Validation of a Model for the Complex of HIV-1 Reverse Transcriptase with Sustiva through Computation of Resistance Profiles Robert C. Rizzo, De-Ping Wang, Julian Tirado-Rives, and William L. Jorgensen* Department of Chemistry, Yale UniVersity New HaVen, Connecticut 06520-8107 ReceiVed August 21, 2000 ReVised Manuscript ReceiVed October 24, 2000 All retroviruses depend on a virally encoded reverse tran- scriptase enzyme (RT) to convert viral RNA into DNA for subsequent incorporation into the host cell genome. 1 Drug-design efforts to arrest reverse transcription in HIV have led to the FDA approval of three non-nucleoside reverse transcriptase inhibitors (NNRTIs), nevirapine, delavaridine, and efavirenz (Sustiva). Additional compounds, including MKC-442, are in clinical trials (Table 1). Because of the low fidelity of HIVRT, the mutation rate in the encoded proteins including HIVRT is great. 2,3 As a result, all HIVRT inhibitors incur resistance problems that adversely affect their clinical value. 4,5 A measure of a drug’s effectiveness against a mutation is given by the fold resistance, which is the ratio of mutant to wild-type activities. Sustiva has been shown to remain notably active against several common HIVRT point mutations including Val f Ala at position 106 (V106A) and Tyr f Cys at position 181 (Y181C) (Table 1). No HIVRT structure with Sustiva has been reported that may help explain its improved resistance profile. Herein, we have (a) computed a structure for the Sustiva/HIVRT complex, (b) validated the structure through computations of the effects of the V106A and Y181C mutations on binding affinities for four drugs, and (c) obtained structural insights on the improved effectiveness of Sustiva. A binding site model 6 was constructed from the 2.55-Å crystal structure of the MKC-442/HIVRT complex (pdb 1rt1) 7 with MKC-442 removed including only those residues within ∼15 Å of MKC-442. The MATADOR program 8 was then used to dock Sustiva into the NNRTI site. 9 The other complexes were prepared analogously starting with coordinates from the X-ray structures of nevirapine (pdb 1vrt), 10 HEPT (pdb 1rti), 7 and 9-Cl TIBO (pdb 1rev) 11 bound to HIVRT. The docking calculations placed Sustiva in a reasonable position and orientation in the binding site in comparison with the crystal structures for the complexes with MKC-442, nevirapine, and 9-Cl TIBO (Figure 1). As controls, MKC-442, nevirapine, 9-Cl TIBO, and HEPT were also docked back into their binding sites to verify that the docking protocol could reproduce experimental structures. The lowest-energy structure generated during the docking runs was taken as the “best” structure and was found in all cases to reproduce closely the position and orientation observed in the crystal structures; the rmsd for the non-hydrogen atoms of the four ligands between the X-ray and docked structures was 0.43-0.60 Å. These low rmsd values and the limited flexibility of Sustiva are favorable for the accuracy of the docking calculations. The best docked structure of Sustiva reveals that it makes interactions that are consistent with those for other NNRTIs and that it overlays well with the “butterfly” shape of nevirapine. Unlike nevirapine, hydrogen bonds are present between Sustiva and the protein backbone at position Lys101 that are similar to those observed in the crystal structures with 9-Cl TIBO and MKC- 442 (Figure 1). Nevirapine makes no formal ligand-protein hydrogen bonds, but it does form a π-type hydrogen bond between the secondary amide hydrogen and Tyr188 12 and water-mediated hydrogen bonds. 10,12 The cyclopropyl ethynyl group of Sustiva is positioned toward aromatic residues Tyr181 and Tyr188 in the same fashion as the methylpyridine fragment of nevirapine, the benzyl ring of MKC-442, and the dimethylallyl group of 9-Cl TIBO (Figure 1). Presumably, these aryl-π interactions all contribute favorably to binding. 4,5 Superposition based on the HIVRT CR atoms shows that these π fragments of the inhibitors coincide spatially in the binding site and that Sustiva’s π fragment is the smallest (Figure 2). An alternative binding mode suggested by Maga et al. was based on an alignment of nevirapine and Sustiva in which the (1) Katz, R. A.; Skalka, A. M. Annu. ReV. Biochem. 1994, 63, 133-173. (2) Preston, B. D.; Poiesz, B. J.; Loeb, L. A. Science 1988, 242, 1168- 1171. (3) Roberts, J. D.; Bebenek, K.; Kunkel, T. A. Science 1988, 242, 1171- 1173. (4) Tantillo, C.; Ding, J. P.; Jacobomolina, A.; Nanni, R. G.; Boyer, P. L.; Hughes, S. H.; Pauwels, R.; Andries, K.; Janssen, P. A. J.; Arnold, E. J. Mol. Biol. 1994, 243, 369-387. (5) De Clercq, E. AntiViral Res. 1998, 38, 153-179. (6) Protein residues included in the binding site model were 91-110A, 161-205A, 222-242A, 316-321A, 343-349A, 381-383A, and 134-140B. (7) Hopkins, A. L.; Ren, J. S.; Esnouf, R. M.; Willcox, B. E.; Jones, E. Y.; Ross, C.; Miyasaka, T.; Walker, R. T.; Tanaka, H.; Stammers, D. K.; Stuart, D. I. J. Med. Chem. 1996, 39, 1589-1600. (8) Plount Price, M. L. Ph.D. Thesis, Yale University, 2000. (9) MATADOR uses a Monte Carlo-based Tabu search algorithm. To keep the Tabu search focused on the known NNRTI binding site during the docking runs, a 50 kcal/mol Å 2 half-harmonic restraining force was applied if the distance between the ligand and the binding site center was greater than 5 Å. The defined binding site was roughly centered on the alkyne group of Sustiva. The Tabu list was set to be 25 and constructed from unique structures considering energetic as well as geometric criteria. In total, 100 Tabu cycles were performed with each Tabu search generating 100 randomly placed ligand positions around the binding site. The decision to accept a new structure onto the Tabu lists is made after an intermolecular energy minimization and is based on both energetic and geometric criteria. The protein and ligand were rigid during the docking. The CM1P augmented OPLS-AA force field [Duffy, E. M.; Jorgensen, W. L. J. Am. Chem. Soc. 2000, 122, 2878-2888] provided the initial structure of Sustiva; it was also used to determine the nonbonded energies, which were stored on a spherical grid for efficiency. The total intermolecular interactions between the ligand and protein amount to a measure of both steric and electrostatic complimentarity; the lowest energy structure found during the simulations was taken as the “best” docked system. A distance-dependent dielectric constant of 4 (ǫ ) 4r) was used for all docking calculations. (10) Ren, J.; Esnouf, R.; Garman, E.; Somers, D.; Ross, C.; Kirby, I.; Keeling, J.; Darby, G.; Jones, Y.; Stuart, D.; et al. Nat. Struct. Biol. 1995, 2, 293-302. (11) Ren, J.; Esnouf, R.; Hopkins, A.; Ross, C.; Jones, Y.; Stammers, D.; Stuart, D. Structure 1995, 3, 915-26. (12) Rizzo, R. C.; Tirado-Rives, J.; Jorgensen, W. L. J. Med. Chem. 2000, 0000, 0000-0000. In press. Figure 1. Orientation of the four NNRTIs in the HIVRT binding site. (A) Best docked structure of Sustiva. (B) Nevirapine from pdb entry 1vrt. (C) MKC-442 from pdb entry 1rt1. (D) 9-Cl TIBO from pdb entry 1rev. 12898 J. Am. Chem. Soc. 2000, 122, 12898-12900 10.1021/ja003113r CCC: $19.00 © 2000 American Chemical Society Published on Web 12/06/2000