Drug-resistant HIV-1 Protease Research Articles

Evolving Mutational Buildup in HIV-1 Protease Shifts Conformational Dynamics to Gain Drug Resistance.

Drug resistance in antiviral treatments is a serious public health problem. Viral proteins mutate very fast, giving them a way to escape drugs by lowering drug binding affinity but with compromised function. Human immunodeficiency virus type I (HIV-1) protease, a critical antiretroviral therapeutic target, represents a model for such viral regulation under inhibition. Drug inhibitors of HIV-1 protease lose effectiveness as the protein evolves through several variants to become more resistant. However, the detailed mechanism of drug resistance in HIV-1 protease is still unclear. Here, we test the hypothesis that mutations throughout the protease alter the protein conformational ensemble to weaken protein-inhibitor binding, resulting in an inefficient protease but still viable virus. Comparing conformational ensembles between variants and the wild type helps detect these function-related dynamical changes. All analyses of over 30 μs simulations converge to the conclusion that conformational dynamics of more drug-resistant variants are more different from that of the wild type. Distinct roles of mutations during viral evolution are discussed, including a mutation predominantly contributing to the increase of drug resistance and a mutation that is responsible (synergistically) for restoring catalytic efficiency. Drug resistance is mainly due to altered flap dynamics that hinder the access to the active site. The mutant variant showing the highest drug resistance has the most ″collapsed″ active-site pocket and hence the largest magnitude of hindrance of drug binding. An enhanced difference contact network community analysis is applied to understand allosteric communications. The method summarizes multiple conformational ensembles in one community network and can be used in future studies to detect function-related dynamics in proteins.

Design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors containing pyrrolidine-derived P2 ligands to combat drug-resistant variant

The design, synthesis, and biological evaluation of a novel series of HIV-1 protease inhibitors containing pyrrolidines with diverse linkers as the P2 ligands and various aromatic derivatives as the P2’ ligands were described. A number of inhibitors demonstrated potent efficacy in both enzyme and cellular assays, as well as relatively low cytotoxicity. In particular, inhibitor 34b with a (R)-pyrrolidine-3-carboxamide P2 ligand and a 4-hydroxyphenyl P2’ ligand displayed exceptional enzyme inhibitory activity with an IC50 value of 0.32 nM. Furthermore, 34b also exhibited robust antiviral activity against both wild-type HIV-1 and drug-resistant variant with low micromolar EC50 values. In addition, the molecular modelling studies revealed the extensive interactions between inhibitor 34b and the backbone residues of both wild-type and drug-resistant HIV-1 protease. These results suggested the feasibility of utilizing pyrrolidine derivatives as the P2 ligands and provided valuable information for further design and optimization of highly potent HIV-1 protease inhibitors.

HIV-1 Protease as DNA Immunogen against Drug Resistance in HIV-1 Infection: DNA Immunization with Drug Resistant HIV-1 Protease Protects Mice from Challenge with Protease-Expressing Cells

DNA immunization with HIV-1 protease (PR) is advanced for immunotherapy of HIV-1 infection to reduce the number of infected cells producing drug-resistant virus. A consensus PR of the HIV-1 FSU_A strain was designed, expression-optimized, inactivated (D25N), and supplemented with drug resistance (DR) mutations M46I, I54V, and V82A common for FSU_A. PR variants with D25N/M46I/I54V (PR_Ai2mut) and with D25N/M46I/I54V/V82A (PR_Ai3mut) were cloned into the DNA vaccine vector pVAX1, and PR_Ai3mut, into a lentiviral vector for the transduction of murine mammary adenocarcinoma cells expressing luciferase 4T1luc2. BALB/c mice were DNA-immunized by intradermal injections of PR_Ai, PR_Ai2mut, PR_Ai3mut, vector pVAX1, or PBS with electroporation. All PR variants induced specific CD8+ T-cell responses revealed after splenocyte stimulation with PR-derived peptides. Splenocytes of mice DNA-immunized with PR_Ai and PR_Ai2mut were not activated by peptides carrying V82A, whereas splenocytes of PR_Ai3mut-immunized mice recognized both peptides with and without V82A mutation. Mutations M46I and I54V were immunologically silent. In the challenge study, DNA immunization with PR_Ai3mut protected mice from the outgrowth of subcutaneously implanted adenocarcinoma 4T1luc2 cells expressing PR_Ai3mut; a tumor was formed only in 1/10 implantation sites and no metastases were detected. Immunizations with other PR variants were not protective; all mice formed tumors and multiple metastasis in the lungs, liver, and spleen. CD8+ cells of PR_Ai3mut DNA-immunized mice exhibited strong IFN-γ/IL-2 responses against PR peptides, while the splenocytes of mice in other groups were nonresponsive. Thus, immunization with a DNA plasmid encoding inactive HIV-1 protease with DR mutations suppressed the growth and metastatic activity of tumor cells expressing PR identical to the one encoded by the immunogen. This demonstrates the capacity of T-cell response induced by DNA immunization to recognize single DR mutations, and supports the concept of the development of immunotherapies against drug resistance in HIV-1 infection. It also suggests that HIV-1-infected patients developing drug resistance may have a reduced natural immune response against DR HIV-1 mutations causing an immune escape.

Drug Resistance Mechanism of M46I-Mutation-Induced Saquinavir Resistance in HIV-1 Protease Using Molecular Dynamics Simulation and Binding Energy Calculation.

Drug-resistance-associated mutation in essential proteins of the viral life cycle is a major concern in anti-retroviral therapy. M46I, a non-active site mutation in HIV-1 protease has been clinically associated with saquinavir resistance in HIV patients. A 100 ns molecular dynamics (MD) simulation and MM-PBSA calculations were performed to study the molecular mechanism of M46I-mutation-based saquinavir resistance. In order to acquire deeper insight into the drug-resistance mechanism, the flap curling, closed/semi-open/open conformations, and active site compactness were studied. The M46I mutation significantly affects the energetics and conformational stability of HIV-1 protease in terms of RMSD, RMSF, Rg, SASA, and hydrogen formation potential. This mutation significantly decreased van der Waals interaction and binding free energy (∆G) in the M46I–saquinavir complex and induced inward flap curling and a wider opening of the flaps for most of the MD simulation period. The predominant open conformation was reduced, but inward flap curling/active site compactness was increased in the presence of saquinavir in M46I HIV-1 protease. In conclusion, the M46I mutation induced structural dynamics changes that weaken the protease grip on saquinavir without distorting the active site of the protein. The produced information may be utilized for the discovery of inhibitor(s) against drug-resistant HIV-1 protease.

Molecular basis for reduced cleavage activity and drug resistance in D30N HIV-1 protease

Nelfinavir is one of the FDA-approved HIV-1 protease inhibitors and a part of highly active anti-retroviral therapy (HAART) for the treatment of HIV-AIDS. Nelfinavir was the first HIV-1 protease inhibitor to be approved as a paediatric formulation. The application of HAART had resulted in significant improvement in the lives of AIDS patients. However, the emergence of drug resistance in HIV-1 protease has limited the use of many of these drugs including nelfinavir. A unique mutation observed frequently in patients treated with nelfinavir is D30N as it is selected exclusively by nelfinavir. The D30N mutation imparts very high resistance to nelfinavir but unlike other primary mutations does not give cross-resistance to the majority of other drugs. D30N mutation also significantly reduces cleavage activity of HIV-1 protease and affects viral fitness. Here, we have determined crystal structures of D30N HIV-1 protease in unliganded form and in complex with nelfinavir. These structures provide the rationale for reduced cleavage activity and the molecular basis of drug resistance induced by D30N mutation. The loss of coulombic interaction part of a crucial hydrogen bond between the drug and the protease is likely to play a major role in reduced affinity and resistance towards nelfinavir. The decreased catalytic activity of D30N HIV-1 protease due to altered interaction with the substrates and reduced stability of folding core may be the reason for the reduced replicative capacity of the virus harboring mutant HIV-1 protease. Communicated by Ramaswamy H. Sarma

Characterizing Protein-Ligand Binding Using Atomistic Simulation and Machine Learning: Application to Drug Resistance in HIV-1 Protease.

Over the past several decades, atomistic simulations of biomolecules, whether carried out using molecular dynamics or Monte Carlo techniques, have provided detailed insights into their function. Comparing the results of such simulations for a few closely related systems has guided our understanding of the mechanisms by which changes such as ligand binding or mutation can alter the function. The general problem of detecting and interpreting such mechanisms from simulations of many related systems, however, remains a challenge. This problem is addressed here by applying supervised and unsupervised machine learning techniques to a variety of thermodynamic observables extracted from molecular dynamics simulations of different systems. As an important test case, these methods are applied to understand the evasion by human immunodeficiency virus type-1 (HIV-1) protease of darunavir, a potent inhibitor to which resistance can develop via the simultaneous mutation of multiple amino acids. Complex mutational patterns have been observed among resistant strains, presenting a challenge to developing a mechanistic picture of resistance in the protease. In order to dissect these patterns and gain mechanistic insight into the role of specific mutations, molecular dynamics simulations were carried out on a collection of HIV-1 protease variants, chosen to include highly resistant strains and susceptible controls, in complex with darunavir. Using a machine learning approach that takes advantage of the hierarchical nature in the relationships among the sequence, structure, and function, an integrative analysis of these trajectories reveals key details of the resistance mechanism, including changes in the protein structure, hydrogen bonding, and protein-ligand contacts.

Mechanism of inhibition of drug-resistant HIV-1 protease clinical isolates by TMC310911: A molecular dynamics study

HIV-1 protease continues to be a major target for therapy against AIDS. Although there are ten FDA approved drugs available, long term use of these drugs elicit drug resistant mutations leading to major challenges in therapy. Recently there have been reports of a new inhibitor, TMC310911 (TMC) that has shown significant activity against a wide spectrum of HIV clinical isolates that are resistant to even darunavir (DRV), the best HIV-1 protease drug so far. However the mechanism of action of TMC is unknown. In this work, we have employed all-atom molecular dynamics simulation to understand how TMC can be a potential drug candidate against mulidrug-resistant protease variants. Our results suggest that TMC has a dual mode of action. It acts as a conventional peptidomimetic inhibitor as well as a dimerisation inhibitor. It can bind to the active site cavity of dimeric protease in an extended conformation similar to the available crystal structure pose. In parallel, it can also bind to the monomeric protease and block the dimerisation interface of variant monomers. The detailed mechanism of action of TMC and the underlined mode of interaction could pave way for designing other potential HIV-1 protease inhibitors.

NMR and MD studies combined to elucidate inhibitor and water interactions of HIV-1 protease and their modulations with resistance mutations.

Over the last two decades, both the sensitivity of NMR and the time scale of molecular dynamics (MD) simulation have increased tremendously and have advanced the field of protein dynamics. HIV-1 protease has been extensively studied using these two methods, and has presented a framework for cross-evaluation of structural ensembles and internal dynamics by integrating the two methods. Here, we review studies from our laboratories over the last several years, to understand the mechanistic basis of protease drug-resistance mutations and inhibitor responses, using NMR and crystal structure-based parallel MD simulations. Our studies demonstrate that NMR relaxation experiments, together with crystal structures and MD simulations, significantly contributed to the current understanding of structural/dynamic changes due to HIV-1 protease drug resistance mutations.

Highly Drug-Resistant HIV-1 Protease Mutant PRS17 Shows Enhanced Binding to Substrate Analogues.

We report the structural analysis of highly drug-resistant human immunodeficiency virus protease (PR) variant PRS17, rationally selected by machine learning, in complex with substrate analogues. Crystal structures were solved of inhibitor-free inactive PRS17-D25N, wild-type PR/CA-p2 complex, and PRS17 in complex with substrate analogues, CA-p2 and p2-NC. Peptide analogues p2-NC and CA-p2 exhibit inhibition constants of 514 and 22 nM, respectively, for PRS17 or approximately 3-fold better than for PR. CA-p2 is a better inhibitor of PRS17 than are clinical inhibitors (Ki = 50–8390 nM) except for amprenavir (Ki = 11 nM). G48V resistance mutation induces curled flap tips in PRS17-D25N structure. The inner P2–P2′ residues of substrate analogues in PRS17 complexes maintain similar conformations to those of wild-type complex, while significant conformational changes are observed in the peripheral residues P3, P4′ of CA-p2 and P3, P4, and P3′ of p2-NC. The loss of β-branched side chain by V82S mutation initiates a shift in 80’s loop and reshapes the S3/S3′ subsite, which enhances substrate binding with new hydrogen bonds and van der Waals interactions that are absent in the wild-type structures. The steric hindrance caused by G48V mutation in the flap of PRS17 contributes to altered binding interactions of P3 Arg, P4′ norleucine of CA-p2, and P4 and P3′ of p2-NC with the addition of new hydrogen bonds and van der Waals contacts. The enhanced interaction of PRS17 with substrate analogues agrees with their relative inhibition, suggesting that this mutant improves substrate binding while decreasing affinity for clinical inhibitors.

Inference of Epistatic Effects Leading to Entrenchment and Drug Resistance in HIV-1 Protease.

Understanding the complex mutation patterns that give rise to drug resistant viral strains provides a foundation for developing more effective treatment strategies for HIV/AIDS. Multiple sequence alignments of drug-experienced HIV-1 protease sequences contain networks of many pair correlations which can be used to build a (Potts) Hamiltonian model of these mutation patterns. Using this Hamiltonian model, we translate HIV-1 protease sequence covariation data into quantitative predictions for the probability of observing specific mutation patterns which are in agreement with the observed sequence statistics. We find that the statistical energies of the Potts model are correlated with the fitness of individual proteins containing therapy-associated mutations as estimated by in vitro measurements of protein stability and viral infectivity. We show that the penalty for acquiring primary resistance mutations depends on the epistatic interactions with the sequence background. Primary mutations which lead to drug resistance can become highly advantageous (or entrenched) by the complex mutation patterns which arise in response to drug therapy despite being destabilizing in the wildtype background. Anticipating epistatic effects is important for the design of future protease inhibitor therapies.

Room Temperature Neutron Crystallography of Drug Resistant HIV-1 Protease Uncovers Limitations of X-ray Structural Analysis at 100 K.

HIV-1 protease inhibitors are crucial for treatment of HIV-1/AIDS, but their effectiveness is thwarted by rapid emergence of drug resistance. To better understand binding of clinical inhibitors to resistant HIV-1 protease, we used room-temperature joint X-ray/neutron (XN) crystallography to obtain an atomic-resolution structure of the protease triple mutant (V32I/I47V/V82I) in complex with amprenavir. The XN structure reveals a D+ ion located midway between the inner Oδ1 oxygen atoms of the catalytic aspartic acid residues. Comparison of the current XN structure with our previous XN structure of the wild-type HIV-1 protease-amprenavir complex suggests that the three mutations do not significantly alter the drug-enzyme interactions. This is in contrast to the observations in previous 100 K X-ray structures of these complexes that indicated loss of interactions by the drug with the triple mutant protease. These findings, thus, uncover limitations of structural analysis of drug binding using X-ray structures obtained at 100 K.

187 Drug resistance: crystallography of drug-resistant HIV-1 protease mutant

187 Drug resistance: crystallography of drug-resistant HIV-1 protease mutant

Genetic Changes in HIV-1 Gag-Protease Associated with Protease Inhibitor-Based Therapy Failure in Pediatric Patients.

Studies have shown a low frequency of HIV-1 protease drug resistance mutations in patients failing protease inhibitor (PI)-based therapy. Recent studies have identified mutations in Gag as an alternate pathway for PI drug resistance in subtype B viruses. We therefore genotyped the Gag and protease genes from 20 HIV-1 subtype C-infected pediatric patients failing a PI-based regimen. Major protease resistance mutations (M46I, I54V, and V82A) were identified in eight (40%) patients, as well as Gag cleavage site (CS) mutations (at codons 373, 374, 378, 428, 431, 449, 451, and 453) in nine (45%) patients. Four of these Gag CS mutations occurred in the absence of major protease mutations at PI failure. In addition, amino acid changes were noted at Gag non-CS with some predicted to be under HLA/KIR immune-mediated pressure and/or drug selection pressure. Changes in Gag during PI failure therefore warrant further investigation of the Gag gene and its role in PI failure in HIV-1 subtype C infection.

Structural Basis of Why Nelfinavir-Resistant D30N Mutant of HIV-1 Protease Remains Susceptible to Saquinavir.

Although anti-HIV-1 protease drugs nelfinavir (NFV) and saquinavir (SQV) share common functional groups, D30N is a major resistance mutation against NFV but remains susceptible to SQV. We have determined the crystal structure of D30N mutant-tethered HIV-1 protease in complex with SQV to 1.79 Å resolution. Structural analysis showed that SQV forms two direct hydrogen bonds with the main chain atoms of the residues Asp29 and Asp30 that are not observed in the D30N-NFV complex. Apart from maintaining these two main chain hydrogen bonds, the P2-asparagine of SQV forms an additional hydrogen bond to the mutated side chain of the residue 30. These could be the reasons why D30N is not a drug resistance mutation against SQV. This structure supports the previous studies showing that the interactions between a potential inhibitor and backbone atoms of the enzyme are important to maintain potency against drug-resistant HIV-1 protease.

Combatting Drug Resistance: Lessons from the viral proteases of HIV and HCV

Drug resistance negatively impacts the lives of millions of patients and costs our society billions of dollars by limiting the longevity of many of our most potent drugs. Drug resistance can be caused by a change in the balance of molecular recognition events that selectively weakens inhibitor binding but maintains the biological function of the target. To reduce the likelihood of drug resistance, a detailed understanding of the target's function is necessary. Both structure at atomic resolution and evolutionarily constraints on its variation is required. "Resilient" targets are less susceptible to drug resistance due to their key location in a particular pathway. This rationale was derived through crystallographic studies elucidating substrate recognition and drug resistance in HIV-1 protease and Hepatitis C (HCV) NS3/4A protease. Both are key therapeutic targets and are potentially "resilient" targets where resistant mutations occur outside of the substrate binding site. To reduce the probability of drug resistance inhibitors should be designed to fit within what we define as the "substrate envelope". These principals are likely more generally applicable to other quickly evolving diseases where drug resistance is quickly evolving. http://www.umassmed.edu/schifferlab/index.aspx

Drug Resistance Mutations Alter Dynamics of Inhibitor-Bound HIV-1 Protease.

Under the selective pressure of therapy, HIV-1 protease mutants resistant to inhibitors evolve to confer drug resistance. Such mutations can impact both the dynamics and structures of the bound and unbound forms of the enzyme. Flap+ is a multidrug-resistant variant of HIV-1 protease with a combination of primary and secondary resistance mutations (L10I, G48V, I54V, V82A) and a strikingly altered thermodynamic profile for darunavir (DRV) binding relative to the wild-type protease. We elucidated the impact of these mutations on protein dynamics in the DRV-bound state using molecular dynamics simulations and NMR relaxation experiments. Both methods concur in that the conformational ensemble and dynamics of protease are impacted by the drug resistance mutations in Flap+ variant. Surprisingly this change in ensemble dynamics is different from that observed in the unliganded form of the same variant (Cai, Y. et al. J. Chem. Theory Comput.2012, 8, 3452–3462). Our comparative analysis of both inhibitor-free and bound states presents a comprehensive picture of the altered dynamics in drug-resistant mutant HIV-1 protease and underlies the importance of incorporating dynamic analysis of the whole system, including the unliganded state, into revealing drug resistance mechanisms.

HIV-1 drug resistance-associated mutations among HIV-1 infected drug-naïve antenatal clinic attendees in rural Kenya

BackgroundAccess to antiretroviral therapy (ART) has increased dramatically in Sub-Saharan Africa. In Kenya, 560,000 people had access to ART by the end of 2011. This scaling up of ART has raised challenges to the Kenyan health system due to emergence of drug resistant viruses among those on treatment and possible onward transmission. To counter this, and come up with an effective treatment strategy, it has become vital to determine baseline mutations associated with drug resistance among the circulating strains of HIV-1in Kenya.MethodsThe prevalence of mutations associated with drug resistance in HIV-1 protease (PR) and reverse transcriptase (RT) regions from 188 HIV-1 infected treatment-naïve pregnant women was investigated in Kapsabet, Nandi Hills and Kitale district hospitals of Kenya. Blood samples were collected between April 2005 and June 2006. The HIV-1 pol gene was amplified using primers for HIV-1 PR and RT and sequenced using the BigDye chemistry. The mutations were analyzed based on the IAS algorithm as well as the Stanford University HIV Drug Resistance Database.ResultsBased on the PR and RT sequences, HIV-1 subtypes A1 (n=117, 62.2%), A2 (n=2, 1.1%), D (n=27, 14.4%), C (n=13, 6.9%), G (n=3, 1.6%), and possible recombinants (n=26, 13.8%) were detected. Mutations associated with nucleoside reverse transcriptase inhibitors (NRTI) and non-nucleoside RTI (NNRTI)-resistance were detected in 1.6% (3 of 188) and 1.1% (2 of 188), respectively. Mutations associated with PI resistance were detected in 0.5% (1 of 188) of the study population.ConclusionThe prevalence of drug resistance among drug-naïve pregnant women in rural North Rift, Kenya in 2006 was 3.2%. Major drug resistance mutations associated with PIs, NRTIs and NNRTIs do exist among treatment-naïve pregnant women in North Rift, Kenya. There is a need for consistent follow-up of drug-naïve individuals in this region to determine the impact of mutations on treatment outcomes.

Enhanced Stability of Monomer Fold Correlates with Extreme Drug Resistance of HIV-1 Protease

During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20(T26A) and dimeric PR20 exhibit Tm values 6-7.5 °C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PR(T26A) by ~8 °C, similar to PR20(T26A). However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR(D25N) in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ΔTm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.

Elucidating a Relationship between Conformational Sampling and Drug Resistance in HIV-1 Protease

Enzyme targets in rapidly replicating systems, such as retroviruses, commonly respond to drug-selective pressure with mutations arising in the active site pocket that limit inhibitor effectiveness by introducing steric hindrance or by eliminating essential molecular interactions. However, these primary mutations are disposed to compromising pathogenic fitness. Emerging secondary mutations, which are often found outside of the binding cavity, may or can restore fitness while maintaining drug resistance. The accumulated drug pressure selected mutations could have an indirect effect in the development of resistance, such as altering protein flexibility or the dynamics of protein-ligand interactions. Here, we show that accumulation of mutations in a drug-resistant HIV-1 protease (HIV-1 PR) variant, D30N/M36I/A71V, changes the fractional occupancy of the equilibrium conformational sampling ensemble. Correlations are made among populations of the conformational states, namely, closed-like, semiopen, and open-like, with inhibition constants, as well as kinetic parameters. Mutations that stabilize a closed-like conformation correlate with enzymes of lowered activity and with higher affinity for inhibitors, which is corroborated by a further increase in the fractional occupancy of the closed state upon addition of inhibitor or substrate-mimic. Cross-resistance is found to correlate with combinations of mutations that increase the population of the open-like conformations at the expense of the closed-like state while retaining native-like occupancy of the semiopen population. These correlations suggest that at least three states are required in the conformational sampling model to establish the emergence of drug resistance in HIV-1 PR. More importantly, these results shed light on a possible mechanism whereby mutations combine to impart drug resistance while maintaining catalytic activity.

Transition states of native and drug-resistant HIV-1 protease are the same

HIV-1 protease is an important target for the treatment of HIV/AIDS. However, drug resistance is a persistent problem and new inhibitors are needed. An approach toward understanding enzyme chemistry, the basis of drug resistance, and the design of powerful inhibitors is to establish the structure of enzymatic transition states. Enzymatic transition structures can be established by matching experimental kinetic isotope effects (KIEs) with theoretical predictions. However, the HIV-1 protease transition state has not been previously resolved using these methods. We have measured primary (14)C and (15)N KIEs and secondary (3)H and (18)O KIEs for native and multidrug-resistant HIV-1 protease (I84V). We observed (14)C KIEs ((14)V/K) of 1.029±0.003 and 1.025±0.005, (15)N KIEs ((15)V/K) of 0.987±0.004 and 0.989±0.003, (18)O KIEs ((18)V/K) of 0.999±0.003 and 0.993±0.003, and (3)H KIEs ((3)V/K) KIEs of 0.968±0.001 and 0.976±0.001 for the native and I84V enzyme, respectively. The chemical reaction involves nucleophilic water attack at the carbonyl carbon, proton transfer to the amide nitrogen leaving group, and C-N bond cleavage. A transition structure consistent with the KIE values involves proton transfer from the active site Asp-125 (1.32Å) with partial hydrogen bond formation to the accepting nitrogen (1.20Å) and partial bond loss from the carbonyl carbon to the amide leaving group (1.52Å). The KIEs measured for the native and I84V enzyme indicate nearly identical transition states, implying that a true transition-state analogue should be effective against both enzymes.