotspot 260 -1.10 kcal/mol  53.6% IPRO  34.8% ACET  11.6% ACAM
Hotspot 261 -1.10 kcal/mol  65.9% IPAM  29.8% IPRO   3.0% ACAM   1.3% IBUT
Hotspot 262 -1.09 kcal/mol  50.8% IPRO  31.2% IPAM  17.9% ACAM
Hotspot 263 -1.09 kcal/mol  78.3% ACET  21.7% IPRO
Hotspot 264 -1.09 kcal/mol  44.7% IBUT  40.3% IPRO  13.3% ACAM   1.3% ACET   0.3% IPAM
Hotspot 265 -1.09 kcal/mol  97.3% IPAM   2.7% IPRO
Hotspot 266 -1.09 kcal/mol  96.0% IPRO   2.7% IBUT   1.3% ACAM
Hotspot 267 -1.09 kcal/mol  84.2% IPRO   7.7% ACAM   7.0% IPAM   1.0% ACET
Hotspot 268 -1.09 kcal/mol  67.1% ACET  31.9% IPRO   1.0% ACAM
Hotspot 269 -1.09 kcal/mol  48.0% IBUT  28.5% IPRO  12.4% IPAM  10.7% ACAM   0.3% ACET
Hotspot 270 -1.09 kcal/mol  59.1% IPRO  35.2% IBUT   4.4% IPAM   1.3% ACAM
Hotspot 271 -1.09 kcal/mol  80.8% IPRO  19.2% IBUT
Hotspot 272 -1.08 kcal/mol  84.8% IPRO  10.1% IPAM   2.0% IBUT   1.7% ACAM   1.4% ACET
Hotspot 273 -1.08 kcal/mol  89.9% ACET   9.5% IPRO   0.7% ACAM
Hotspot 274 -1.08 kcal/mol  39.2% IPAM  31.1% ACAM  29.1% IPRO   0.7% ACET
Hotspot 275 -1.08 kcal/mol  67.8% IPRO  31.9% IBUT   0.3% ACAM
Hotspot 276 -1.08 kcal/mol  59.5% IPRO  40.5% IPAM
Hotspot 277 -1.08 kcal/mol  95.2% IPRO   3.1% ACAM   1.7% IPAM
Hotspot 278 -1.08 kcal/mol  56.5% ACET  38.1% IPRO   4.8% IBUT   0.7% ACAM
Hotspot 279 -1.08 kcal/mol  99.3% IPRO   0.7% ACET
Hotspot 280 -1.08 kcal/mol  73.5% IPRO  16.7% IPAM   7.8% ACAM   2.0% ACET
Hotspot 281 -1.08 kcal/mol  74.1% IPRO  25.9% IBUT
Hotspot 282 -1.08 kcal/mol  98.3% IPRO   1.4% ACAM   0.3% ACET
Hotspot 283 -1.08 kcal/mol  95.9% ACET   2.7% IPRO   0.7% ACAM   0.7% IPAM
Hotspot 284 -1.08 kcal/mol  68.2% IPRO  31.5% IBUT   0.3% ACAM
Hotspot 285 -1.08 kcal/mol  44.2% IPRO  39.4% IBUT  16.1% IPAM   0.3% ACAM
Hotspot 286 -1.07 kcal/mol  86.9% IPAM  11.3% IPRO   1.7% ACAM
Hotspot 287 -1.07 kcal/mol 100.0% IPRO
Hotspot 288 -1.07 kcal/mol  73.2% IPRO  18.9% IBUT   4.8% ACAM   3.1% ACET
Hotspot 289 -1.07 kcal/mol  83.8% IPRO  10.7% IBUT   5.5% ACAM
Hotspot 290 -1.07 kcal/mol 100.0% ACAM
Hotspot 291 -1.06 kcal/mol  77.7% IPRO  19.9% ACAM   1.7% IBUT   0.7% ACET
Hotspot 292 -1.06 kcal/mol  93.3% IPRO   6.4% IPAM   0.4% ACAM
Hotspot 293 -1.05 kcal/mol  61.7% IPRO  37.9% IBUT   0.4% ACAM
Hotspot 294 -1.05 kcal/mol  95.7% IPRO   2.8% IBUT   1.4% ACAM
Hotspot 295 -1.05 kcal/mol  98.2% IPAM   1.1% ACAM   0.4% ACET   0.4% IPRO
Hotspot 296 -1.05 kcal/mol 100.0% IPRO
Hotspot 297 -1.05 kcal/mol  99.3% ACET   0.7% IPRO
Hotspot 298 -1.05 kcal/mol  86.4% ACET   8.6% IPRO   2.9% ACAM   2.1% IBUT
Hotspot 299 -1.05 kcal/mol  83.6% ACET  12.1% IPRO   2.1% IPAM   1.4% IBUT   0.7% ACAM
Hotspot 300 -1.05 kcal/mol 100.0% ACET
Hotspot 301 -1.05 kcal/mol  84.3% IPRO  11.1% IBUT   4.3% ACAM   0.4% ACET
Hotspot 302 -1.05 kcal/mol 100.0% ACET
Hotspot 303 -1.05 kcal/mol  98.6% IPAM   1.4% IPRO
Hotspot 304 -1.05 kcal/mol  98.2% ACET   1.8% IPRO
Hotspot 305 -1.05 kcal/mol  86.4% ACET   9.7% IPAM   3.6% ACAM   0.4% IPRO
Hotspot 306 -1.04 kcal/mol  99.3% IPRO   0.7% IBUT
Hotspot 307 -1.04 kcal/mol  97.1% IPAM   2.6% ACAM   0.4% IPRO
Hotspot 308 -1.04 kcal/mol 100.0% IPRO
Hotspot 309 -1.04 kcal/mol  98.9% ACET   0.7% IBUT   0.4% IPRO
Hotspot 310 -1.04 kcal/mol  99.6% IPAM   0.4% ACAM
Hotspot 311 -1.03 kcal/mol  65.1% IPRO  21.0% IBUT  14.0% ACAM
Hotspot 312 -1.03 kcal/mol  68.3% IPRO  15.5% ACAM  11.4% IBUT   4.8% IPAM
Hotspot 313 -1.03 kcal/mol  51.1% IPRO  33.7% IBUT  15.2% ACAM
Hotspot 314 -1.02 kcal/mol  77.9% ACET  15.4% IPRO   6.4% IPAM   0.4% ACAM
Hotspot 315 -1.02 kcal/mol  96.3% IPRO   3.4% ACAM   0.4% ACET
Hotspot 316 -1.02 kcal/mol  79.2% IPRO  13.6% IPAM   6.8% IBUT   0.4% ACAM
Hotspot 317 -1.01 kcal/mol  61.5% IPRO  36.3% IBUT   2.3% ACAM
Hotspot 318 -1.01 kcal/mol  79.3% IPRO  11.1% ACAM   7.7% IBUT   1.9% IPAM
Hotspot 319 -1.01 kcal/mol  92.3% IPRO   5.7% ACAM   1.9% IBUT
Hotspot 320 -1.01 kcal/mol  48.7% ACET  37.5% IPRO  13.8% ACAM
Hotspot 321 -1.01 kcal/mol  84.6% IPRO  15.4% ACAM
Hotspot 322 -1.01 kcal/mol  91.9% ACET   8.1% IPRO
Hotspot 323 -1.01 kcal/mol  70.8% IPRO  16.2% IBUT  13.1% ACET
Hotspot 324 -1.01 kcal/mol  96.5% ACET   2.7% IPRO   0.8% ACAM
Hotspot 325 -1.00 kcal/mol  96.5% ACET   3.5% IPRO
Hotspot 326 -1.00 kcal/mol  95.4% ACET   1.9% ACAM   1.5% IPRO   1.2% IPAM
Hotspot 327 -1.00 kcal/mol  97.3% ACET   2.3% IPRO   0.4% IPAM
Hotspot 328 -1.00 kcal/mol  56.8% IBUT  35.9% IPRO   6.9% ACAM   0.4% ACET
Hotspot 329 -1.00 kcal/mol  67.1% IBUT  30.2% IPRO   2.7% ACAM
Hotspot 330 -1.00 kcal/mol  53.9% ACET  40.3% IPRO   5.8% IBUT
IPRO: 153 isopropanol binding hotspots were identified.
IPRO: lowest binding free energy is -2.43 kcal/mol.
IBUT: 27 isobutane binding hotspots were identified.
IBUT: lowest binding free energy is -2.11 kcal/mol.
IPAM: 32 isopropylamine binding hotspots were identified.
IPAM: lowest binding free energy is -2.57 kcal/mol.
ACAM: 14 acetamide binding hotspots were identified.
ACAM: lowest binding free energy is -2.06 kcal/mol.
ACET: 104 acetate binding hotspots were identified.
ACET: lowest binding free energy is -2.67 kcal/mol.
Clustering probe binding hotspots.
Clustering completed in 1.74ms.
Table S2: Druggability analysis of JEV NS5
9 potential sites are identified.
Calculating achievable affinity ranges.
Site 1: 27 probe binding hotspots
Site 1: Lowest probe binding free energy -2.67 kcal/mol
Site 1: Average probe binding free energy-1.52 kcal/mol
Site 1: Total of 259 solutions.
            Achievable affinities for site 1          
-log10(affinity)
     #------------------------------------------------#
9.01 |-o                                              |
8.75 |-----------o                                    |
8.49 |-----------o                                    |
8.24 |------------------------------------------o     |
7.98 |----------------------------------------------o |
7.72 |---------------------------------------------o  |
7.47 |-----------------------------------------o      |
7.21 |----------------------------------o             |
6.95 |-----------------o                              |
6.70 |-o                                              |
     #------------------------------------------------#
     0    5   10   15   20   25   30   35   40   45
Site 1: Lowest drug-like binding free energy -12.36 kcal/mol
Site 1: Highest drug-like affinity 0.982 nM
Site 1: Solution 1 binding free energy -12.36 kcal/mol
Site 1: Solution 1 affinity 0.982 nM
Site 1: Solution 1 total charge -1.92 e
Site 1: Solution 1 number of hotspots 7
Site 1: Solution 1 approximate volume 428.57 A^3
Site 1: Solution 2 binding free energy -12.01 kcal/mol
Site 1: Solution 2 affinity 1.759 nM
Site 1: Solution 2 total charge -1.92 e
Site 1: Solution 2 number of hotspots 7
Site 1: Solution 2 approximate volume 431.26 A^3
Site 1: Solution 3 binding free energy -11.95 kcal/mol
Site 1: Solution 3 affinity 1.946 nM
Site 1: Solution 3 total charge -1.93 e
Site 1: Solution 3 number of hotspots 7
Site 1: Solution 3 approximate volume 447.06 A^3
Site 2: 9 probe binding hotspots
Site 2: Lowest probe binding free energy -2.15 kcal/mol
Site 2: Average probe binding free energy-1.54 kcal/mol
Site 2: Total of 18 solutions.
Achievable affinities for site 2
-log10(affinity)
     #----#
8.26 |--o |
8.16 |-o  |
8.06 |--o |
7.97 |o   |
7.87 |    |
7.77 |    |
7.68 |-o  |
7.58 |--o |
7.48 |-o  |
7.38 |-o  |
     #----#
     0
Site 2: Lowest drug-like binding free energy -11.33 kcal/mol
Site 2: Highest drug-like affinity 5.504nM
Site 2: Solution 1 binding free energy -11.33 kcal/mol
Site 2: Solution 1 affinity 5.504 nM
Site 2: Solution 1 total charge -0.42 e
Site 2: Solution 1 number of hotspots 7
Site 2: Solution 1 approximate volume 434.83 A^3
Site 2: Solution 2 binding free energy -11.28 kcal/mol
Site 2: Solution 2 affinity 5.986 nM
Site 2: Solution 2 total charge -0.42 e
Site 2: Solution 2 number of hotspots 7
Site 2: Solution 2 approximate volume 437.02 A^3
Site 2: Solution 3 binding free energy -11.20 kcal/mol
Site 2: Solution 3 affinity 6.846 nM
Site 2: Solution 3 total charge -0.42 e
Site 2: Solution 3 number of hotspots 7
Site 2: Solution 3 approximate volume 428.28 A^3
Site 3: 10 probe binding hotspots
Site 3: Lowest probe binding free energy -2.16 kcal/mol
Site 3: Average probe binding free energy-1.49 kcal/mol
Site 3: Lowest drug-like binding free energy -11.16 kcal/mol
Site 3: Highest drug-like affinity 7.380 nM
Site 3: Solution 1 binding free energy -11.16 kcal/mol
Site 3: Solution 1 affinity 7.380 nM
Site 3: Solution 1 total charge -1.59 e
Site 3: Solution 1 number of hotspots 7
Site 3: Solution 1 approximate volume 465.81 A^3
Site 4: 19 probe binding hotspots
Site 4: Lowest probe binding free energy -2.45 kcal/mol
Site 4: Average probe binding free energy-1.48 kcal/mol
Site 4: Lowest drug-like binding free energy -10.59 kcal/mol
Site 4: Highest drug-like affinity 19.109 nM
Site 4: Solution 1 binding free energy -10.59 kcal/mol
Site 4: Solution 1 affinity 19.109 nM
Site 4: Solution 1 total charge 1.05 e
Site 4: Solution 1 number of hotspots 7
Site 4: Solution 1 approximate volume 469.24 A^3
Site 5: 11 probe binding hotspots
Site 5: Lowest probe binding free energy -2.07 kcal/mol
Site 5: Average probe binding free energy-1.32 kcal/mol
Site 5: Total of 11 solutions.
Achievable affinities for site 5
-log10(affinity)
     #-----#
7.36 |---o |
7.28 |o    |
7.19 |     |
7.11 |     |
7.02 |     |
6.94 |     |
6.85 |o    |
6.77 |o    |
6.68 |-o   |
6.60 |-o   |
     #-----#
     0
Site 5: Lowest drug-like binding free energy -10.10 kcal/mol
Site 5: Highest drug-like affinity 43.311 nM
Site 5: Solution 1 binding free energy -10.10 kcal/mol
Site 5: Solution 1 affinity 43.311 nM
Site 5: Solution 1 total charge 0.08 e
Site 5: Solution 1 number of hotspots 7
Site 5: Solution 1 approximate volume 446.84 A^3
Site 5: Solution 2 binding free energy -10.06 kcal/mol
Site 5: Solution 2 affinity 46.783 nM
Site 5: Solution 2 total charge 0.02 e
Site 5: Solution 2 number of hotspots 7
Site 5: Solution 2 approximate volume 463.89 A^3
Site 5: Solution 3 binding free energy -10.00 kcal/mol
Site 5: Solution 3 affinity 51.689 nM
Site 5: Solution 3 total charge 1.05 e
Site 5: Solution 3 number of hotspots 7
Site 5: Solution 3 approximate volume 459.74 A^3
Site 6: 6 probe binding hotspots
Site 6: Lowest probe binding free energy -1.99 kcal/mol
Site 6: Average probe binding free energy-1.59 kcal/mol
Site 6: Lowest drug-like binding free energy -9.52 kcal/mol
Site 6: Highest drug-like affinity 0.115 uM
Site 6: Solution 1 binding free energy -9.52 kcal/mol
Site 6: Solution 1 affinity 0.115 uM
Site 6: Solution 1 total charge -1.99 e
Site 6: Solution 1 number of hotspots 6
Site 6: Solution 1 approximate volume 333.92 A^3
Site 7: 13 probe binding hotspots
Site 7: Lowest probe binding free energy -1.68 kcal/mol
Site 7: Average probe binding free energy-1.21 kcal/mol
Site 7: Total of 57 solutions.
Achievable affinities for site 7 -log10(affinity)
     #-----------#
6.80 |---o       |
6.71 |---------o |
6.63 |------o    |
6.55 |---o       |
6.47 |-----o     |
6.39 |---------o |
6.30 |---------o |
6.22 |--o        |
6.14 |           |
6.06 |--o        |
     #-----------#
     0    5   10
Site 7: Lowest drug-like binding free energy -9.32 kcal/mol
Site 7: Highest drug-like affinity 0.160 uM
Site 7: Solution 1 binding free energy -9.32 kcal/mol
Site 7: Solution 1 affinity 0.160 uM
Site 7: Solution 1 total charge 0.00 e
Site 7: Solution 1 number of hotspots 7
Site 7: Solution 1 approximate volume 460.21 A^3
Site 7: Solution 2 binding free energy -9.31 kcal/mol
Site 7: Solution 2 affinity 0.162 uM
Site 7: Solution 2 total charge 0.00 e
Site 7: Solution 2 number of hotspots 7
Site 7: Solution 2 approximate volume 450.33 A^3
Site 7: Solution 3 binding free energy -9.27 kcal/mol
Site 7: Solution 3 affinity 0.175 uM
Site 7: Solution 3 total charge 0.00 e
Site 7: Solution 3 number of hotspots 7
Site 7: Solution 3 approximate volume 439.12 A^3
Site 8: 6 probe binding hotspots
Site 8: Lowest probe binding free energy -2.12 kcal/mol
Site 8: Average probe binding free energy-1.55 kcal/mol
Site 8: Lowest drug-like binding free energy -9.29 kcal/mol
Site 8: Highest drug-like affinity 0.170 uM
Site 8: Solution 1 binding free energy -9.29 kcal/mol
Site 8: Solution 1 affinity 0.170 uM
Site 8: Solution 1 total charge 1.21 e
Site 8: Solution 1 number of hotspots 6
Site 8: Solution 1 approximate volume 360.82 A^3
Site 9: 7 probe binding hotspots
Site 9: Lowest probe binding free energy -1.67 kcal/mol
Site 9: Average probe binding free energy-1.29 kcal/mol
Site 9: Lowest drug-like binding free energy -9.04 kcal/mol
Site 9: Highest drug-like affinity 0.256 uM
Site 9: Solution 1 binding free energy -9.04 kcal/mol
Site 9: Solution 1 affinity 0.256 uM
Site 9: Solution 1 total charge 0.00 e
Site 9: Solution 1 number of hotspots 7
Site 9: Solution 1 approximate volume 391.65