Can transcription factors in the intervertebral disc of lopinavir/ritonavir prevent degeneration in the nucleus pulposus by mediating the regulation of inflammation through signaling pathways?

OBJECTIVE: This study was conducted to examine whether lopinavir/ritonavir (Lop/r), an HIV protease inhibitor, can improve disc physiology and slow down intervertebral disc (IVD) degeneration through in vitro experimental methods, as well as whether it can suppress inflammation with interleukin-1 beta (IL-1ß) and sex-determining region Y (SRY) protein-related high-mobility group box genes-9 (SOX9) through hypoxia-inducible factor 1-alpha (HIF-1α) and the nuclear factor kappa B (NF-κB) signaling pathway. The aim was to investigate whether Lop/r application is toxic to IVD cells and the microenvironment simultaneously. PATIENTS AND METHODS: Human primary cell cultures were prepared using herniated IVD tissues obtained from patients with lumbar disc hernia who were unresponsive to conservative and medical treatment, and thereby, were operated on. The untreated culture samples served as control group, and the samples treated with Lop/r served as study group. Microscopic evaluations were performed simultaneously using fluorescent and supravital dyes in all groups. In addition to cell viability, toxicity, and proliferation analysis through a commercial kit, IL-1ß, SOX9, HIF-1α, and NF-κB protein expressions were evaluated using Western blotting. In the statistical comparison of the obtained data, an alpha value less than 0.05 was considered significant. RESULTS: Cell proliferation decreased in the Lop/r group, but no cell death was observed (p < 0.05). Moreover, at the end of 72 hours after Lop/r application, IL-1ß and NF-kB protein expressions decreased by 40% and 52%, respectively, while HIF-1α and SOX9 protein expressions increased by 4% and 59%, respectively (p< 0.05). CONCLUSIONS: Although these data were obtained from an in vitro experimental study, it is believed that these findings could make significant contributions to the pharmaco-regenerative treatment modalities of IVD degeneration. Lop/r suppresses the IL-1ß and NF-κB and induces SOX9 and HIF-1α, since these signaling pathways may be related to human IVD degeneration.

Short-Term Administration of HIV Protease Inhibitor Saquinavir Improves Skull Bone Healing with Enhanced Osteoclastogenesis.

BACKGROUND: Using immunomodulatory methods to address the challenging issue of craniofacial bone repair may be a potentially effective approach. The protease inhibitor saquinavir has been shown to inhibit the inflammatory response by targeting the toll-like receptor 4/myeloid differentiation primary response complex. Independently, inhibition of toll-like receptor 4 or myeloid differentiation primary response led to enhanced skull bone repair. Therefore, the authors aimed to investigate the effects of saquinavir on skull bone healing. METHODS: The effects of saquinavir on skull bone healing were assessed by means of gene expression, histology, immunohistochemistry, and tomography in a mouse calvarial defect model. Subsequently, the role of saquinavir in cell viability, migration, and osteogenic and osteoclastogenic differentiation was also evaluated in vitro. RESULTS: One-week saquinavir administration improved skull bone healing based on micro-computed tomographic and histomorphometric analyses. Compared to the vehicle control, 1-week saquinavir treatment (1) enhanced osteoclast infiltration (tartrate-resistant acid phosphatase staining) at day 7, but not at days 14 and 28; (2) induced more CD206 + M2 macrophage infiltration, but not F4/80 + M0 macrophages at days 7, 14, and 28; and (3) elevated osteoclastogenic gene RANKL (quantitative polymerase chain reaction) expression and other osteogenic and cytokine expression. Furthermore, in vitro data showed that saquinavir administration did not influence MC3T3-E1 cell migration or mineralization, whereas higher concentrations of saquinavir inhibited cell viability. Saquinavir treatment also enhanced the osteoclastic differentiation of bone marrow-derived precursors, and partially reversed high-mobility group box 1-driven osteoclastogenesis inhibition and elevated proinflammatory cytokine expression. CONCLUSION: The improved skull bone repair following short-term saquinavir treatment may involve enhanced osteoclastogenesis and modulated inflammatory response following skull injury. CLINICAL RELEVANCE STATEMENT: The authors' work demonstrates improved skull bone healing by short-term application of saquinavir, a drug traditionally used in the treatment of acquired immunodeficiency syndrome. As such, saquinavir may be repurposed for skeletal repair.

In silico evaluation of atazanavir as a potential HIV main protease inhibitor and its comparison with new designed analogs.

Starting three decades ago and spreading rapidly around the world, acquired immunodeficiency syndrome (AIDS) is an infectious disease distinct from other contagious diseases by its unique ways of transmission. Over the past few decades, research into new drug compounds has been accompanied by extensive advances, and the design and manufacture of drugs that inhibit virus enzymes is one way to combat the AIDS virus. Since blocking enzyme activity can kill a pathogen or correct a metabolic imbalance, the design and use of enzyme inhibitors is a new approach against viruses. We carried out an in-depth analysis of the efficacy of atazanavir and its newly designed analogs as human immunodeficiency virus (HIV) protease inhibitors using molecular docking. The best-designed analogs were then compared with atazanavir by the molecular dynamics simulation. The most promising results were ultimately found based on the docking analysis for HIV protease. Several exhibited an estimated free binding energy lower than -9.45 kcal/mol, indicating better prediction results than the atazanavir. ATV7 inhibitor with antiviral action may be more beneficial for infected patients with HIV. Molecular dynamics analysis and binding energy also showed that the ATV7 drug had more inhibitory ability than the atazanavir drug.

Multiple Molecular Dynamics Simulations and Energy Analysis Unravel the Dynamic Properties and Binding Mechanism of Mutants HIV-1 Protease with DRV and CA-p2.

PRS17, a variant of human immunodeficiency virus type I protease (HIV-1 PR), has 17 mutated residues showing high levels of multidrug resistance. To describe the effects of these mutated residues on the dynamic properties and the binding mechanism of PR with substrate and inhibitor, focused on six systems (two complexes of WT PR and PRS17 with inhibitor Darunavir (DRV), two complexes of WT PR and PRS17 with substrate analogue CA-p2, two unligand WT PR and PRS17), we performed multiple molecular dynamics (MD) simulations combined with MM-PBSA and solvated interaction energy (SIE) methods. For both the unligand PRs and ligand-PR complexes, the results from simulations revealed 17 mutated residues alter the flap-flap distance, the distance from flap regions to catalytic sites, and the curling degree of the flap tips. These mutated residues changed the flexibility of the flap region in PR, and thus affected its binding energy with DRV and CA-p2, resulting in differences in sensitivity. Hydrophobic cavity makes an important contribution to the binding of PR and ligands. And most noticeable of all, the binding of the guanidine group in CA-p2 and Arg8' of PRS17 is useful for increasing their binding ability. These results have important guidance for the further design of drugs against multidrug resistant PR. IMPORTANCE Developing effective anti-HIV inhibitors is the current requirement to cope with the emergence of the resistance of mutants. Compared with the experiments, MD simulations along with energy calculations help reduce the time and cost of designing new inhibitors. Based on our simulation results, we propose two factors that may help design effective inhibitors against HIV-1 PR: (i) importance of hydrophobic cavity, and (ii) introduction of polar groups similar to the guanidine group.

Comparative analysis of the unbinding pathways of antiviral drug Indinavir from HIV and HTLV1 proteases by supervised molecular dynamics simulation.

Determining the unbinding pathways of potential small molecule compounds from their target proteins is of great significance for designing efficacious treatment solutions. One of these potential compounds is the approved HIV-1 protease inhibitor, Indinavir, which has a weak effect on the HTLV-1 protease. In this work, by employing the SuMD method, we reconstructed the unbinding pathways of Indinavir from HIV and HTLV-1 proteases to compare and understand the mechanism of the unbinding and to discover the reasons for the lack of inhibitory activity of Indinavir against the HTLV-1 protease. We achieved multiple unbinding events from both HIV and HTLV-1 proteases in which the RMSD values of Indinavir reached over 40 Å. Also, we found that the mobility and fluctuations of the flap region are higher in the HTLV-1 protease, making the drug less stable. We realized that critically positioned aromatic residues such as Trp98/Trp98' and Phe67/Phe67' in the HTLV-1 protease could make strong π-Stacking interactions with Indinavir in the unbinding pathway, which are unfavorable for the stability of Indinavir in the active site. The details found in this study can make a reasonable explanation for the lack of inhibitory activity of this drug against HTLV-1 protease. We believe the details discovered in this work can help design more effective and selective inhibitors for the HTLV-1 protease.

Prediction of HIV drug resistance based on the 3D protein structure: Proposal of molecular field mapping.

A method for predicting HIV drug resistance by using genotypes would greatly assist in selecting appropriate combinations of antiviral drugs. Models reported previously have had two major problems: lack of information on the 3D protein structure and processing of incomplete sequencing data in the modeling procedure. We propose obtaining the 3D structural information of viral proteins by using homology modeling and molecular field mapping, instead of just their primary amino acid sequences. The molecular field potential parameters reflect the physicochemical characteristics associated with the 3D structure of the proteins. We also introduce the Bayesian conditional mutual information theory to estimate the probabilities of occurrence of all possible protein candidates from an incomplete sequencing sample. This approach allows for the effective use of uncertain information for the modeling process. We applied these data analysis techniques to the HIV-1 protease inhibitor dataset and developed drug resistance prediction models with reasonable performance.

Molecular, Solid-State and Surface Structures of the Conformational Polymorphic Forms of Ritonavir in Relation to their Physicochemical Properties.

PURPOSE: Application of multi-scale modelling workflows to characterise polymorphism in ritonavir with regard to its stability, bioavailability and processing. METHODS: Molecular conformation, polarizability and stability are examined using quantum mechanics (QM). Intermolecular synthons, hydrogen bonding, crystal morphology and surface chemistry are modelled using empirical force fields. RESULTS: The form I conformation is more stable and polarized with more efficient intermolecular packing, lower void space and higher density, however its shielded hydroxyl is only a hydrogen bond donor. In contrast, the hydroxyl in the more open but less stable and polarized form II conformation is both a donor and acceptor resulting in stronger hydrogen bonding and a more stable crystal structure but one that is less dense. Both forms have strong 1D networks of hydrogen bonds and the differences in packing energies are partially offset in form II by its conformational deformation energy difference with respect to form I. The lattice energies converge at shorter distances for form I, consistent with its preferential crystallization at high supersaturation. Both forms exhibit a needle/lath-like crystal habit with slower growing hydrophobic side and faster growing hydrophilic capping habit faces with aspect ratios increasing from polar-protic, polar-aprotic and non-polar solvents, respectively. Surface energies are higher for form II than form I and increase with solvent polarity. The higher deformation, lattice and surface energies of form II are consistent with its lower solubility and hence bioavailability. CONCLUSION: Inter-relationship between molecular, solid-state and surface structures of the polymorphic forms of ritonavir are quantified in relation to their physical-chemical properties.

A synergy of activity, stability, and inhibitor-interaction of HIV-1 protease mutants evolved under drug-pressure.

A clinically-relevant, drug-resistant mutant of HIV-1 protease (PR), termed Flap+(I54V) and containing L10I, G48V, I54V and V82A mutations, is known to produce significant changes in the entropy and enthalpy balance of drug-PR interactions, compared to wild-type PR. A similar mutant, Flap+(I54A) , which evolves from Flap+(I54V) and contains the single change at residue 54 relative to Flap+(I54V) , does not. Yet, how Flap+(I54A) behaves in solution is not known. To understand the molecular basis of V54A evolution, we compared nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and enzymatic assay data from four PR proteins: PR (pWT), Flap+(I54V) , Flap+(I54A) , and Flap+(I54) , a control mutant that contains only L10I, G48V and V82A mutations. Our data consistently show that selection to the smaller side chain at residue 54, not only decreases inhibitor affinity, but also restores the catalytic activity.

(Carbonyl)oxyalkyl linker-based amino acid prodrugs of the HIV-1 protease inhibitor atazanavir that enhance oral bioavailability and plasma trough concentration.

We describe the design, synthesis and pharmacokinetic (PK) evaluation of a series of amino acid-based prodrugs of the HIV-1 protease inhibitor atazanavir (1) derivatized on the pharmacophoric secondary alcohol using a (carbonyl)oxyalkyl linker. Prodrugs of 1 incorporating simple (carbonyl)oxyalkyl-based linkers and a primary amine in the promoiety were found to exhibit low chemical stability. However, chemical stability was improved by modifying the primary amine moiety to a tertiary amine, resulting in a 2-fold enhancement of exposure in rats following oral dosing compared to dosing of the parent drug 1. Further refinement of the linker resulted in the discovery of 22 as a prodrug that delivered the parent 1 to rat plasma with a 5-fold higher AUC and 67-fold higher C24 when compared to oral administration of the parent drug. The PK profile of 22 indicated that plasma levels of this prodrug were higher than that of the parent, providing a more sustained release of 1 in vivo.

Drug binding dynamics of the dimeric SARS-CoV-2 main protease, determined by molecular dynamics simulation.

We performed molecular dynamics simulation of the dimeric SARS-CoV-2 (severe acute respiratory syndrome corona virus 2) main protease (Mpro) to examine the binding dynamics of small molecular ligands. Seven HIV inhibitors, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir, were used as the potential lead drugs to investigate access to the drug binding sites in Mpro. The frequently accessed sites on Mpro were classified based on contacts between the ligands and the protein, and the differences in site distributions of the encounter complex were observed among the ligands. All seven ligands showed binding to the active site at least twice in 28 simulations of 200 ns each. We further investigated the variations in the complex structure of the active site with the ligands, using microsecond order simulations. Results revealed a wide variation in the shapes of the binding sites and binding poses of the ligands. Additionally, the C-terminal region of the other chain often interacted with the ligands and the active site. Collectively, these findings indicate the importance of dynamic sampling of protein-ligand complexes and suggest the possibilities of further drug optimisations.

Conformational landscape of non-B variants of HIV-1 protease: A pulsed EPR study.

HIV infection is a global health epidemic with current FDA-approved HIV-1 Protease inhibitors (PIs) designed against subtype B protease, yet they are used in HIV treatment world-wide regardless of patient HIV classification. In this study, double electron-electron resonance (DEER) electron paramagnetic resonance (EPR) spectroscopy was utilized to gain insights in how natural polymorphisms in several African and Brazilian protease (PR) variants affect the conformational landscape both in the absence and presence of inhibitors. Findings show that Subtypes F and H HIV-1 PR adopt a primarily closed conformation in the unbound state with two secondary mutations, D60E and I62V, postulated to be responsible for the increased probability for closed conformation. In contrast, subtype D, CRF_AG, and CRF_BF HIV-1 PR adopt a primarily semi-open conformation, as observed for PI-naïve-subtype B when unbound by substrate or inhibitor. The impact that inhibitor binding has on shifting the conformational land scape of these variants is also characterized, where analysis provides classification of inhibitor induced shifts away from the semi-open state into weak, moderate and strong effects. The findings are compared to those for prior studies of inhibitor induced conformational shifts in PI-naïve Subtype B, C and CRF_AE.

Computational Determination of Potential Inhibitors of SARS-CoV-2 Main Protease.

The novel coronavirus (SARS-CoV-2) has infected several million people and caused thousands of deaths worldwide since December 2019. As the disease is spreading rapidly all over the world, it is urgent to find effective drugs to treat the virus. The main protease (Mpro) of SARS-CoV-2 is one of the potential drug targets. Therefore, in this context, we used rigorous computational methods, including molecular docking, fast pulling of ligand (FPL), and free energy perturbation (FEP), to investigate potential inhibitors of SARS-CoV-2 Mpro. We first tested our approach with three reported inhibitors of SARS-CoV-2 Mpro, and our computational results are in good agreement with the respective experimental data. Subsequently, we applied our approach on a database of ∼4600 natural compounds, as well as 8 available HIV-1 protease (PR) inhibitors and an aza-peptide epoxide. Molecular docking resulted in a short list of 35 natural compounds, which was subsequently refined using the FPL scheme. FPL simulations resulted in five potential inhibitors, including three natural compounds and two available HIV-1 PR inhibitors. Finally, FEP, the most accurate and precise method, was used to determine the absolute binding free energy of these five compounds. FEP results indicate that two natural compounds, cannabisin A and isoacteoside, and an HIV-1 PR inhibitor, darunavir, exhibit a large binding free energy to SARS-CoV-2 Mpro, which is larger than that of 13b, the most reliable SARS-CoV-2 Mpro inhibitor recently reported. The binding free energy largely arises from van der Waals interaction. We also found that Glu166 forms H-bonds to all of the inhibitors. Replacing Glu166 by an alanine residue leads to ∼2.0 kcal/mol decreases in the affinity of darunavir to SARS-CoV-2 Mpro. Our results could contribute to the development of potential drugs inhibiting SARS-CoV-2.

Structure-Based Design of Highly Potent HIV-1 Protease Inhibitors Containing New Tricyclic Ring P2-Ligands: Design, Synthesis, Biological, and X-ray Structural Studies.

We describe here design, synthesis, and biological evaluation of a series of highly potent HIV-1 protease inhibitors containing stereochemically defined and unprecedented tricyclic furanofuran derivatives as P2 ligands in combination with a variety of sulfonamide derivatives as P2' ligands. These inhibitors were designed to enhance the ligand-backbone binding and van der Waals interactions in the protease active site. A number of inhibitors containing the new P2 ligand, an aminobenzothiazole as the P2' ligand and a difluorophenylmethyl as the P1 ligand, displayed very potent enzyme inhibitory potency and also showed excellent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The tricyclic P2 ligand has been synthesized efficiently in an optically active form using enzymatic desymmetrization of meso-1,2-(dihydroxymethyl)cyclohex-4-ene as the key step. We determined high-resolution X-ray structures of inhibitor-bound HIV-1 protease. These structures revealed extensive interactions with the backbone atoms of HIV-1 protease and provided molecular insights into the binding properties of these new inhibitors.

Development of Darunavir proliposome powder for oral delivery by using Box-Bhenken design.

The aim of this study is to develop Darunavir (DRV) proliposome powder for oral delivery. Darunavir-loaded oral proliposome powder (OPP) was prepared by a solvent evaporation technique with varying independent variables at three different levels. Based on different levels, proliposome powder formulation was optimized by using Box-Behnken design. The formulations were analyzed for its size distribution, entrapment efficiency, and surface morphology. Optimized proliposome batch A was evaluated for physical parameter, morphological parameters, entrapment efficiency, followed by in vitro, ex vivo, and in vivo studies. Oral proliposome powder showed good micromeritic properties with angle of repose was less than 30°, Carr's index and Hausner's ratio were also less than 21 and 1.25, respectively. The mean size of the vesicles was in the range of 180-290 nm. The assay and entrapment efficiency of pro-liposome powder formulations were 79.00 ± 0.2 and 93.46 ± 0.2%, respectively. In vitro release of DRV proliposome powder was 78.17 ± 0.1% after 24 h which shows good release from the vesicle of proliposome. Ex vivo permeation study shows 58.11% enhancement which shows good permeation. The optimize batch A of proliposome powder indicated 50% enhancement in the relative bioavailability as compared to the DRV suspension. The results showed that proliposome powder containing DRV can efficiently deliver in to the blood stream. This drug delivery system has been designed as a novel platform for potential oral delivery of drugs having poor water solubility and high first-pass metabolism.

Design, synthesis and biological evaluation of HIV-1 protease inhibitors with morpholine derivatives as P2 ligands in combination with cyclopropyl as P1' ligand.

A series of novel HIV-1 protease inhibitors has been designed and synthesized, which contained morpholine derivatives as the P2 ligands and hydrophobic cyclopropyl as the P1' ligand at the meantime in this study, with the aim of improving the interactions between the active sites of HIV-1 protease and the inhibitors. Twenty-eight compounds were synthesized and assessed, among which inhibitors m18 and m1 exhibited excellent inhibitory effect on the activity of HIV-1 protease with IC50 value of 47 nM and 53 nM, respectively. The molecular modeling of m1 revealed possible hydrogen bondings or van der Waals between the inhibitor and the protease, worthy of in-depth study.

Accessory mutations balance the marginal stability of the HIV-1 protease in drug resistance.

The HIV-1 protease is a major target of inhibitor drugs in AIDS therapies. The therapies are impaired by mutations of the HIV-1 protease that can lead to resistance to protease inhibitors. These mutations are classified into major mutations, which usually occur first and clearly reduce the susceptibility to protease inhibitors, and minor, accessory mutations that occur later and individually do not substantially affect the susceptibility to inhibitors. Major mutations are predominantly located in the active site of the HIV-1 protease and can directly interfere with inhibitor binding. Minor mutations, in contrast, are typically located distal to the active site. A central question is how these distal mutations contribute to resistance development. In this article, we present a systematic computational investigation of stability changes caused by major and minor mutations of the HIV-1 protease. As most small single-domain proteins, the HIV-1 protease is only marginally stable. Mutations that destabilize the folded, active state of the protease therefore can shift the conformational equilibrium towards the unfolded, inactive state. We find that the most frequent major mutations destabilize the HIV-1 protease, whereas roughly half of the frequent minor mutations are stabilizing. An analysis of protease sequences from patients in treatment indicates that the stabilizing minor mutations are frequently correlated with destabilizing major mutations, and that highly resistant HIV-1 proteases exhibit significant fractions of stabilizing mutations. Our results thus indicate a central role of minor mutations in balancing the marginal stability of the protease against the destabilization induced by the most frequent major mutations.

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.

In silico Tools at Early Stage of Pharmaceutical Development: Data Needs and Software Capabilities.

In early drug development, the selection of a formulation platform and decisions on formulation strategies have to be made within a short timeframe and often with minimal use of the active pharmaceutical ingredient (API). The current work evaluated the various physicochemical parameters required to improve the prediction accuracy of simulation software for immediate release tablets in early drug development. DDDPlus™ was used in simulating dissolution test profiles of immediate release tablets of ritonavir and all simulations were compared with experimental results. The minimum data requirements to make useful predictions were assessed using the ADMET predictor (part of DDDPlus) and Chemicalize (an online resource). A surfactant model was developed to estimate the solubility enhancement in media containing surfactant and the software's transfer model based on the USP two-tiered dissolution test was assessed. One measured data point was shown to be sufficient to make predictive simulations in DDDPlus. At pH 2.0, the software overestimated drug release while at pH 1.0 and 6.8, simulations were close to the measured values. A surfactant solubility model established with measured data gave good dissolution predictions. The transfer model uses a single-vessel model and was unable to predict the two in vivo environments separately. For weak bases like ritonavir, a minimum of three solubility data points is recommended for in silico predictions in buffered media. A surfactant solubility model is useful when predicting dissolution behavior in surfactant media and in silico predictions need measured solubility data to be predictive.

Picomolar to Micromolar: Elucidating the Role of Distal Mutations in HIV-1 Protease in Conferring Drug Resistance.

Drug resistance continues to be a growing global problem. The efficacy of small molecule inhibitors is threatened by pools of genetic diversity in all systems, including antibacterials, antifungals, cancer therapeutics, and antivirals. Resistant variants often include combinations of active site mutations and distal "secondary" mutations, which are thought to compensate for losses in enzymatic activity. HIV-1 protease is the ideal model system to investigate these combinations and underlying molecular mechanisms of resistance. Darunavir (DRV) binds wild-type (WT) HIV-1 protease with a potency of <5 pM, but we have identified a protease variant that loses potency to DRV 150 000-fold, with 11 mutations in and outside the active site. To elucidate the roles of these mutations in DRV resistance, we used a multidisciplinary approach, combining enzymatic assays, crystallography, and molecular dynamics simulations. Analysis of protease variants with 1, 2, 4, 8, 9, 10, and 11 mutations showed that the primary active site mutations caused ∼50-fold loss in potency (2 mutations), while distal mutations outside the active site further decreased DRV potency from 13 nM (8 mutations) to 0.76 µM (11 mutations). Crystal structures and simulations revealed that distal mutations induce subtle changes that are dynamically propagated through the protease. Our results reveal that changes remote from the active site directly and dramatically impact the potency of the inhibitor. Moreover, we find interdependent effects of mutations in conferring high levels of resistance. These mechanisms of resistance are likely applicable to many other quickly evolving drug targets, and the insights may have implications for the design of more robust inhibitors.