Proc. Natl. Acad. Sci. USA Vol. 88, pp. 10287-10291, November 1991 Biophysics Relative differences in the binding free energies of human immunodeficiency virus 1 protease inhibitors: A thermodynamic cycle-perturbation approach M. RAMI REDDYt*, VELLARKAD N. VISWANADHANt§, AND JOHN N. WEINSTEIN§ tAgouron Pharmaceuticals, Inc., 3565 General Atomics Court, San Diego, CA 92121; and 1National Cancer Institute, Laboratory of Mathematical Biology, Building 10, Room 4B-56, National Institutes of Health, Bethesda, MD 20892 Communicated by Robert G. Parr, August 12, 1991

ABSTRACT Peptidomimetic inhibitors of the human im- personal communication) and its analogs, binding constants munodeficiency virus 1 protease show considerable promise for are also available (8, 9). This presents us with an opportunity treatment of AIDS. We have, therefore, been seeking comput- to perform free-energy simulations that might aid in system- er-assisted drug design methods to aid in the systematic design atic design of peptide-based HIV-1-PR inhibitors. of such inhibitors from a lead compound. Here we report Free-energy simulation techniques have been used to thermodynamic cycle-perturbation calculations (using molec- probe a variety ofchemical and biochemical factors including ular dynamics simulations) to compute the relative difference solvation and binding of ions and small molecules (10, 11), in free energy of binding that results when one entire residue relative binding free-energy differences between similar in- (valine) is deleted from one such inhibitor. In particular, we hibitors (13, 14), antigen-antibody complex formation (15), studied the "alchemic" mutation of the inhibitor Ac-Ser-Leu- and subunit association in oligomeric proteins (16). Although Asn-(Phe-Hea-Pro)-Ile-Val-OMe (S1) to Ac-Ser-Leu-Asn-(Phe- the results of many of these simulations show remarkable Hea-Pro)-Ile-OMe (S2), where Hea is hydroxyethylamine, in concordance with experimental measurements, insight into two different (R and S) diastereomeric configurations of the the nature of the interactions has not come easily. hydroxyethylene group. The calculated (averaged for R and S) The objective of the present work is to rationalize the difference in binding free energy [3.3 + 1.1 kcal/mol (mean ± specificities and free energies of binding for peptide-based SD); 1 cal = 4.184 J] is in good agreement with the experi- inhibitors of the HIV-1-PR using a free-energy simulation mental value of 3.8 ± 1.3 kcal/mol, obtained from the method, the thermodynamic cycle-perturbation (TCP) ap- measured K; values for an equilibrium mixture of R and S proach (17-19). Validation of the TCP approach and algo- configurations. Precise testing of our predictions will be pos- rithms, particularly for computing large changes in the ligand sible when binding data become available for the two disaste- structure, is important to computer-assisted drug design reomers separately. The observed binding preference for S1 is because binding data spanning the desired range of chemical explained by the stronger ligand-protein interaction, which structures of interest is usually unavailable. In the present dominates an opposing contribution arising from the large work, we used the TCP approach to simulate a large change desolvation penalty of S1 relative to S2. This calculation in an inhibitor ligand, deletion of the hydrophobic residue suggests that the thermodynamic cycle-perturbation approach valine in the heptapeptide inhibitor Ac-Ser-Leu-Asn-(Phe- can be useful even when a relatively large change in the ligand Hea-Pro)-Ile-Val-OMe (Si) to convert it to the hexapeptide is simulated and supports the use of the thermodynamic Ac-Ser-Leu-Asn-(Phe-Hea-Pro)-Ile-OMe (S2) (where Hea is cycle-perturbation algorithm for screening proposed deriva- hydroxyethylamine), using the TCP approach. Such a sys- tives of a lead inhibitor/drug prior to their synthesis. tematic reduction of the peptide sequence by one amino acid residue at a time is often necessary to determine the minimum The human immunodeficiency virus 1 aspartic protease sequence required for bioactivity (20). (HIV-1-PR) (1) is an important target for anti-AIDS drug Earlier, a model of the dynamical structure of the HIV- design because it mediates a crucial step in the life cycle of 1-PR dimer was developed using "dynamical cross- the HIV-1 retrovirus, namely, the proteolytic processing of correlation" maps (21). In the present free-energy simula- polyprotein precursors encoded by its gag and pol genes. tion, we explore the origins of free-energy differences in Crystallographic structures for recombinant and synthetic binding between two peptidomimetic inhibitors of the HIV- HIV-1-PRs (2-4), some complexes of the HIV-1-PR (refs. 5 1-PR and make predictions of their relative binding affinities and 6 and A. Wlodower, personal communication), and for two possible diastereomeric configurations. related proteases (7) are available. Since the enzymatic mechanism of the PRs requires a transient intermediate to be formed during hydrolysis of the peptide bond, a chemically THEORY stable structure that mimics this tetrahedral intermediate can The TCP approach (17-19, 22) provides a computationally potently inhibit the enzyme's action. This principle has tractable way to evaluate complex thermodynamic free en- motivated inhibitor design efforts in which a hydrolyzable ergies associated with solvation and binding of a ligand in the dipeptide bond within an oligopeptide substrate is replaced aqueous and enzyme-bound states. Fig. 1 shows the schema with a reduced amide, statine analog, hydroxyethylene isos- for computing relative changes in free energy of binding by tere, or hydroxyethyl amine analog [refs. 8 and 9 and refer- construction of a nonphysical path connecting the desired ences therein]. Crystal structures oftwo complexes with such initial and terminal (mutated) states. For two substrates S1 "designer" inhibitors have recently been determined (refs. 5 and S2, the relation between experimentally measured bind- and 6). For one of these complexes (ref. 6 and A. Wlodower, Abbreviations: HIV-1-PR, human immunodeficiency virus 1 aspartic The publication costs of this article were defrayed in part by page charge protease; TCP, thermodynamic cycle-perturbation; Hea, hydroxy- payment. This article must therefore be hereby marked "advertisement" ethylamine; MD, molecular dynamics. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

10287 Downloaded by guest on September 30, 2021 10288 Biophysics: Reddy et al. Proc. Natl. Acad. Sci. USA 88 (1991) All equilibrium bond lengths, bond angles, and dihedral -G4 S1(aq) + AG1 S1:HIV1-PR(aq) S1(gas) HlV1-PR(aq) angles for nonstandard residues were taken from ab initio (GAUSSIAN88) quantum mechanically optimized geometries. Missing force-field parameters were estimated from similar AGgas CYCLE 1 AGaq CYCLE 2 AGcom chemical species in the AMBER database. (These parameters and the charges on the inhibitor are available from the authors upon request.) To describe the water interactions, we used S2s -AG3 AG2 S2(gas) S2(aq) +HlVl-PR(aq) P- S2:HIV1-PR(aq) the SPC/E rigid geometry model potential (27), which repro- duces bulk properties of water quite accurately (28). FIG. 1. Thermodynamic cycles used in this study. Each "reac- Molecular Dynamics (MD) Calculations. All MD simula- tion" shown in the two cycles is reversible, but the direction of the tions were performed with the GIBBS module of the AMBER arrow indicates that the change in free energy is computed by taking program (23, 24). Newton's equations ofmotion for all atoms the difference between free energy ofthe state at the end ofthe arrow were solved using the Verlet algorithm (29) for integration and that at the point. and the SHAKE algorithm to constrain all bond lengths (30). ing constants (k1 and k2) and free energies (AG1, AG2, AGaq Constant temperature (at T = 298 K) was maintained by and AGcom) is velocity scaling. The initial phase of equilibration consisted of 20 ps of MD simulation. All nonbonded interactions -kBT ln(k2/k1) = AG2 - AG1 involving the inhibitor were computed without any cutoff limit. However, to reduce the computation time, a 10.0-A nonbonded interaction residue-based cutoff was used for = AGcom -AGaq = AAGbind, [1] other interactions that do not directly involve the inhibitor. The nonbonded pair list was updated every 10 MD steps for where kB is the Boltzmann constant and T is the absolute the solvent or 20 MD steps for the ligand-protein complex temperature. simulations. The relative solvation free-energy change for two sub- Mutation of S1 to S2. Fig. 2 shows the atom conversions strates, computed from cycle 1 of Fig. 1, is involved in mutating S1 through an intermediate state S1* to S2. In all free-energy results reported here, the mutation was AG3 -AG4 = AGaq - AGgas = [2] AAGso. accomplished using the GIBBS module ofAMBER, in two steps: The free-energy change for converting S1 into S2 is computed (i) during the first step ofthe transformation (S1 -* S1*) only by transforming or "perturbing" the Hamiltonian of reactant the partial charges were mutated; (it) during the second (S1* state S1 into that of product state S2. This transformation is -- S2), the van der Waal's parameters were mutated and, in a of the terms com- addition, bond stretching, bond angle changes, and torsional accomplished through parametrization were The prising the interaction potentials of the system with a change changes accompanying the mutation simulated. total free-energy change in each step was computed by of state variable A that maps onto reactant and product states summing these incremental free-energy changes in each when A is 0 and 1, respectively. The incremental free-energy window between A = 0 and A = 1. In each step, a total of 51 change between any two successive windows is given by windows was used (AA = 0.02) for the complete mutation. At each A, free-energy changes were evaluated both in forward G(A + AA) - G(A) = - kBT InA, [3] when A 0 or 1 (because, at these values of A, change possible only in one direction). Sequential decomposition of where the ensemble average in angle brackets is computed as electrostatic and van der Waal's contributions to free-energy a time average over the trajectory of Hamiltonian H(A). AA is changes avoids certain sampling difficulties, as noted (14), kept small enough to enhance convergence. The total free- and is entirely consistent with TCP theory. In all free-energy energy change for the mutation from the initial state to the simulations, the system was initially equilibrated for 20 ps at final state is computed by summing these incremental free- A = 0. Then, in each window, the system was equilibrated for energy changes in each window encompassing A from 0 to 1, 1 ps, and the data were collected for 2 ps. Thus, data collected for the complete mutation included both forward (A from 0 to AG = [G(Ai + AA) - G(A)]. [4] 1) and reverse (A from 1 to 0) simulations. A reverse simu- lation (A from 1 to 0) is, however, different from a reverse mutation (S2 -+ S1). COMPUTATIONAL DETAILS Since deletion of a valine residue involves the loss of 5 heavy atoms and 10 hydrogens, changes in the last windows Model and Parameters. The x-ray structure of the HIV- 1-PR complex with Ac-Ser-Leu-Asn-(Phe-Hea-Pro)-Ile-Val- H(D) 0(D) H(D) OMe (Si) was made available to us by A. Wlodower (per- I II 1-00 sonal communication). This 2.8-A resolution structure of the N() C'(H) C(D) HIV-1-PR-Si complex was used as the starting configura- tion. The protein, the inhibitor, and the solvent were modeled (Cjc O(D) \ (D) using the AMBER (23, 24) all-atom force field, which takes all hydrogens explicitly into account. The hydrogens for the (D)H (H)CQ H(D) inhibitor S1, crystallographic water, and protein dimer were added using the EDIT module ofAMBER. Electrostatic charges (D)Cy1 (D)CY2 and parameters for the standard residues of the inhibitor (D)Ho | (D) model were taken from the AMBER database. The total charge (D)H H(D) on the HIV-1-PR dimer was +5 e. For nonstandard residues FIG. 2. Description of atom conversions used in simulating the in the inhibitor, electrostatic charges were fitted with CHELP mutation of S1 to S2. The conversion at each atomic location is (25) from ab initio 3-21G*//6-31G* wave functions calculated shown in the form A(B) where A belongs to S1 and B belongs to S2. with GAUSSIAN88 (26). One of the aspartic acids in the D, dummy atom, an atom with no charge or radius; X, common part catalytic dyad (Asp-124) was protonated in all simulations (1). of both ligands (S1 and S2). Downloaded by guest on September 30, 2021 Biophysics: Reddy et al. Proc. Natl. Acad. Sci. USA 88 (1991) 10289 will be dramatic and usually lead to difficulties in the con- followed by 2000 cycles ofconjugate gradient minimization of vergence of free energies. Hence, for the last five windows the entire complex (11, 14). Other specifics of the enzyme- (between A values 0.90 and 1.0), we used a longer simulation inhibitor complex simulation were identical to those of the time (6 ps ofequilibration and 8 ps ofdata collection for each solvent simulation. window) to enhance convergence ofthe free-energy changes. Thus, a total of361 ps was required for the complete mutation AND in each state. RESULTS DISCUSSION Free-Energy Simulations. For simulation of the inhibitor The x-ray structure of the HIV-1-PR complex with Ac-Ser- mutation (S1 to S2) in solvent (in the absence ofthe protein), Leu-Asn-(Phe-Hea-Pro)-Ile-Val-OMe (Si) (ref. 6 and A. the solute (Si) was immersed in a large water bath con- Wlodower, personal communication) reports the S config- structed from repeated cubes of SPC/E water molecules, uration for the hydroxyethyl moiety in S1. We have, how- which were a snapshot from an MD simulation ofliquid water ever, simulated mutations for both possible configurations, S (28). Any water molecules located less than 2.5 A from any and R, and calculated the free-energy differences. The latter solute atom were removed. The aqueous-phase simulations configuration of the complex was generated from the crystal were performed in a rectangular box whose dimensions structure of the original complex. These three-dimensional (40.38 A x 33.8 A x 29.36 A) allowed a 10.0-A layer ofwater structures were the starting points of the TCP calculations. to surround the solute atoms. Initially, the system was Binding constants for this and a few other complexes have minimized using the steepest descent method (500 steps); this been measured experimentally (8, 9). In the following sec- was followed by the conjugate gradient method (2000 steps). tions, we first discuss the simulations involving the S con- MD simulations were carried out using periodic boundary figuration ofthe inhibitor (hydroxyethylene moiety). We then conditions in all directions. The system was kept at a pressure compare the results with those of the corresponding calcu- of 1 atm (1 atm = 101.3 kPa). lations for the R configuration. Finally, we offer a rational- We have also performed a reverse mutation (S2 - SiP ization of kinetic experimental results and make predictions Si) in solvent to compare the corresponding free-energy in light of our computational analysis. differences in the two mutations (S1 to S2 and S2 to Si). Structural Comparison. Initially, an energy minimization Thus, a total of 722 (2 x 361) ps was required for the solvent (500 steps of steepest descent followed by 2000 steps of part of the simulation. The gas-phase simulations were sim- conjugate gradient optimization) of the HIV-i-PR-S1 com- ilar in detail to the solvent-phase simulation, except for the plex was performed. This was followed by a 20-ps MD absence of solvent. The reported solvation free energy is the simulation for equilibration. The average dynamical structure average of four calculations: both forward and reverse sim- of the complex was computed from the MD simulation. For ulations for the two mutations-Si -- S2 and S2 -. S1. time steps of 1 fs and 2 fs in MD simulations, the rms For the macromolecular simulations, protein residues be- deviations from the crystal structure were 1.03 A and 1.10 A yond 25 A from the Ca ofvaline (mutated group) were frozen. for backbone atoms and 1.55 A and 1.65 A for side-chain Solvent (SPC/E water) was placed (using the SOL option of atoms, respectively. Fig. 3 shows the structural comparison AMBER) within a sphere of radius 25 A from the Ca of the of the averaged dynamical structure from a 20-ps dynamics valine in S1. As in the solvent simulations, water molecules trajectory (with a time step of 2 fs) with the x-ray structure located less than 2.5 A away from any of the solute atoms of the dimer. Since both time steps (of 1 fs and 2 fs) yielded were removed. Half-harmonic restraint (with aforce constant good agreement with the x-ray structure, in the interest of of 1.0 kcal/A; 1 cal = 4.184 J) was applied near the boundary saving computer time, we used the larger time step of 2 fs for ofthe solvent (26 A away from the C. ofthe valine) to prevent all free-energy calculations reported here. solvent evaporation (13, 14). This restraining force is felt by Relative Solvation Free-Energy Change (AAG..a. Solvation all solvent atoms at the boundary and originates at the C. of free-energy changes ofthe uncomplexed substrates S1 and S2 the valine. Initially, only the solvent was minimized (freezing were computed using MD in conjunction with the TCP the protein complex) for 500 cycles using the steepest descent approach for the first cycle shown in Fig. 1. To complete the method to relieve any bad contacts in the solvent; this was cycle in Fig. 1, we performed two independent simulations,

FIG. 3. Comparison of the dynamics-averaged structure of the HIV-1-PR-S1 complex (shown in green) with the original x-ray structure (shown in red). Downloaded by guest on September 30, 2021 10290 Biophysics: Reddy et al. Proc. Natl. Acad. Sci. USA 88 (1991)

one in the gas phase and the other in solvent water. Both tional Details), no significant differences in any of the trends simulations involved mutation of S1 to S2, corresponding to reported here are seen between the two sets of results. the deletion of a valine. The results are shown in Table 1. One Relative Change in Binding Free Energy (AAGm). The may estimate the relative solvation free-energy change due to relative difference in binding free energy upon complexation intermolecular interactions (i.e., interaction of each ligand with the HIV-1-PR for the substrates S1 and S2 was com- with the solvent) by assuming that the magnitudes of intra- puted using the second cycle (cycle 2) shown in Fig. 1. molecular interaction for the two simulations (S1 to S2 in gas Enthalpic and entropic changes are given in Table 1. Quali- and in solvent) are approximately equal and separating the tatively, the results are similar to those we obtained for the intermolecular and intramolecular interaction energy contri- solvation free-energy changes. The enthalpic contribution is butions in the solvent simulation. A value of 8.93 kcal/mol is larger than the opposing entropic contribution. Detailed obtained for this estimate. analyses of free energy and enthalpic components and mo- A more rigorous estimate of the same contribution is the lecular dynamics trajectories will be presented elsewhere. difference between total (intra + inter) free-energy changes Some of the intermolecular interactions (in HIV-1-PR-S1 in the two simulations (see Eq. 2); AGaq - AGgas). The and HIV-1-PR-S2 complexes) are easily identified using aqueous-phase free-energy change from the simulation is computer graphics and modeling tools. One ofthe hydrogens 11.88 kcal/mol, and the figure for the gas-phase simulation is in the NH2 group of Arg-8 in the first monomer strongly 3.93 kcal/mol, resulting in a value of 7.95 kcal/mol for the hydrogen-bonds to the carbonyl oxygen of the main chain at change in free energy. Wolfenden's (31) measured value of the valine residue in S1. The second hydrogen also has good free-energy change corresponding to the transfer of a valine electrostatic interaction with that oxygen. In S1, the side residue (and the corresponding backbone atoms) from dilute chain of valine has hydrophobic contacts with side chains of vapor phase to liquid water is estimated as the sum of the Ile-146, Phe-152, and Lys-144 (the hydrophobic part) in transfer free energy for that side chain (1.99 kcal/mol) plus HIV-1-PR. These hydrophobic interactions would not be the backbone contribution (-10.0 kcal/mol), which is -8.01 present in HIV-1-PR-S2 or in solvent simulations of S1 and kcal/mol. From our calculation of the difference in free- S2. Thus, the loss of enthalpy in solvent is smaller than the to the loss of strong energy change, the comparable value (AGgaS - AGaq = -7.95 corresponding loss in the complex, due kcal/mol) indicates excellent agreement. interaction of valine with the protein dimer. Enthalpic and entropic contributions to the relative differ- We also performed a second set of simulations on the R same ences in free energy were calculated by using numerical configuration ofthe hydroxyethyl moiety, assuming the temperature derivatives (32). Consequently, the uncertain- binding mode as in the crystal structure complex (S config- second set of simulations were ties (as reflected by the computed error bars) in the enthalpic uration). Conditions for this identical to those for the S configuration. The free-energy and entropic contributions were larger than the correspond- ing uncertainties in the relative free-energy differences. The changes we calculated were quite similar to those we ob- tained with the S configuration. (7.5 ± 0.85 kcal/mol) temperature step in the numerical differentiation was ±2K. AAG,01 is 0.45 kcal lower and AAGbind (3.55 ± 1.1) is 0.6 kcal/mol Relative solvation and binding enthalpy, entropy, and total higher than the corresponding values of the S configuration are listed in Table 1. All energy changes free-energy changes of the complex. Both these changes are within the computed are as energy free energy or a reported the (total component error bounds. This suggests that for an equilibrium mixture of thereof) of the mutated ligand (S2) minus the corresponding R and S (considering the error bounds for the differences in the energy ofthe original (Si). Data from Table 1 indicate that free energies), AAGbind can vary from 2 to 4.5 kcal/mol, relative change in free energy is the result of opposing depending on the dominant diastereomer. contributions from enthalpic and entropic effects. The larger Prediction of Relative Binding Affinity. Our calculated enthalpic contribution [AAH(sol) = 23.77 kcal/mol] is due to predictions of relative binding affinities S1 and S2 (R and S the loss of electrostatic and van der Waal's interactions of diastereomers) for HIV-1-PR, which indicate stronger bind- valine with water. This dominates the gain in entropy ing of S1 than of S2, are summarized in Table 2. The average = [TAAS(sol) 15.82 kcal/mol] stemming from the loss of value of AAGbind (3.25 ± 1.06 kcal/mol) we obtained from hydrogen bonds and the removal of a hydrophobic group calculations compares favorably with the "experimental" (valine) from water. However, the increase in entropy can be value of 3.8 ± 1.3 kcal/mol. Experimental measures are ascribed mostly to reversal of hydrophobic hydration upon available separately for R and S diastereomer complexes of loss ofthe valine residue from aqueous solution (33). The net S1 (9). When similar data become available for S2, our loss of free energy upon deletion of valine in solvent is 7.95 predictions will be tested more precisely. Present results kcal/mol. Error bars were estimated for each window by show that the observed binding preference of S1 relative to dividing the window statistics into four groups and computing S2 (about 4 kcal/mol) stems from strong (hydrogen bonding the standard deviation. The rms values of the errors in each and hydrophobic) interaction of the valine residue with of the windows are reported in Table 1 as a measure of the HIV-1-PR. This strong interaction is not fully compensated statistical uncertainty in the result for each complete muta- upon deletion of valine, because the deletion eliminates only tion. Though the solvation free-energy changes are the av- a part of the opposing contribution due to the larger desol- erages from forward and reverse mutations (see Computa- vation penalty of the bigger ligand (S1). Table 1. Relative enthalpy, entropy, and free-energy differences (in kcal/mol) for the S configuration of the inhibitor Change of state AAH TAAS AAG AMG (expt) Intra- and intermolecular interactions* S1(aq) + S2(g) =S1(g) + S2(aq) 23.77 + 8.4 15.82 ± 8.45 7.95 + 0.85 8.10 HIV-1-S1(aq+S2(aq) = Sl(aq) + HIV-1-S2(aq) 14.09 ± 8.7 11.14 ± 8.76 2.95 ± 1.02 3.82 ± 1.2 Intermolecular interactions onlyt S1(aq) + S2(g) = S1(g) + S2(aq) 28.16 ± 7.60 19.23 ± 7.62 8.93 + 0.6 8.10 HIV-1-S1(aq) + S2(aq) = S1(aq) + HIV-1-S2(aq) 23.37 ± 7.90 20.58 ± 7.93 2.79 + 0.7 3.82 ± 1.2 Expt., experimental; HIV-1, HIV-1-PR. *Sum of intramolecular interactions of the ligand and its intermolecular interactions with the protein and solvent. tOnly intermolecular interactions of the ligand with the protein and solvent. Downloaded by guest on September 30, 2021 Biophysics: Reddy et al. Proc. Natl. Acad. Sci. USA 88 (1991) 10291 Table 2. Relative solvation and binding free-energy differences 5. Erickson, J., Neidhart, D. J., VanDrie, J., Kempf, D., Wang, (in kcal/mol) for the S and R configurations of the X. C., Norbeck, D. W., Plattner, J. J., Rittenhouse, J. W., hydroxyethylene moiety in S1 Turon, M., Wideburg, N., Kohlbrenner, W. E., Simmer, R., Helfrich, R., Paul, D. A. & Knigge, M. (1990) Science 249, AAGbind AAGbind 527-533. Configuration(s) AAGsol (calc.) (expt.) 6. Miller, M., Swain, A. L., Saskolski, M., Sathyanarayana, S 7.95 ± 0.85 2.95 ± 1.02 B. K., Marshall, G. R., Rich, D. H., Kent, S. B. H. & Wlodower, A. (1990) in Retroviral Proteases: Control ofMat- R 7.50 ± 0.90 3.55 ± 1.10 uration & Morphogenesis, ed. Pearl, L. (MacMillan, New R + S 7.73 ± 0.88 3.25 ± 1.06* 3.8 ± 1.3t York), pp. 93-106. Calc., calculated; expt., experimental. 7. Davies, D. R. (1990) Annu. Rev. Biophys. Biophys. Chem. 19, *Value assuming an equimolar mixture of S1 and S2. 189-215. tExperimental value available is that for the equilibrium mixture of 8. Rich, D. H., Green, J., Toth, M. V., Marshall, G. R. & Kent, R and S configurations, whose ratio is undetermined. Some exper- S. B. H. (1990) J. Med. Chem. 33, 1288-1295. iments indicate that the S is dominant. 9. Rich, D. H., Sun, C.-Q., Vara Prasad, J. V. N., Pathiasseril, (9) configuration A., Toth, M. V., Marshall, G. R., Clare, M., Mueller, R. A. & Houseman, K. (1991) J. Med. Chem. 34, 1222-1225. CONCLUSIONS 10. Jorgenson, W. L. & Ravimohan, C. (1985) J. Chem. Phys. 83, The present work offers an explanation for the stronger 3050-3054. 11. Rao, B. G. & Singh, U. C. (1989) J. Am. Chem. Soc. 111, inhibition of the HIV-1-PR by Ac-Ser-Leu-Asn-(Phe-Hea- 3125-3133. Pro)-Ile-Val-OMe (Si) relative to Ac-Ser-Leu-Asn-(Phe- 12. Wong, C. F. & McCammon, J. A. (1986) J. Am. Chem. Soc. Hea-Pro)-Ile-OMe (S2). The interaction energy (relative 108, 3830-3832. binding enthalpy) of S1 with the dimer is stronger than that 13. Fleishman, S. H. & Brooks, C. A., III (1990) Proteins 7, 52-61. of S2, and this contribution dominates an opposing contri- 14. Singh, U. C. & Benkovic, S. J. (1988) Proc. Natl. Acad. Sci. bution arising from the larger desolvation USA 85, 9519-9523. penalty of S1. 15. Novotny, J., Bruccoleri, R. E. & Saul, F. A. (1989) Biochem- These conclusions are valid for both possible diastereomers istry 28, 4735-4749. (R and S configurations of the hydroxyethylene moiety) and 16. Gao, J., Kuczera, K., Tidor, B. & Karplus, M. (1989) Science hence also for their equilibrium mixture. The computed 244, 1069-1072. relative solvation free-energy change is consistent with the 17. Van Gunsteren, W. F. & Weiner, P. K., eds. (1989) Computer experimental solvation free-energy change for transfer of Simulation of Biomolecular Systems (ESCOM Science, Lei- valine den, The Netherlands). from the gas phase to the aqueous phase. These 18. Hermans, J., ed. (1985) Molecular Dynamics & Protein Struc- calculations suggest that the TCP approach in conjunction ture (Polycrystal, West Springs, IL). with MD simulations can be useful in simulating the effect of 19. Beveridge, D. L. & DiCapua, D. L. (1989) Annu. Rev. Biophys. a relatively large change in a substrate and they support the Biophys. Chem. 18, 431-492. use ofthis methodology for screening proposed derivatives of 20. Hruby, V. J., Al-Obeidi, F. & Kazmierski, W. (1990) Biochem. a lead inhibitor/drug prior to their synthesis. J. 268, 249-262. 21. Harte, W. E., Jr., Swaminathan, S., Mansuri, M. M., Martin, We thank the Advanced Scientific Computation Laboratory, Na- J. C., Rosenberg, I. E. & Beveridge, D. L. (1990) Proc. Natl. tional Cancer Institute for use of the CRAY/XMP supercomputer Acad. Sci. USA 87, 8864-8868. and for staff 22. Zwanzig, R. W. (1954) J. Chem. Phys. 22, 1420-1426. support. Dr. Alex Wlodower kindly provided the 23. Weiner, S. J., Kollman, P. A., Case, D. A., Singh, U. C., coordinates of the HIV-1-PR complex used in the present work. We Ghio, C., Alagona, G., Profeta, S. & Weiner, P. K. (1984) J. are grateful to Mr. Richard Venable for help with molecular graphics. Am. Chem. Soc. 106, 765-784. We also thank Drs. Dominic Zichi, Russel Bacquet, David Mat- 24. Singh, U. C., Weiner, P. K., Caldwell, J. K. & Kollman, P. K. thews, Michael Varney, Siegfried Reich, Krzysztof Appelt, and (1986) AMBER (Univ. ofCalifornia, San Francisco), Version 3.0. Biman Bagchi for helpful suggestions. M.R.R. thanks his colleagues 25. Chirlian, L. E. & Francl, M. M. (1987) J. Comput. Chem. 8, at Agouron Pharmaceuticals, Inc., particularly Mr. Peter Johnson 894-905. and Drs. David Henry and Robert Jackson for their support and 26. Frisch, M. J. Head-Gordon, M., Trucks, G. W., Foresman, encouragement of this work. We thank the referees and Dr. U. C. H. B., Schlegel, H. B., Raghavachari, K., Robb, M. A., Bink- Singh for their useful comments. 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