SUPPORTING INFORMATION

Structure-based Methods to Predict Mutational Resistance to Diarylpyrimidine NNRTIs

Syeda Maryam Azeem≠; Alecia N. Muwonge≠;; Nehaben Thakkar; Kristina W. Lam; Kathleen M. Frey*

≠ These authors contributed equally.*Corresponding Author

Affiliation: Long Island University, Arnold & Marie Schwartz College of Pharmacy and Health Sciences, Brooklyn, NY 11201

Table of Contents Page NumberMolecular Docking

Structural Comparison between DAPY crystal structures and RT (WT):DPV docking model

Table of RMSD values from structural alignment

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Correlation Plots for Residue Scanning ParametersTable of Correlation Plot Data

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Molecular Dynamics Supporting Information

Root Mean Square Fluctuation (RMSF) Plots Root Mean Square Deviation (RMSD) Plots Interaction Bar Graphs Interaction Diagrams

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Enzyme Inhibition Assays RT (WT): RPV - Non-Linear Regression Plots:

% Relative Inhibition vs. Concentration RT (K101P): RPV - Non-Linear Regression

Plots: % Relative Inhibition vs. Concentration RT (WT): ETV - Non-Linear Regression Plots:

% Relative Inhibition vs. Concentration RT (K101P): ETV - Non-Linear Regression

Plots: % Relative Inhibition vs. Concentration RT (WT): DPV - Non-Linear Regression Plots:

% Relative Inhibition vs. Concentration RT (K101P): DPV - Non-Linear Regression

Plots: % Relative Inhibition vs. Concentration

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References S12

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MOLECULAR DOCKING:

Figure 1: Alignment of RT (WT):DPV docking model (Green) with crystal structures RT (WT):RPV (Cyan; PDB code: 2ZD1) and RT (WT):ETV (Pink; PDB code: 3MEC). Root mean square deviations (RMSD) values for all residues in the binding site were used for the alignment. RMSD value comparing RPV crystal structure with DPV docking model is 0 Å. The RMSD value comparing ETV crystal structure with DPV docking model is 1.504 Å. A slight change in binding mode for ETV is observed due to the pyrimidine Br group.

Table 1: Root mean square deviation (RMSD) values from structural alignment:

Aligned RT Structures RMSD (Å)

RPV-WT/K101P 0.065

ETV-WT/K101P 0.112

DPV-WT/K101P 0.041

RPV-WT/DPV-WT 0.000

ETV-WT/DPV-WT 1.504

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CORRELATION PLOTS:

Figure 2: The above plots represent LOG of Fold Resistance against dA, dS and dA+dS for RPV, ETV, and DPV.

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Figure 3: The above plots represent resistance scores from Stanford HIV Database against dA+dS for RPV and ETV. Table 2: Values used for Log(Fold Resistance) correlation plots.Rilpivirine ΔS ΔA ΔS+ΔA Fold-Resistance LOG(Fold Resistance)WT 0 0 0 1 [1] 0L100I 10.426 -4.05 10.021 1.1 [1] 0.041K101P 24.904 16.071 40.975 40.1 [1] 1.603K103N -1.785 1.542 -0.243 1.6 [1] 0.204V108I 13.798 0.126 13.924 1.5 [1] 0.176E138K 4.247 8.561 12.808 2.9 [1] 0.462Y181C 14.62 -3.306 11.314 1.8 [1] 0.255G190A 12.364 0.01 12.374 1.3 [1] 0.114M230L 7.198 0.146 7.344 7.9 [1] 0.898Etravirine ΔS ΔA ΔS+ΔA Fold-Resistance LOG(Fold Resistance)WT 0 0 0 1 [2] 0L100I 28.322 -1.183 27.139 2 [2] 0.301029996K101P 42.851 10.407 53.258 5.2 [2] 0.716003344K103N 1.815 0.351 2.166 1 [2] 0V108I -4.413 0.05 -4.363 1 [2] 0E138K 1.909 11.815 13.724 2.6 [2] 0.414973348Y181C 14.959 -1.134 13.825 5.5 [2] 0.740362689G190A 7.025 0.004 7.029 1 [2] 0M230L 11.112 0.004 11.116 10 [2] 1Dapivirine ΔS ΔA ΔS+ΔA Fold-Resistance LOG(Fold Resistance)WT 0 0 0 1 [1] 0L100I 16.862 0.946 17.808 14.7 [1] 1.167K101P 16.409 12.914 29.323 100 [1] 2K103N -1.005 2.283 1.278 4.8 [1] 0.681V108I 17.317 -0.387 16.93 1.5 [1] 0.176E138K 4.097 11.599 15.696 4.4 [1] 0.643Y181C 6.4 0.035 6.435 7.9 [1] 0.898G190A 13.96 -1.876 12.084 1.2 [1] 0.079M230L 8.028 0.267 8.295 15.8 [1] 1.199

Table 3: Values used for Mutation Resistance Score (HIV Stanford Database) correlation plots.Rilpivirine ΔS ΔA ΔS+ΔA Resistance ScoreWT 0 0 0 0L100I 10.426 -4.05 10.021 60K101P 24.904 16.071 40.975 60K103N -1.785 1.542 -0.243 0V108I 13.798 0.126 13.924 0E138K 4.247 8.561 12.808 45Y181C 14.62 -3.306 11.314 45G190A 12.364 0.01 12.374 15M230L 7.198 0.146 7.344 60Etravirine ΔS ΔA ΔS+ΔA Resistance ScoreWT 0 0 0 0L100I 28.322 -1.183 27.139 30K101P 42.851 10.407 53.258 60K103N 1.815 0.351 2.166 0V108I -4.413 0.05 -4.363 0E138K 1.909 11.815 13.724 10Y181C 14.959 -1.134 13.825 30G190A 7.025 0.004 7.029 10M230L 11.112 0.004 11.116 0

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MOLECULAR DYNAMICS SUPPORTING INFORMATION:

RMSF

RPV (WT) RPV (K101P)

Figure 4: The RMSF helps in characterizing local changes along the protein chain in the reference backbone. In the above plot, peaks indicate areas of the protein that fluctuate the most during the simulation.

ETV (WT) ETV (K101P)

Figure 5: The RMSF helps in characterizing local changes along the protein chain in the reference backbone. In the above plot, peaks indicate areas of the protein that fluctuate the most during the simulation.

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DPV (WT) DPV (K101P)

Figure 6: The RMSF helps in characterizing local changes along the protein chain in the reference backbone. In the above plot, peaks indicate areas of the protein that fluctuate the most during the simulation.

RMSD PLOTS:

RPV (WT) RPV (K101P)

Figure 7: The above plot shows the RMSD of protein in blue (left Y-axis) and ligand RMSD in red (right Y-axis). The RMSD of the protein is calculated based on the atom selection after it is aligned to the reference backbone. Ligand RMSD which is calculated indicates how stable the ligand is with respect to the protein and its binding pocket. Changes between 1-3 Å are acceptable.

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ETV (WT) ETV (K101P)

Figure 8: The above plot shows the RMSD of protein in blue (left Y-axis) and ligand RMSD in red (right Y-axis). The RMSD of the protein is calculated based on the atom selection after it is aligned to the reference backbone. Ligand RMSD which is calculated indicates how stable the ligand is with respect to the protein and its binding pocket. Changes between 1-3 Å are acceptable.

DPV (WT) DPV (K101P)

Figure 9: The above plot shows the RMSD of protein in blue (left Y-axis) and ligand RMSD in red (right Y-axis). The RMSD of the protein is calculated based on the atom selection after it is aligned to the reference backbone. Ligand RMSD which is calculated indicates how stable the ligand is with respect to the protein and its binding pocket. Changes between 1-3 Å are acceptable.

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INTERACTION BAR GRAPHS FROM MOLECULAR DYNAMICS FOR RT-NNRTI COMPLEXES

RPV (WT) RPV (K101P)

Figure 10: The above bar chart represents the different protein-ligand interactions which are normalized over the course of trajectory. Values greater than 1.0 indicate that protein residues make more than one contact with the ligand.

ETV (WT) ETV (K101P)

Figure 11: The above bar chart represents the different protein-ligand interactions which are normalized over the course of trajectory. Values greater than 1.0 indicate that protein residues make more than one contact with the ligand.

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DPV (WT) DPV (K101P)

Figure 12: The above bar chart represents the different protein-ligand interactions which are normalized over the course of trajectory. Values greater than 1.0 indicate that protein residues make more than one contact with the ligand.

INTERACTION DIAGRAMS:

RPV (WT) RPV (K101P)

Figure 13: The above protein-ligand interaction diagram depicts the interactions that occur more than 30% of the simulation time (2.4 ns) of the specified trajectory (9.6 unit).

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ETV (WT) ETV (K101P)

Figure 14: The above protein-ligand interaction diagram depicts the interactions that occur more than 30% of the simulation time (2.4 ns) of the specified trajectory (9.6 unit).

DPV (WT) DPV (K101P)

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Figure 15: The above protein-ligand interaction diagram depicts the interactions that occur more than 30% of the simulation time (2.4 ns) of the specified trajectory (9.6 unit).

ENZYME INHIBITION ASSAYS PLOTTED TO NON-LINEAR REGRESSION:

IC50: 4.7nM (±0.4) IC50: 327.8nM (±91.7)A B

IC50: 5.23nM (±1.4) IC50: 375nM (±62.5)

C D

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IC50: 6nM (±1.0) IC50: 347.2nM (±69) E F

Figure 15: Enzymatic Inhibition Non-Linear Regression plots of NNRTI Concentration (nM) vs. Percentage (%) Relative Inhibition showing A: RT (WT): RPV. B: RT(K101P): RPV. C: RT(WT): ETV. D: RT (K101P): ETV E: RT(WT): DPV. F: RT (K101P): DPV. Experiments were done in triplicates with error bars representing deviations.

REFERENCES

[1] Giacobbi, N.S., Sluis-Cremer, N. In Vitro Cross-Resistance Profiles of Rilpivirine, Dapivirine, and MIV-150, Nonnucleoside Reverse Transcriptase Inhibitor Microbicides in Clinical Development for the Prevention of HIV-1 Infection. Antimicrob Agents Chemother. 2017, 61.[2] Basson, A.E., Rhee, S.Y., Parry, C.M., El-Khatib, Z., Charalambous, S., De Oliveira, T., et al. Impact of drug resistance-associated amino acid changes in HIV-1 subtype C on susceptibility to newer nonnucleoside reverse transcriptase inhibitors. Antimicrob Agents Chemother. 2015, 59, 960-71.

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