Proc. Natl. Acad. Sci. USAVol. 88, pp. 10287-10291, November 1991Biophysics

Relative differences in the binding free energies of humanimmunodeficiency virus 1 protease inhibitors: Athermodynamic cycle-perturbation approachM. 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-munodeficiency virus 1 protease show considerable promise fortreatment of AIDS. We have, therefore, been seeking comput-er-assisted drug design methods to aid in the systematic designof such inhibitors from a lead compound. Here we reportthermodynamic cycle-perturbation calculations (using molec-ular dynamics simulations) to compute the relative differencein free energy of binding that results when one entire residue(valine) is deleted from one such inhibitor. In particular, westudied the "alchemic" mutation of the inhibitor Ac-Ser-Leu-Asn-(Phe-Hea-Pro)-Ile-Val-OMe (S1) to Ac-Ser-Leu-Asn-(Phe-Hea-Pro)-Ile-OMe (S2), where Hea is hydroxyethylamine, intwo different (R and S) diastereomeric configurations of thehydroxyethylene group. The calculated (averaged for R and S)difference in binding free energy [3.3 + 1.1 kcal/mol (mean ±SD); 1 cal = 4.184 J] is in good agreement with the experi-mental value of 3.8 ± 1.3 kcal/mol, obtained from themeasured K; values for an equilibrium mixture of R and Sconfigurations. Precise testing of our predictions will be pos-sible when binding data become available for the two disaste-reomers separately. The observed binding preference for S1 isexplained by the stronger ligand-protein interaction, whichdominates an opposing contribution arising from the largedesolvation penalty of S1 relative to S2. This calculationsuggests that the thermodynamic cycle-perturbation approachcan be useful even when a relatively large change in the ligandis simulated and supports the use of the thermodynamiccycle-perturbation algorithm for screening proposed deriva-tives of a lead inhibitor/drug prior to their synthesis.

The human immunodeficiency virus 1 aspartic protease(HIV-1-PR) (1) is an important target for anti-AIDS drugdesign because it mediates a crucial step in the life cycle ofthe HIV-1 retrovirus, namely, the proteolytic processing ofpolyprotein precursors encoded by its gag and pol genes.Crystallographic structures for recombinant and syntheticHIV-1-PRs (2-4), some complexes of the HIV-1-PR (refs. 5and 6 and A. Wlodower, personal communication), andrelated proteases (7) are available. Since the enzymaticmechanism of the PRs requires a transient intermediate to beformed during hydrolysis of the peptide bond, a chemicallystable structure that mimics this tetrahedral intermediate canpotently inhibit the enzyme's action. This principle hasmotivated inhibitor design efforts in which a hydrolyzabledipeptide bond within an oligopeptide substrate is replacedwith a reduced amide, statine analog, hydroxyethylene isos-tere, or hydroxyethyl amine analog [refs. 8 and 9 and refer-ences therein]. Crystal structures oftwo complexes with such"designer" inhibitors have recently been determined (refs. 5and 6). For one of these complexes (ref. 6 and A. Wlodower,

personal communication) and its analogs, binding constantsare also available (8, 9). This presents us with an opportunityto perform free-energy simulations that might aid in system-atic design of peptide-based HIV-1-PR inhibitors.

Free-energy simulation techniques have been used toprobe a variety of chemical and biochemical factors includingsolvation and binding of ions and small molecules (10, 11),relative binding free-energy differences between similar in-hibitors (13, 14), antigen-antibody complex formation (15),and subunit association in oligomeric proteins (16). Althoughthe results of many of these simulations show remarkableconcordance with experimental measurements, insight intothe nature of the interactions has not come easily.The objective of the present work is to rationalize the

specificities and free energies of binding for peptide-basedinhibitors of the HIV-1-PR using a free-energy simulationmethod, the thermodynamic cycle-perturbation (TCP) ap-proach (17-19). Validation of the TCP approach and algo-rithms, particularly for computing large changes in the ligandstructure, is important to computer-assisted drug designbecause binding data spanning the desired range of chemicalstructures of interest is usually unavailable. In the presentwork, we used the TCP approach to simulate a large changein an inhibitor ligand, deletion of the hydrophobic residuevaline in the heptapeptide inhibitor Ac-Ser-Leu-Asn-(Phe-Hea-Pro)-Ile-Val-OMe (Si) to convert it to the hexapeptideAc-Ser-Leu-Asn-(Phe-Hea-Pro)-Ile-OMe (S2) (where Hea ishydroxyethylamine), using the TCP approach. Such a sys-tematic reduction of the peptide sequence by one amino acidresidue at a time is often necessary to determine the minimumsequence required for bioactivity (20).

Earlier, a model of the dynamical structure of the HIV-1-PR dimer was developed using "dynamical cross-correlation" maps (21). In the present free-energy simula-tion, we explore the origins of free-energy differences inbinding between two peptidomimetic inhibitors of the HIV-1-PR and make predictions of their relative binding affinitiesfor two possible diastereomeric configurations.

THEORYThe TCP approach (17-19, 22) provides a computationallytractable way to evaluate complex thermodynamic free en-ergies associated with solvation and binding of a ligand in theaqueous and enzyme-bound states. Fig. 1 shows the schemafor computing relative changes in free energy of binding byconstruction of a nonphysical path connecting the desiredinitial and terminal (mutated) states. For two substrates S1and S2, the relation between experimentally measured bind-

Abbreviations: HIV-1-PR, human immunodeficiency virus 1 asparticprotease; TCP, thermodynamic cycle-perturbation; Hea, hydroxy-ethylamine; MD, molecular dynamics.tTo whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 88 (1991)

S1(gas) -G4 S1(aq) + HlV1-PR(aq) AG1 S1:HIV1-PR(aq)

AGgas CYCLE 1 AGaq

S2s -AG3S2(gas) S2(aq) +HlVl-PR(aq)

CYCLE 2 AGcom

AG2P- S2:HIV1-PR(aq)

FIG. 1. Thermodynamic cycles used in this study. Each "reac-tion" shown in the two cycles is reversible, but the direction of thearrow indicates that the change in free energy is computed by takingthe difference between free energy of the state at the end ofthe arrowand that at the point.

ing constants (k1 and k2) and free energies (AG1, AG2, AGaqand AGcom) is

-kBT ln(k2/k1) = AG2 - AG1

= AGcom -AGaq = AAGbind, [1]

where kB is the Boltzmann constant and T is the absolutetemperature.The relative solvation free-energy change for two sub-

strates, computed from cycle 1 of Fig. 1, is

AG3 -AG4 = AGaq - AGgas = AAGso. [2]

The free-energy change for converting S1 into S2 is computedby transforming or "perturbing" the Hamiltonian of reactantstate S1 into that of product state S2. This transformation isaccomplished through a parametrization of the terms com-prising the interaction potentials of the system with a changeof state variable A that maps onto reactant and product stateswhen A is 0 and 1, respectively. The incremental free-energychange between any two successive windows is given by

G(A + AA) - G(A) = - kBT In<exp{-[H(A + AA)

- H(A)]/kB7T>A, [3]

where the ensemble average in angle brackets is computed asa time average over the trajectory of Hamiltonian H(A). AA iskept small enough to enhance convergence. The total free-energy change for the mutation from the initial state to thefinal state is computed by summing these incremental free-energy changes in each window encompassing A from 0 to 1,

AG = [G(Ai + AA) - G(A)]. [4]

COMPUTATIONAL DETAILS

Model and Parameters. The x-ray structure of the HIV-1-PR complex with Ac-Ser-Leu-Asn-(Phe-Hea-Pro)-Ile-Val-OMe (Si) was made available to us by A. Wlodower (per-sonal communication). This 2.8-A resolution structure of theHIV-1-PR-Si complex was used as the starting configura-tion. The protein, the inhibitor, and the solvent were modeledusing the AMBER (23, 24) all-atom force field, which takes allhydrogens explicitly into account. The hydrogens for theinhibitor S1, crystallographic water, and protein dimer wereadded using the EDIT module ofAMBER. Electrostatic chargesand parameters for the standard residues of the inhibitormodel were taken from the AMBER database. The total chargeon the HIV-1-PR dimer was +5 e. For nonstandard residuesin the inhibitor, electrostatic charges were fitted with CHELP(25) from ab initio 3-21G*//6-31G* wave functions calculatedwith GAUSSIAN88 (26). One of the aspartic acids in thecatalytic dyad (Asp-124) was protonated in all simulations (1).

All equilibrium bond lengths, bond angles, and dihedralangles for nonstandard residues were taken from ab initio(GAUSSIAN88) quantum mechanically optimized geometries.Missing force-field parameters were estimated from similarchemical species in the AMBER database. (These parametersand the charges on the inhibitor are available from the authorsupon request.) To describe the water interactions, we usedthe SPC/E rigid geometry model potential (27), which repro-duces bulk properties of water quite accurately (28).

Molecular Dynamics (MD) Calculations. All MD simula-tions were performed with the GIBBS module of the AMBERprogram (23, 24). Newton's equations ofmotion for all atomswere solved using the Verlet algorithm (29) for integrationand the SHAKE algorithm to constrain all bond lengths (30).Constant temperature (at T = 298 K) was maintained byvelocity scaling. The initial phase of equilibration consistedof 20 ps of MD simulation. All nonbonded interactionsinvolving the inhibitor were computed without any cutofflimit. However, to reduce the computation time, a 10.0-Anonbonded interaction residue-based cutoff was used forother interactions that do not directly involve the inhibitor.The nonbonded pair list was updated every 10 MD steps forthe solvent or 20 MD steps for the ligand-protein complexsimulations.Mutation of S1 to S2. Fig. 2 shows the atom conversions

involved in mutating S1 through an intermediate state S1* toS2. In all free-energy results reported here, the mutation wasaccomplished using the GIBBS module ofAMBER, in two steps:(i) during the first step of the transformation (S1 -* S1*) onlythe partial charges were mutated; (it) during the second (S1*-- S2), the van der Waal's parameters were mutated and, inaddition, bond stretching, bond angle changes, and torsionalchanges accompanying the mutation were simulated. Thetotal free-energy change in each step was computed bysumming these incremental free-energy changes in eachwindow between A = 0 and A = 1. In each step, a total of 51windows was used (AA = 0.02) for the complete mutation. Ateach A, free-energy changes were evaluated both in forward(A to A + AA) and backward (A to A - AA) directions, exceptwhen A = 0 or 1 (because, at these values of A, change ispossible only in one direction). Sequential decomposition ofelectrostatic and van der Waal's contributions to free-energychanges avoids certain sampling difficulties, as noted (14),and is entirely consistent with TCP theory. In all free-energysimulations, the system was initially equilibrated for 20 ps atA = 0. Then, in each window, the system was equilibrated for1 ps, and the data were collected for 2 ps. Thus, data collectedfor the complete mutation included both forward (A from 0 to1) and reverse (A from 1 to 0) simulations. A reverse simu-lation (A from 1 to 0) is, however, different from a reversemutation (S2 -+ S1).

Since deletion of a valine residue involves the loss of 5heavy atoms and 10 hydrogens, changes in the last windows

H(D) 0(D) H(D)I II 1-00N() C'(H) C(D)

(Cjc O(D) \ (D)

(D)H (H)CQ H(D)(D)Cy1 (D)CY2

(D)Ho | (D)(D)H H(D)

FIG. 2. Description of atom conversions used in simulating themutation of S1 to S2. The conversion at each atomic location isshown in the form A(B) where A belongs to S1 and B belongs to S2.D, dummy atom, an atom with no charge or radius; X, common partof both ligands (S1 and S2).

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Proc. Natl. Acad. Sci. USA 88 (1991) 10289

will be dramatic and usually lead to difficulties in the con-vergence of free energies. Hence, for the last five windows(between A values 0.90 and 1.0), we used a longer simulationtime (6 ps of equilibration and 8 ps of data collection for eachwindow) to enhance convergence of the free-energy changes.Thus, a total of361 ps was required for the complete mutationin each state.Free-Energy Simulations. For simulation of the inhibitor

mutation (S1 to S2) in solvent (in the absence of the protein),the solute (Si) was immersed in a large water bath con-structed from repeated cubes of SPC/E water molecules,which were a snapshot from an MD simulation ofliquid water(28). Any water molecules located less than 2.5 A from anysolute atom were removed. The aqueous-phase simulationswere performed in a rectangular box whose dimensions(40.38 A x 33.8 A x 29.36 A) allowed a 10.0-A layer of waterto surround the solute atoms. Initially, the system wasminimized using the steepest descent method (500 steps); thiswas followed by the conjugate gradient method (2000 steps).MD simulations were carried out using periodic boundaryconditions in all directions. The system was kept at a pressureof 1 atm (1 atm = 101.3 kPa).We have also performed a reverse mutation (S2 - SiP

Si) in solvent to compare the corresponding free-energydifferences in the two mutations (S1 to S2 and S2 to Si).Thus, a total of 722 (2 x 361) ps was required for the solventpart of the simulation. The gas-phase simulations were sim-ilar in detail to the solvent-phase simulation, except for theabsence of solvent. The reported solvation free energy is theaverage of four calculations: both forward and reverse sim-ulations for the two mutations-Si -- S2 and S2 -. S1.For the macromolecular simulations, protein residues be-

yond 25 A from the Ca of valine (mutated group) were frozen.Solvent (SPC/E water) was placed (using the SOL option ofAMBER) within a sphere of radius 25 A from the Ca of thevaline in S1. As in the solvent simulations, water moleculeslocated less than 2.5 A away from any of the solute atomswere removed. Half-harmonic restraint (with a force constantof 1.0 kcal/A; 1 cal = 4.184 J) was applied near the boundaryofthe solvent (26A away from the C. ofthe valine) to preventsolvent evaporation (13, 14). This restraining force is felt byall solvent atoms at the boundary and originates at the C. ofthe valine. Initially, only the solvent was minimized (freezingthe protein complex) for 500 cycles using the steepest descentmethod to relieve any bad contacts in the solvent; this was

followed by 2000 cycles ofconjugate gradient minimization ofthe entire complex (11, 14). Other specifics of the enzyme-inhibitor complex simulation were identical to those of thesolvent simulation.

RESULTS AND DISCUSSIONThe x-ray structure of the HIV-1-PR complex with Ac-Ser-Leu-Asn-(Phe-Hea-Pro)-Ile-Val-OMe (Si) (ref. 6 and A.Wlodower, personal communication) reports the S config-uration for the hydroxyethyl moiety in S1. We have, how-ever, simulated mutations for both possible configurations, Sand R, and calculated the free-energy differences. The latterconfiguration of the complex was generated from the crystalstructure of the original complex. These three-dimensionalstructures were the starting points of the TCP calculations.Binding constants for this and a few other complexes havebeen measured experimentally (8, 9). In the following sec-tions, we first discuss the simulations involving the S con-figuration ofthe inhibitor (hydroxyethylene moiety). We thencompare the results with those of the corresponding calcu-lations for the R configuration. Finally, we offer a rational-ization of kinetic experimental results and make predictionsin light of our computational analysis.

Structural Comparison. Initially, an energy minimization(500 steps of steepest descent followed by 2000 steps ofconjugate gradient optimization) of the HIV-i-PR-S1 com-plex was performed. This was followed by a 20-ps MDsimulation for equilibration. The average dynamical structureof the complex was computed from the MD simulation. Fortime steps of 1 fs and 2 fs in MD simulations, the rmsdeviations from the crystal structure were 1.03 A and 1.10 Afor backbone atoms and 1.55 A and 1.65 A for side-chainatoms, respectively. Fig. 3 shows the structural comparisonof the averaged dynamical structure from a 20-ps dynamicstrajectory (with a time step of 2 fs) with the x-ray structureof the dimer. Since both time steps (of 1 fs and 2 fs) yieldedgood agreement with the x-ray structure, in the interest ofsaving computer time, we used the larger time step of 2 fs forall free-energy calculations reported here.

Relative Solvation Free-Energy Change (AAG..a. Solvationfree-energy changes ofthe uncomplexed substrates S1 and S2were computed using MD in conjunction with the TCPapproach for the first cycle shown in Fig. 1. To complete thecycle 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).

Biophysics: Reddy et al.

Proc. Natl. Acad. Sci. USA 88 (1991)

one in the gas phase and the other in solvent water. Bothsimulations involved mutation of S1 to S2, corresponding tothe deletion ofa valine. The results are shown in Table 1. Onemay estimate the relative solvation free-energy change due tointermolecular interactions (i.e., interaction of each ligandwith the solvent) by assuming that the magnitudes of intra-molecular interaction for the two simulations (S1 to S2 in gasand in solvent) are approximately equal and separating theintermolecular and intramolecular interaction energy contri-butions in the solvent simulation. A value of 8.93 kcal/mol isobtained for this estimate.A more rigorous estimate of the same contribution is the

difference between total (intra + inter) free-energy changesin the two simulations (see Eq. 2); AGaq - AGgas). Theaqueous-phase free-energy change from the simulation is11.88 kcal/mol, and the figure for the gas-phase simulation is3.93 kcal/mol, resulting in a value of 7.95 kcal/mol for thechange in free energy. Wolfenden's (31) measured value offree-energy change corresponding to the transfer of a valineresidue (and the corresponding backbone atoms) from dilutevapor phase to liquid water is estimated as the sum of thetransfer free energy for that side chain (1.99 kcal/mol) plusthe backbone contribution (-10.0 kcal/mol), which is -8.01kcal/mol. From our calculation of the difference in free-energy change, the comparable value (AGgaS - AGaq = -7.95kcal/mol) indicates excellent agreement.

Enthalpic and entropic contributions to the relative differ-ences in free energy were calculated by using numericaltemperature derivatives (32). Consequently, the uncertain-ties (as reflected by the computed error bars) in the enthalpicand entropic contributions were larger than the correspond-ing uncertainties in the relative free-energy differences. Thetemperature step in the numerical differentiation was ±2K.Relative solvation and binding enthalpy, entropy, and totalfree-energy changes are listed in Table 1. All energy changesare reported as the energy (total free energy or a componentthereof) of the mutated ligand (S2) minus the correspondingenergy ofthe original (Si). Data from Table 1 indicate that therelative change in free energy is the result of opposingcontributions from enthalpic and entropic effects. The largerenthalpic contribution [AAH(sol) = 23.77 kcal/mol] is due tothe loss of electrostatic and van der Waal's interactions ofvaline with water. This dominates the gain in entropy[TAAS(sol) = 15.82 kcal/mol] stemming from the loss ofhydrogen bonds and the removal of a hydrophobic group(valine) from water. However, the increase in entropy can beascribed mostly to reversal of hydrophobic hydration uponloss of the valine residue from aqueous solution (33). The netloss of free energy upon deletion of valine in solvent is 7.95kcal/mol. Error bars were estimated for each window bydividing the window statistics into four groups and computingthe standard deviation. The rms values of the errors in eachof the windows are reported in Table 1 as a measure of thestatistical uncertainty in the result for each complete muta-tion. Though the solvation free-energy changes are the av-

erages from forward and reverse mutations (see Computa-

tional Details), no significant differences in any of the trendsreported here are seen between the two sets of results.

Relative Change in Binding Free Energy (AAGm). Therelative difference in binding free energy upon complexationwith the HIV-1-PR for the substrates S1 and S2 was com-

puted using the second cycle (cycle 2) shown in Fig. 1.Enthalpic and entropic changes are given in Table 1. Quali-tatively, the results are similar to those we obtained for thesolvation free-energy changes. The enthalpic contribution islarger than the opposing entropic contribution. Detailedanalyses of free energy and enthalpic components and mo-

lecular dynamics trajectories will be presented elsewhere.Some of the intermolecular interactions (in HIV-1-PR-S1

and HIV-1-PR-S2 complexes) are easily identified usingcomputer graphics and modeling tools. One of the hydrogensin the NH2 group of Arg-8 in the first monomer stronglyhydrogen-bonds to the carbonyl oxygen of the main chain atthe valine residue in S1. The second hydrogen also has goodelectrostatic interaction with that oxygen. In S1, the sidechain of valine has hydrophobic contacts with side chains ofIle-146, Phe-152, and Lys-144 (the hydrophobic part) inHIV-1-PR. These hydrophobic interactions would not bepresent in HIV-1-PR-S2 or in solvent simulations of S1 andS2. Thus, the loss of enthalpy in solvent is smaller than thecorresponding loss in the complex, due to the loss of stronginteraction of valine with the protein dimer.We also performed a second set of simulations on the R

configuration ofthe hydroxyethyl moiety, assuming the samebinding mode as in the crystal structure complex (S config-uration). Conditions for this second set of simulations wereidentical to those for the S configuration. The free-energychanges we calculated were quite similar to those we ob-tained with the S configuration. AAG,01 (7.5 ± 0.85 kcal/mol)is 0.45 kcal lower and AAGbind (3.55 ± 1.1) is 0.6 kcal/molhigher than the corresponding values of the S configurationof the complex. Both these changes are within the computederror bounds. This suggests that for an equilibrium mixture ofR and S (considering the error bounds for the differences infree energies), AAGbind can vary from 2 to 4.5 kcal/mol,depending on the dominant diastereomer.

Prediction of Relative Binding Affinity. Our calculatedpredictions of relative binding affinities S1 and S2 (R and Sdiastereomers) for HIV-1-PR, which indicate stronger bind-ing of S1 than of S2, are summarized in Table 2. The averagevalue of AAGbind (3.25 ± 1.06 kcal/mol) we obtained fromcalculations compares favorably with the "experimental"value of 3.8 ± 1.3 kcal/mol. Experimental measures areavailable separately for R and S diastereomer complexes ofS1 (9). When similar data become available for S2, ourpredictions will be tested more precisely. Present resultsshow that the observed binding preference of S1 relative toS2 (about 4 kcal/mol) stems from strong (hydrogen bondingand hydrophobic) interaction of the valine residue withHIV-1-PR. This strong interaction is not fully compensatedupon deletion of valine, because the deletion eliminates onlya part of the opposing contribution due to the larger desol-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 8.10HIV-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 onlytS1(aq) + S2(g) = S1(g) + S2(aq) 28.16 ± 7.60 19.23 ± 7.62 8.93 + 0.6 8.10HIV-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.

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Proc. Natl. Acad. Sci. USA 88 (1991) 10291

Table 2. Relative solvation and binding free-energy differences(in kcal/mol) for the S and R configurations of thehydroxyethylene moiety in S1

AAGbind AAGbindConfiguration(s) AAGsol (calc.) (expt.)

S 7.95 ± 0.85 2.95 ± 1.02R 7.50 ± 0.90 3.55 ± 1.10R + S 7.73 ± 0.88 3.25 ± 1.06* 3.8 ± 1.3t

Calc., calculated; expt., experimental.*Value assuming an equimolar mixture of S1 and S2.tExperimental value available is that for the equilibrium mixture ofR and S configurations, whose ratio is undetermined. Some exper-iments (9) indicate that the S configuration is dominant.

CONCLUSIONSThe present work offers an explanation for the strongerinhibition of the HIV-1-PR by Ac-Ser-Leu-Asn-(Phe-Hea-Pro)-Ile-Val-OMe (Si) relative to Ac-Ser-Leu-Asn-(Phe-Hea-Pro)-Ile-OMe (S2). The interaction energy (relativebinding enthalpy) of S1 with the dimer is stronger than thatof S2, and this contribution dominates an opposing contri-bution arising from the larger desolvation penalty of S1.These conclusions are valid for both possible diastereomers(R and S configurations of the hydroxyethylene moiety) andhence also for their equilibrium mixture. The computedrelative solvation free-energy change is consistent with theexperimental solvation free-energy change for transfer ofvaline from the gas phase to the aqueous phase. Thesecalculations suggest that the TCP approach in conjunctionwith MD simulations can be useful in simulating the effect ofa relatively large change in a substrate and they support theuse of this methodology for screening proposed derivatives ofa lead inhibitor/drug prior to their synthesis.

We thank the Advanced Scientific Computation Laboratory, Na-tional Cancer Institute for use of the CRAY/XMP supercomputerand for staff support. Dr. Alex Wlodower kindly provided thecoordinates of the HIV-1-PR complex used in the present work. Weare grateful to Mr. Richard Venable for help with molecular graphics.We also thank Drs. Dominic Zichi, Russel Bacquet, David Mat-thews, Michael Varney, Siegfried Reich, Krzysztof Appelt, andBiman Bagchi for helpful suggestions. M.R.R. thanks his colleaguesat Agouron Pharmaceuticals, Inc., particularly Mr. Peter Johnsonand Drs. David Henry and Robert Jackson for their support andencouragement of this work. We thank the referees and Dr. U. C.Singh for their useful comments. The work of J.N.W. and V.N.V.was supported in part by the National Institutes of Health IntramuralTargeted AIDS Antiviral Program.

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