Protease Dimer Research Articles

Carbonylhydrazide-Based Molecular Tongs Inhibit Wild-Type and Mutated HIV-1 Protease Dimerization

We have designed and synthesized new molecular tongs based on a rigid naphthalene scaffold and evaluated their antidimer activity on HIV-1 protease (PR). We inserted carbonylhydrazide and oligohydrazide (azatide) fragments into their peptidomimetic arms to reduce hydrophobicity and increase metabolic stability. These fragments are designed to disrupt the protein-protein interactions by reproducing the hydrogen bond pattern found in the antiparallel β-sheet formed between the N- and C-ends of the two monomers in the native PR. Kinetic analyses and fluorescent probe binding studies showed that several molecular tongs can inhibit PR dimerization. The best nonpeptidic molecular tongs to date were obtained with an inhibition constant K(id) of 50 nM for PR and 80 nM for the multimutated protease ANAM-11. The PR inhibition was selective, the aspartic proteases renin and pepsin were not inhibited.

NMR study of xenotropic murine leukemia virus-related virus protease in a complex with amprenavir

Xenotropic murine leukemia virus-related virus (XMRV) is a virus created through recombination of two murine leukemia proviruses under artificial conditions during the passage of human prostate cancer cells in athymic nude mice. The homodimeric protease (PR) of XMRV plays a critical role in the production of functional viral proteins and is a prerequisite for viral replication. We synthesized XMRV PR using the wheat germ cell-free expression system and carried out structural analysis of XMRV PR in a complex with an inhibitor, amprenavir (APV), by means of NMR. Five different combinatorially 15N-labeled samples were prepared and backbone resonance assignments were made by applying Otting’s method, with which the amino acid types of the [1H, 15N] HSQC resonances were automatically identified using the five samples (Wu et al., 2006) [14]. A titration experiment involving APV revealed that one APV molecule binds to one XMRV PR dimer. For many residues, two distinct resonances were observed, which is thought to be due to the structural heterogeneity between the two protomers in the APV:XMRV PR=1:2 complex. PR residues at the interface with APV have been identified on the basis of chemical shift perturbation and identification of the intermolecular NOEs by means of filtered NOE experiments. Interestingly, chemical shift heterogeneity between the two protomers of XMRV PR has been observed not only at the interface with APV but also in regions apart from the interface. This indicates that the structural heterogeneity induced by the asymmetry of the binding of APV to the XMRV PR dimer is transmitted to distant regions. This is in contrast to the case of the APV:HIV-1 PR complex, in which the structural heterogeneity is only localized at the interface. Long-range transmission of the structural change identified for the XMRV PR complex might be utilized for the discovery of a new type of drug.

How Does Darunavir Prevent HIV-1 Protease Dimerization?

The drug Darunavir (DRV) is a potent inhibitor of HIV-1 protease (PR), a homodimeric essential enzyme of the AIDS virus. Recent experimental data suggest that DRV is able to prevent dimerization of HIV-1 PR, which, together with its high affinity for the mature enzyme, has been linked to the high genetic barrier to the development of viral resistance. The mechanism of dimerization inhibition and the binding mode(s) of DRV to monomeric HIV-1 PR are unknown. Here, multiple molecular dynamics simulations with explicit solvent (for a total of 11 μs with the CHARMM force field and 1 μs with the Amber force field) show that the monomer of HIV-1 PR is structurally stable and reveal a major binding mode of DRV. This binding mode is stabilized by favorable interactions between the apolar groups of DRV and the hydrophobic residues Ile32, Ile47, Ile50, Ile54, Pro79, Val82, and Ile84. The binding mode to monomeric HIV-1 PR identified by molecular dynamics is different from the two binding modes observed in the crystal structure of the complex with dimeric HIV-1 PR. As an example, there are no interactions between DRV and the catalytic Asp25 in the binding mode to monomeric HIV-1 PR revelead by the simulations. In contrast, the simulations show extensive and stable interactions between DRV and the flap (residues 46-55), which are likely to sterically hinder the formation of the flap interface as observed in the dimeric structure. Which of the two mechanisms of inhibition (dimerization inhibition by association with the flap or binding to the active site of the mature enzyme) dominates might depend on the HIV-1 PR mutations, and it is likely that dimerization inhibition is predominant for multiple mutations at the active site in multidrug resistant strains.

Activity of Human Immunodeficiency Virus Type 1 Protease Inhibitors against the Initial Autocleavage in Gag-Pol Polyprotein Processing

Inhibitors of HIV protease have proven to be important drugs in combination anti-HIV therapy. These inhibitors were designed to target mature protease and prevent viral particle maturation by blocking Gag and Gag-Pol processing by mature protease. Currently there are few data assessing the ability of these protease inhibitors to block the initial step in autoproteolytic processing of Gag-Pol. This unique step involves the dimerization of two Gag-Pol polyproteins and autocleavage of the Gag-Pol polyprotein by the embedded dimeric protease. We developed a plasmid encoding a modified form of Gag-Pol that can undergo autoprocessing only at the initial cleavage site between p2 and nucleocapsid. Using an in vitro transcription/translation system, we assessed the ability of six different approved protease inhibitors (darunavir, indinavir, nelfinavir, ritonavir, saquinavir, and tipranavir) to block this initial autocleavage step. Of these inhibitors, darunavir and saquinavir were the most effective. Darunavir and saquinavir were also the most effective at blocking the initial autoprocessing of full-length Gag-Pol in HIV-1-infected T cells. Thus, we have identified at least two HIV-1 protease inhibitors that have activity against the primary autocatalytic step of the embedded HIV-1 protease in Gag-Pol at concentrations that may be attained in HIV-1-infected patients. Due to unique aspects of the initial processing step, it may be possible to develop inhibitors with greater potency against this step, thus halting viral maturation at the earliest stages. The transcription/translation assay could be used to develop more potent inhibitors of this essential first step in viral maturation.

Placement of Leucine Zipper Motifs at the Carboxyl Terminus of HIV-1 Protease Significantly Reduces Virion Production

Natural HIV-1 protease (PR) is homodimeric. Some researchers believe that interactions between HIV-1 Gag-Pol molecules trigger the activation of embedded PR (which mediates Gag and Gag-Pol cleavage), and that Gag-Pol assembly domains outside of PR may contribute to PR activation by influencing PR dimer interaction in a Gag-Pol context. To determine if the enhancement of PR dimer interaction facilitates PR activation, we placed single or tandem repeat leucine zippers (LZ) at the PR C-terminus, and looked for a correlation between enhanced Gag processing efficiency and increased Gag-PR-LZ multimerization capacity. We found significant reductions in virus-like particles (VLPs) produced by HIV-1 mutants, with LZ fused to the end of PR as a result of enhanced Gag cleavage efficiency. Since VLP production can be restored to wt levels following PR activity inhibition, this assembly defect is considered PR activity-dependent. We also found a correlation between the LZ enhancement effect on Gag cleavage and enhanced Gag-PR multimerization. The results suggest that PR dimer interactions facilitated by forced Gag-PR multimerization lead to premature Gag cleavage, likely a result of premature PR activation. Our conclusion is that placement of a heterologous dimerization domain downstream of PR enhances PR-mediated Gag cleavage efficiency, implying that structural conformation, rather than the primary sequence outside of PR, is a major determinant of HIV-1 PR activation.

Inhibition of caspase-9 by stabilized peptides targeting the dimerization interface.

Caspases comprise a family of dimeric cysteine proteases that control apoptotic programmed cell death and are therefore critical in both organismal development and disease. Specific inhibition of individual caspases has been repeatedly attempted, but has not yet been attained. Caspase-9 is an upstream or initiator caspase that is regulated differently from all other caspases, as interaction with natural inhibitor X-linked inhibitor of apoptosis protein (XIAP)-baculovirus inhibitory repeat 3 (BIR3) occurs at the dimer interface maintaining caspase-9 in an inactive monomeric state. One route to caspase-9-specific inhibition is to mimic this interaction, which has been localized to the α5 helix of XIAP-BIR3. We have developed three types of stabilized peptides derived from the α5 helix, using incorporation of aminoisobutyric acid, the avian pancreatic polypeptide (aPP)-scaffold or aliphatic staples. The stabilized peptides are helical in solution and achieve up to 32 μM inhibition, indicating that this allosteric site at the caspase-9 dimerization interface is regulatable with low-molecular weight synthetic ligands and is thus a druggable site. The most potent peptides against caspase-9 activity are the aPP-scaffolded peptides. Other caspases, which are not regulated by dimerization, should not be inactivated by these peptides. Given that all of the peptides attain helical structures but cannot recapitulate the high-affinity inhibition of the intact BIR3 domain, it has become clear that interactions of caspase-9 with the BIR3 exosite are essential for high-affinity binding. These results explain why the full XIAP-BIR3 domain is required for maximal inhibition and suggest a path forward for achieving allosteric inhibition at the dimerization interface using peptides or small molecules.

Neriifolin S, a dimeric serine protease from Euphorbia neriifolia Linn.: Purification and biochemical characterisation

A dimeric serine protease Neriifolin S of molecular mass 94kDa with milk clotting activity has been purified from the latex of Euphorbia neriifolia by anion exchange and size-exclusion chromatography. It hydrolyses peptidyl substrates l-Ala-pNA with highest affinity (Km of 0.195mM) and physiological efficiency (Kcat/Km of 144.5mMs). Enzyme belongs to the class of neutral proteases with pI value of 6.8, optimal proteolytic activity displayed at pH 9.5 and temperature 45°C. Its proteolytic activity is strongly stimulated in the presence of Ca+2 ions and exclusively inhibited by serine protease inhibitors. Enzyme is fairly stable toward chemical denaturants, pH and temperature. The apparent Tm, was found to be 65°C. Thermal inactivation follow first order kinetics with activation energy (Ea), activation enthalpy (ΔH∗), free energy change (ΔG∗) and entropy (ΔS∗) of 27.54kJmol−1, 24.89kJmol−1, −82.34kJmol−1 and 337.20Jmol−1K−1.

Resolution of Discordant HIV-1 Protease Resistance Rankings Using Molecular Dynamics Simulations

The emergence of drug resistance is a major challenge for the effective treatment of HIV. In this article, we explore the application of atomistic molecular dynamics simulations to quantify the level of resistance of a patient-derived HIV-1 protease sequence to the inhibitor lopinavir. A comparative drug ranking methodology was developed to compare drug resistance rankings produced by the Stanford HIVdb, ANRS, and RegaDB clinical decision support systems. The methodology was used to identify a patient sequence for which the three rival online tools produced differing resistance rankings. Mutations at only three positions ( L10I , A71IV, and L90M ) influenced the resistance level assigned to the sequence. We use ensemble molecular dynamics simulations to elucidate the origin of these discrepancies and the mechanism of resistance. By simulating not only the full patient sequences but also systems containing the constituent mutations, we gain insight into why resistance estimates vary and the interactions between the various mutations. In the same way, we also gain valuable knowledge of the mechanistic causes of resistance. In particular, we identify changes in the relative conformation of the two beta sheets that form the protease dimer interface which suggest an explanation of the relative frequency of different amino acids observed in patients at residue 71.

Loss of protease dimerization inhibition activity of darunavir is associated with the acquisition of resistance to darunavir by HIV-1.

Dimerization of HIV protease is essential for the acquisition of protease's proteolytic activity. We previously identified a group of HIV protease dimerization inhibitors, including darunavir (DRV). In the present work, we examine whether loss of DRV's protease dimerization inhibition activity is associated with HIV development of DRV resistance. Single amino acid substitutions, including I3A, L5A, R8A/Q, L24A, T26A, D29N, R87K, T96A, L97A, and F99A, disrupted protease dimerization, as examined using an intermolecular fluorescence resonance energy transfer (FRET)-based HIV expression assay. All recombinant HIV(NL4-3)-based clones with such a protease dimerization-disrupting substitution failed to replicate. A highly DRV-resistant in vitro-selected HIV variant and clinical HIV strains isolated from AIDS patients failing to respond to DRV-containing antiviral regimens typically had the V32I, L33F, I54M, and I84V substitutions in common in protease. None of up to 3 of the 4 substitutions affected DRV's protease dimerization inhibition, which was significantly compromised by the four combined substitutions. Recombinant infectious clones containing up to 3 of the 4 substitutions remained sensitive to DRV, while a clonal HIV variant with all 4 substitutions proved highly resistant to DRV with a 205-fold 50% effective concentration (EC(50)) difference compared to HIV(NL4-3). The present data suggest that the loss of DRV activity to inhibit protease dimerization represents a novel mechanism contributing to HIV resistance to DRV. The finding that 4 substitutions in PR are required for significant loss of DRV's protease dimerization inhibition should at least partially explain the reason DRV has a high genetic barrier against HIV's acquisition of DRV resistance.

Visualization of Early Events in Acetic Acid Denaturation of HIV-1 Protease: A Molecular Dynamics Study

Protein denaturation plays a crucial role in cellular processes. In this study, denaturation of HIV-1 Protease (PR) was investigated by all-atom MD simulations in explicit solvent. The PR dimer and monomer were simulated separately in 9 M acetic acid (9 M AcOH) solution and water to study the denaturation process of PR in acetic acid environment. Direct visualization of the denaturation dynamics that is readily available from such simulations has been presented. Our simulations in 9 M AcOH reveal that the PR denaturation begins by separation of dimer into intact monomers and it is only after this separation that the monomer units start denaturing. The denaturation of the monomers is flagged off by the loss of crucial interactions between the α-helix at C-terminal and surrounding β-strands. This causes the structure to transit from the equilibrium dynamics to random non-equilibrating dynamics. Residence time calculations indicate that denaturation occurs via direct interaction of the acetic acid molecules with certain regions of the protein in 9 M AcOH. All these observations have helped to decipher a picture of the early events in acetic acid denaturation of PR and have illustrated that the α-helix and the β-sheet at the C-terminus of a native and functional PR dimer should maintain both the stability and the function of the enzyme and thus present newer targets for blocking PR function.

Inhibition of autoprocessing of natural variants and multidrug resistant mutant precursors of HIV-1 protease by clinical inhibitors

Self-cleavage at the N terminus of HIV-1 protease from the Gag-Pol precursor (autoprocessing) is crucial for stabilizing the protease dimer required for onset of mature-like catalytic activity, viral maturation, and propagation. Among nine clinical protease inhibitors (PIs), darunavir and saquinavir were the most effective in inhibiting wild-type HIV-1 group M precursor autoprocessing, with an IC(50) value of 1-2 μM, 3-5 orders of magnitude higher than their binding affinities to the corresponding mature protease. Accordingly, both group M and N precursor-PI complexes exhibit T(m)s 17-21 °C lower than those of the corresponding mature protease-PI complexes suggestive of markedly reduced stabilities of the precursor dimer-PI ensembles. Autoprocessing of group N (natural variant) and three group M precursors bearing 11-20 mutations associated with multidrug resistance was either weakly responsive or fully unresponsive to inhibitors at concentrations up to a practical limit of approximately 150 μM PI. This observation parallels decreases of up to 8 × 10(3)-fold (e.g., 5 pM to 40 nM) in the binding affinity of darunavir and saquinavir to mature multidrug resistant proteases relative to wild type, suggesting that inhibition of some of these mutant precursors will occur only in the high μM to mM range in extreme PI-resistance, which is an effect arising from coordinated multiple mutations. An extremely darunavir-resistant mutant precursor is more responsive to inhibition by saquinavir. These findings raise the questions whether clinical failure of PI therapy is related to lack of inhibition of autoprocessing and whether specific inhibitors can be designed with low-nM affinity to target autoprocessing.

Quality Control of Photosystem II: FtsH HEXAMERS ARE LOCALIZED NEAR PHOTOSYSTEM II AT GRANA FOR THE SWIFT REPAIR OF DAMAGE

The reaction center-binding D1 protein of Photosystem II is oxidatively damaged by excessive visible light or moderate heat stress. The metalloprotease FtsH has been suggested as responsible for the degradation of the D1 protein. We have analyzed the distribution and subunit structures of FtsH in spinach thylakoids and various membrane fractions derived from the thylakoids using clear native polyacrylamide gel electrophoresis and Western blot analysis. FtsH was found not only in the stroma thylakoids but also in the Photosystem II-enriched grana membranes. Monomeric, dimeric, and hexameric FtsH proteases were present as major subunit structures in thylakoids, whereas only hexameric FtsH proteases were detected in Triton X-100-solubilized Photosystem II membranes. Importantly, among the membrane fractions examined, hexameric FtsH proteases were most abundant in the Photosystem II membranes. In accordance with this finding, D1 degradation took place in the Photosystem II membranes under light stress. Sucrose density gradient centrifugation analysis of thylakoids and the Photosystem II membranes solubilized with n-dodecyl-β-d-maltoside and a chemical cross-linking study of thylakoids showed localization of FtsH near the Photosystem II light-harvesting chlorophyll-protein supercomplexes in the grana. These results suggest that part of the FtsH hexamers are juxtapositioned to PSII complexes in the grana in darkness, carrying out immediate degradation of the photodamaged D1 protein under light stress.

Interaction between separated consecutive complement control modules of human C1r: Implications for dimerization of the full-length protease

Complement control protein modules (CCP) typically mediate protein:protein interaction during immune response in vertebrates. Using NMR chemical shift perturbation mapping, we present previously lacking experimental evidence for intermolecular interactions between the CCP1 and CCP2 modules of the human C1r serine protease (SP). The identified interface is clearly distinct from that observed in the covalently linked CCP1–CCP2 pair. Structural models of the CCP1–CCP2–SP segments of two C1r molecules built on the basis of shift perturbation data are fully consistent with an extended interaction interface and suggests the possibility of a structural rearrangement as a switch between functional states of human C1r. Structured summary MINT- 8045767: CCP1 (uniprotkb: P00736) and CCP2 (uniprotkb: P00736) bind (MI: 0407) by nuclear magnetic resonance (MI: 0077)

Toward the First Nonpeptidic Molecular Tong Inhibitor of Wild‐Type and Mutated HIV‐1 Protease Dimerization

Herein we describe the synthesis and HIV-1 protease (PR) inhibitory activity of 16 new peptidomimetic molecular tongs with a naphthalene scaffold. Their peptidic character was progressively decreased. Two of these molecules exhibited the best dimerization inhibition activity toward HIV-1 wild-type and multimutated ANAM-11 proteases obtained to date for this class of molecules (∼40 nM for wild-type PR and 100 nM for ANAM-11 PR). Although the peptidic character of one molecular tong was completely suppressed, the mechanism of inhibition and inhibitory potency toward both proteases were maintained.

Probing multidrug-resistance and protein-ligand interactions with oxatricyclic designed ligands in HIV-1 protease inhibitors.

We report the design, synthesis, biological evaluation, and X-ray crystallographic analysis of a new class of HIV-1 protease inhibitors. Compound 4 proved to be an extremely potent inhibitor toward various multidrug-resistant HIV-1 variants, representing a near 10-fold improvement over darunavir (DRV). Compound 4 also blocked protease dimerization with at least 10-fold greater potency than DRV.

In Vitro Selection of Highly Darunavir-Resistant and Replication-Competent HIV-1 Variants by Using a Mixture of Clinical HIV-1 Isolates Resistant to Multiple Conventional Protease Inhibitors

We attempted to select HIV-1 variants resistant to darunavir (DRV), which potently inhibits the enzymatic activity and dimerization of protease and has a high genetic barrier to HIV-1 development of resistance to DRV. We conducted selection using a mixture of 8 highly multi-protease inhibitor (PI)-resistant, DRV-susceptible clinical HIV-1 variants (HIV-1(MIX)) containing 9 to 14 PI resistance-associated amino acid substitutions in protease. HIV-1(MIX) became highly resistant to DRV, with a 50% effective concentration (EC(50)) ∼333-fold greater than that against HIV-1(NL4-3). HIV-1(MIX) at passage 51 (HIV-1(MIX(P51))) replicated well in the presence of 5 μM DRV and contained 14 mutations. HIV-1(MIX(P51)) was highly resistant to amprenavir, indinavir, nelfinavir, ritonavir, lopinavir, and atazanavir and moderately resistant to saquinavir and tipranavir. HIV-1(MIX(P51)) had a resemblance with HIV-1(C) of the HIV-1(MIX) population, and selection using HIV-1(C) was also performed; however, its DRV resistance acquisition was substantially delayed. The H219Q and I223V substitutions in Gag, lacking in HIV-1(C(P51)), likely contributed to conferring a replication advantage on HIV-1(MIX(P51)) by reducing intravirion cyclophilin A content. HIV-1(MIX(P51)) apparently acquired the substitutions from another HIV-1 strain(s) of HIV-1(MIX) through possible homologous recombination. The present data suggest that the use of multiple drug-resistant HIV-1 isolates is of utility in selecting drug-resistant variants and that DRV would not easily permit HIV-1 to develop significant resistance; however, HIV-1 can develop high levels of DRV resistance when a variety of PI-resistant HIV-1 strains are generated, as seen in patients experiencing sequential PI failure, and ensuing homologous recombination takes place. HIV-1(MIX(P51)) should be useful in elucidating the mechanisms of HIV-1 resistance to DRV and related agents.

Development of Anti-HIV-1 Protease Inhibitors against Multi-PIs-Resistant HIV-1 and Study of Inhibition and Dynamics of HIV-1 Protease Dimerization

Development of Anti-HIV-1 Protease Inhibitors against Multi-PIs-Resistant HIV-1 and Study of Inhibition and Dynamics of HIV-1 Protease Dimerization

Assembly and Maturation of the Bacteriophage Lambda Procapsid: gpC Is the Viral Protease

Viral capsids are robust structures designed to protect the genome from environmental insults and deliver it to the host cell. The developmental pathway for complex double-stranded DNA viruses is generally conserved in the prokaryotic and eukaryotic groups and includes a genome packaging step where viral DNA is inserted into a pre-formed procapsid shell. The procapsids self-assemble from monomeric precursors to afford a mature icosahedron that contains a single “portal” structure at a unique vertex; the portal serves as the hole through which DNA enters the procapsid during particle assembly and exits during infection. Bacteriophage λ has served as an ideal model system to study the development of the large double-stranded DNA viruses. Within this context, the λ procapsid assembly pathway has been reported to be uniquely complex involving protein cross-linking and proteolytic maturation events. In this work, we identify and characterize the protease responsible for λ procapsid maturation and present a structural model for a procapsid-bound protease dimer. The procapsid protease possesses autoproteolytic activity, it is required for degradation of the internal “scaffold” protein required for procapsid self-assembly, and it is responsible for proteolysis of the portal complex. Our data demonstrate that these proteolytic maturation events are not required for procapsid assembly or for DNA packaging into the structure, but that proteolysis is essential to late steps in particle assembly and/or in subsequent infection of a host cell. The data suggest that the λ-like proteases and the herpesvirus-like proteases define two distinct viral protease folds that exhibit little sequence or structural homology but that provide identical functions in virus development. The data further indicate that procapsid assembly and maturation are strongly conserved in the prokaryotic and eukaryotic virus groups.

Novel Protease Inhibitors (PIs) Containing Macrocyclic Components and 3( R ),3a( S ),6a( R )- bis -Tetrahydrofuranylurethane That Are Potent against Multi-PI-Resistant HIV-1 Variants In Vitro

Natural products with macrocyclic structural features often display intriguing biological properties. Molecular design incorporating macrocycles may lead to molecules with unique protein-ligand interactions. We generated novel human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing a macrocycle and bis-tetrahydrofuranylurethane. Four such compounds exerted potent activity against HIV-1LAI and had 50% effective concentrations (EC50s) of as low as 0.002 microM with minimal cytotoxicity. GRL-216 and GRL-286 blocked the replication of HIV-1NL4-3 variants selected by up to 5 microM saquinavir, ritonavir, nelfinavir, lopinavir, or atazanavir; they had EC50s of 0.020 to 0.046 microM and potent activities against six multi-PI-resistant clinical HIV-1 (HIVmPIr) variants with EC50s of 0.027 to 0.089 microM. GRL-216 and -286 also blocked HIV-1 protease dimerization as efficiently as darunavir. When HIV-1NL4-3 was selected by GRL-216, it replicated progressively more poorly and failed to replicate in the presence of >0.26 microM GRL-216, suggesting that the emergence of GRL-216-resistant HIV-1 variants is substantially delayed. At passage 50 with GRL-216 (the HIV isolate selected with GRL-216 at up to 0.16 microM [HIV216-0.16 microM]), HIV-1NL4-3 containing the L10I, L24I, M46L, V82I, and I84V mutations remained relatively sensitive to PIs, including darunavir, with the EC50s being 3- to 8-fold-greater than the EC50 of each drug for HIV-1NL4-3. Interestingly, HIV216-0.16 microM had 10-fold increased sensitivity to tipranavir. Analysis of the protein-ligand X-ray structures of GRL-216 revealed that the macrocycle occupied a greater volume of the binding cavity of protease and formed greater van der Waals interactions with V82 and I84 than darunavir. The present data warrant the further development of GRL-216 as a potential antiviral agent for treating individuals harboring wild-type and/or HIVmPIr.

Direct use of 15N relaxation rates as experimental restraints on molecular shape and orientation for docking of protein-protein complexes.

(15)N relaxation rates contain information on overall molecular shape and size, as well as residue specific orientations of N-H bond vectors relative to the axes of the diffusion tensor. Here we describe a pseudopotential E(relax) that permits direct use of (15)N relaxation rates, in the form of R(2)/R(1) ratios, as experimental restraints in structure calculations without requiring prior information to be extracted from a known molecular structure. The elements of the rotational diffusion tensor are calculated from the atomic coordinates at each step of the structure calculation and then used together with the N-H bond vector orientations to compute the (15)N R(2)/R(1) ratios. We show that the E(relax) term can be reliably used for protein-protein docking of complexes and illustrate its applicability to the 40 kDa complex of the N-terminal domain of enzyme I and the histidine phosphocarrier protein HPr and to the symmetric HIV-1 protease dimer.