Computational proteomics analysis of binding mechanisms and molecular signatures of the HIV-1 protease drugs

Artificial Intelligence in Medicine (2009) 45, 197—206

http://www.intl.elsevierhealth.com/journals/aiim

Computational proteomics analysis of binding mechanisms and molecular signatures of the HIV-1 protease drugs Gennady Verkhivker * Department of Pharmaceutical Chemistry, School of Pharmacy and Center for Bioinformatics, The University of Kansas, 2030 Becker Drive, Lawrence, KS 66047, USA Received 28 November 2007; received in revised form 12 August 2008; accepted 19 August 2008

KEYWORDS HIV-1 protease; Monte Carlo simulations; Dimerization inhibitors; Folding inhibition; Structural mimicry; Drug resistance

Summary Objective: Computational proteomics analysis of biomolecular interactions is proposed to determine molecular signatures of the HIV-1 protease inhibitors. A comparative microscopic analysis is conducted for a panel of inhibitors which exemplify a diversity of the HIV-1 PR binding mechanisms, from the active site inhibition to intervening with the protease folding and dimerization. Methods and materials: Replica-exchange Monte Carlo simulations with the conformational ensembles of the HIV-1 PR dimer and monomer structures enable a molecular analysis underlying diversity of the HIV-1 PR binding mechanisms. Results: We have investigated the molecular basis underlying diversity of the HIV-1 PR binding mechanisms. The molecular basis of the HIV-1 PR active site and dimerization inhibition mechanisms has been analyzed for an active site tripeptide inhibitor and a tetrapeptide inhibitor, which can act as both a dimerization inhibitor and a competitive active site inhibitor. We have also simulated a structural mimicry mechanism of the HIV-1 PR folding inhibition and dimerization, according to which the folding inhibitor targets the conserved HIV-1 PR regions by mimicking the interaction network of the active dimer. Conclusions: We have shown that binding interfaces of the studied dimerization and folding HIV-1 PR inhibitors may enable structural mimicry with the hot spot residues of the HIV-1 PR dimer. The proposed structural models of intervening with the HIV-1 PR dimerization and folding support the mechanism of structural mimicry, which may alleviate drug resistance effects. # 2008 Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: +1 785 864 1978; fax: +1 785 864 5558. E-mail address: [email protected]. 0933-3657/$ — see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.artmed.2008.08.011

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1. Introduction 1.1. Structural studies of HIV-1 protease HIV-1 protease (HIV-1 PR) plays an important role in processing the viral polypeptide precursors, critical for the life-cycle of HIV, and continues to present an important target for the design of antiviral agents. The steadily increasing number of crystal structures of the HIV-1 PR complexes has been instrumental in understanding HIV-1 PR flexibility and dynamics, associated with the binding of HIV-1 PR substrates and inhibitors, and continues to play a pivotal role in the structure-based design of novel HIV-1 PR therapeutics [1—4]. Crystal structures of the HIV-1 PR complexes have revealed that the protease flaps can exhibit a spectrum of motions ranging from closed, when the active site is occupied by a ligand, to semi-open, typically observed in the free HIV-1 PR, indicating that the flaps, which control access to the active site, can have a considerable mobility in solution. The solution nuclear magnetic resonance (NMR) experiments of the free HIV-1 PR have discovered that the ensemble of the HIV-1 PR unbound structures can include less structured, ‘‘open flap’’ protease conformations, which permit access to the active site as an outcome of a rare event in the slow conformational exchange [5—8]. Theoretical studies have enabled a detailed molecular picture of the HIV-1 PR motions, providing valuable insights into the enzyme motions and respective function on biologically relevant timescales [9—21]. The increasing body of NMR data and molecular dynamics simulations of the HIV-1 PR motions have demonstrated that functionally relevant HIV-1 PR motions include the initial opening of the flaps that allows the substrate/drug to access the active site, the closing of the flaps and the positioning of the substrate to make the enzyme catalytically competent, and the subsequent reopening of the flaps that allows the products or inhibitor to escape the active site.

1.2. HIV-1 protease folding and dimerization Dimerization of the HIV-1 PR is indispensable for catalytic activity because each subunit contributes one of the two catalytic aspartic acid residues that form the active site. The two major areas that constitute the dimmer interface are the active site region 24—29, that encompasses the triplet Asp-25/ Thr-26/Gly-27 forming the ‘‘fireman’s grip’’ hydrogen bond network, and the four-stranded anti-parallel b-sheet, that is formed by interdigitation of the C- and N-terminal residues of the HIV-1 PR. These interfaces are formed from the evolutionary and

G. Verkhivker structurally conserved segments 24—34 and 83—93 of the HIV-1 PR fold. The protection patterns obtained for the HIV-1 PR units 24—34, 74—78 and 83—93 have shown that these conserved segments of HIV-1 PR constitute the protease folding core [22]. Subsequent NMR analysis of the HIV-1 PR folding propensities have indicated that the extended folding core of the protease may include additional residues from the active site, hinge region, and dimerization domain [23,24]. A systematic understanding of the HIV-1 PR folding and dimerization requires structural analysis of the HIV-1 PR monomer in its precursor and mature forms. NMR studies have revealed that the mature HIV-1 PR lacking its terminal b-sheet residues 1—4 and 96—99 can exhibit a stable monomer fold that is similar to that of the single subunit of the wild-type dimer [25—28]. Furthermore, the HIV-1 PR mutants R87K, D29N and T26A of the active site residues involved in the ‘‘fireman’s grip’’ interfacial network can also form a stable monomeric structure [25,26]. The highly conserved Arg-87 residue in the conserved triad Gly86-Arg87-(Asn/Asp88) of retroviral proteases appeared to play a crucial role in the stability of the HIV-1 PR dimer. Loss of specific intramonomer interactions between Arg-87 and Asp-29 in the HIV-1 PR mutants results in a destabilization of the dimer interfaces, particularly between the C-terminal bstands [25—28]. A comparison of the solution NMR structures of the HIV-1 PR monomers with the subunit of the uninhibited HIV-1 PR dimer has demonstrated that, with the exception of the terminal regions (residues 1—10 and 91—95) that are disordered, the tertiary folds of the HIV-1 PR monomer and a single subunit of the HIV-1 PR dimer are essentially identical [27].

1.3. HIV-1 protease inhibition mechanisms and inhibitors The diversity of the HIV-1 PR inhibition scenarios may include the conventional active site binding and alternative inhibitory mechanisms, based on blocking the assembly of the HIV-1 PR homodimer and disrupting the dimeric interface. The active site inhibitors of the HIV-1 PR typically incorporate a high degree of hydrophobicity and bind symmetrically with both HIV-1 PR subunits. However, the tripeptide inhibitor Glu-Asp-Leu, which was derived from the transframe octapeptide Phe-Leu-Arg-GluAsp-Leu-Ala-Phe (TFP) and forms a crystallographically determined complex with the HIV-1 PR dimer with a K i value of 50 mM, is qualitatively different from known active site inhibitors and elicits a favorable binding enthalpy [29,30]. Design of HIV-PR dimerization inhibitors is typically focused on dis-

Computational proteomics analysis of biomolecular interactions rupting four-stranded b-sheet and targeting flexible N- and C-termini that constitute most of the dimer interface [31—36]. It is important to stress that if the inhibitor can bind to the interface region of a monomeric HIV-1 PR subunit, X-ray crystallography techniques may not be readily applicable, since the necessary high concentrations of the enzyme tend to shift the equilibrium toward the dimer. However, in vivo, active site inhibitors may also act as dimerization inhibitors (53). In particular, the tetrapeptide Ac-Ser-Tyr-Glu-Leu-OH (Ac-SYEL-OH) can exhibit both the dimerization mode of inhibition (K i ¼ 8:7 mM) and the active site mechanism of inhibition (K i ¼ 12:6mM) [37]. The first structural study of the dimerization inhibition of HIV-1 PR by NMR was recently reported [38,39], in which the tetrapeptide inhibitor Ac-SYEL-OH was shown to bind with the monomer using elements of structural mimicry with the other HIV-1 PR monomer. According to the computer simulations of the HIV-1 PR folding, the HIV-1 PR active dimer is likely formed by the association of folded HIV-1 PR monomers rather than by direct coupling between the monomer folding and binding. Consequently, it was suggested that HIV-1 PR dimerization inhibitors should not only focus on the flexible N- and C-termini of the dimer interface, but could be also rationally designed to target structurally conserved segment 24—34 of HIV-1 PR [40]. Discovery of novel classes of the HIV-1 PR therapeutic agents effectively intervening with the HIV-1 PR dimerization and folding have sparkled a renaissance of experimental and theoretical efforts in deciphering the mechanisms of the HIV-1 PR inhibition. It has been recently shown that the peptides with a sequence identical to the evolutionary and structurally conserved segments of the HIV-1 PR folding core may act as nonconventional drugs which can inhibit folding of HIV-1 PR monomer and formation of the active dimer [41— 44]. The biochemical assays have shown that the peptide NIIGRNLLTQI, displaying a sequence identical to that of the HIV-1 PR folding unit (83—93), exhibits biological activity and can inhibit HIV-1 PR folding and dimerization with an appreciable binding affinity (K i ¼ 2:58mM) [42]. Theoretical models and computer simulations have suggested that the detected inhibitory activity of the folding HIV-1 PR inhibitor may be controlled by the stable local elementary structure of the peptide which enables specific interactions with the complementary segment 24—34 of the folding HIV-1 PR core and can prevent folding and subsequent dimerization of the enzyme [41—44]. Despite a growing body of structural and biochemical data, the molecular origins underlying this diversity of the HIV-1 PR inhibition mechanisms are not fully understood. Furthermore,

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the molecular and energetic details of the inhibitor intervention with the HIV-1 PR folding and dimerization remains largely unknown, despite a rapidly increasing experimental and theoretical efforts in this area. In this work, we report the results of a comparative microscopic analysis conducted for a panel of the peptide inhibitors which exemplify a diversity of the HIV-1 PR inhibition mechanisms, ranging from the active site inhibition to intervening with the formation of the active HIV-1 PR dimer and preventing HIV-1 PR folding and dimerization.

2. Methods and materials 2.1. Structural analysis and preparation All available crystal structures of the wild-type and mutant HIV-1 PR complexes (more than 300 structures) [45] were used to categorize structural landscape of the HIV-1 PR conformational states. The conformational ensemble of the HIV-1 monomeric conformations is obtained from the crystal structures of the unliganded HIV-1 PR and HIV-1 PR complexes with the inhibitors, followed by subsequent thermal equilibration and minimization procedures. We have equilibrated the HIV-1 PR dimeric and monomeric structures at 300 K using molecular dynamics simulations performed with the GROMACS package and employing OPLS/AMBER force field within a cubic box of 6.7 nm around the protein, with 9428 water molecules and 2 chloride ions [46,47]. The following steps have been carried out : (1) a steepest descent energy minimization; (2) equilibration of water for 300 ps at 300 K keeping constrained the heavy atoms of the protein; (3) a 300 ps dynamics at 300 K at constant volume to thermalize the system. Although there may exist differences in the time scale of the flap motions in the HIV-1 PR monomeric and dimeric states, the tertiary folds of the HIV-1 PR monomer and a single subunit of the HIV-1 PR dimer are essentially identical which may be captured in simulations with the conformational ensembles of the HIV-1 PR conformations.

2.2. Replica-exchange Monte Carlo binding simulations The hierarchical simulation approach which includes a combination of simplified molecular recognition energy model and sampling of the inhibitor—protein interactions followed by a more detailed MM/GBSA free energy simulations have been extensively documented and used for a variety of biological systems in our previous studies [48,49].

200 Here, we summarize the major ingredients of the computational model used in this particular study. Equilibrium sampling of the peptide conformations is first performed by replica-exchange Monte Carlo simulations with the ensemble of the HIV-1 PR dimeric and monomeric states. In the second stage of the protocol, the inhibitor binding free energies are computed using the obtained equilibrium samples of the inhibitor and respective protein conformations obtained at any given temperature. The molecular recognition energetic model used in this study includes intramolecular energy terms, given by torsional and nonbonded contributions of the DREIDING force field [50], and the intermolecular energy contributions calculated using the AMBER force field [51,52] to describe protein—protein interactions combined with an implicit solvation model [53]. The dispersion—repulsion and electrostatic terms have been modified and include a soft core component that was originally developed in free energy simulations to remove the singularity in the potentials and improve numerical stability of the simulations [54]. A solvation term was added to the interaction potential to account for the free energy of interactions between the explicitly modeled atoms of the system. Equilibrium simulations with the ensembles of HIV-1 conformational states are carried out using

G. Verkhivker parallel simulated tempering dynamics [55—57] with 300 replicas of the ligand—protein system attributed respectively to 300 different temperature levels that are uniformly distributed in the range between 3300 and 300 K. In simulations with ensembles of multiple protein conformations, protein conformations are linearly assigned to each temperature level, that implies a consecutive assignment of protein conformations starting from the highest temperature level and allows each protein conformation from the ensemble at least once be assigned to a certain temperature level. Starting with the highest temperature, every pair of adjacent temperature configurations is tested for swapping until the final lowest value of temperature is reached. This process of swapping configurations is repeated 100 times after each simulation cycle for all replicas. The inhibitor conformations and orientations are sampled in a parallelepiped that encompasses the superimposed HIV-1 PR structures with a ˚ cushion added to every side of the box large 20.0 A surrounding the binding interface. The protein structure of each complex is held fixed in its minimized and equilibrated conformation, while rigid body degrees of freedom and the inhibitor rotatable angles are treated as independent variables. Binding free energies are computed using the molecular mechanics AMBER force field [51,52] and the solva-

Figure 1 Chemical structures of the HIV-1 PR inhibitors studied in this work: (A) a tripeptide analog Glu-Asp-Leu of the transframe octapeptide transframe octapeptide Phe-Leu-Arg-Glu-Asp-Leu-Ala-Phe (TFP), which acts as a conventional active site inhibitor. (B) An acetylated tetrapeptide inhibitor Ac-Ser-Tyr-Glu-Leu-OH, acting as both dimerization inhibitor with a K i value of 8.7 mM and a competitive active site inhibitor with K i value of about 12.6 mM. (C) A folding HIV-1 PR inhibitor, a peptide with a sequence NIIGRNLLTQI of the structurally conserved HIV-1 PR unit 83—93, which can inhibit the HIV-1 PR folding and dimerization. (D) Structure of the HIV-1 PR monomer with the structure of the folding peptide corresponding to the 83—93 segment of the folding core shown in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Computational proteomics analysis of biomolecular interactions tion energy term based on continuum generalized Born and solvent accessible surface area (GB/SA) solvation model [58—60].

3. Results and discussion 3.1. HIV-1 PR binding mechanisms: active site inhibition We have performed a microscopic analysis for a panel of the HIV-1 PR peptide inhibitors exhibiting different inhibition mechanisms, including the active site inhibition and intervening with the HIV-1 PR folding and dimerization. The studied active site tripeptide Glu-Asp-Leu acts as a conventional active site inhibitor with a K i value of 50 mM and forms a crystallographically determined complex with the HIV-1 PR dimer (Fig. 1 A). In contrast to the majority of so-called symmetric HIV-1 PR inhibitors which typically span the entire active site and interact essentially equally with both HIV-1 PR sub-

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units, the interactions of the tripeptide Glu-Asp-Leu are not symmetrically distributed within the two subunits of the HIV-1 PR , with one of the subunits contributing the vast majority of the favorable intermolecular contacts. A network of stable specific interactions is formed during equilibrium simulations between polar residues Glu and Asp of the tripeptide inhibitor with Gly-27, Asp-29, Asp-30, and Gly-48 of the first monomer (Fig. 2). These results are consistent with the structure-based thermodynamic analysis of the Glu-Asp-Leu inhibitor which binds to the HIV-1 protease with a favorable enthalpy, largely due to the favorable polar interactions established by the Glu and Asp peptide residues. Overall, in agreement with the experimentally observed active site inhibition, conformational equilibrium for the tripeptide Glu-Asp-Leu inhibitor binding is predominantly shifted towards the thermodynamically stable crystal structure complex with the HIV-1 PR dimer (Fig. 3). We suggest that the asymmetrical mode of the tripeptide binding with the HIV-1 PR, which is qualitatively differ-

Figure 2 Connolly surface representation of the HIV-1 PR (shown in light blue) bound to the tripeptide inhibitor GluAsp-Leu (shown in CPK model and default color): (A) the thermodynamically stable crystal structure complex formed by the tripeptide inhibitor with the HIV-1 PR dimer; (B) the meta-stable complex formed by the tripeptide inhibitor with the HIV-1 PR monomer; (C) A close-up of the binding interface formed by the tripeptide inhibitor in the crystal structure with the HIV-1 PR dimer. A network of stable specific interactions is predominantly formed with the active site residues of one of the HIV-1 PR subunits. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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G. Verkhivker ent from that of known inhibitors, may give rise to a minor fraction of the inhibitor molecules forming intermediate complexes with the monomeric form of the HIV-1 PR. In complexes with the HIV-1 PR monomers, the tripeptide inhibitor can interact with the residues 1—4 and 96—99 from the antiparallel b-sheet region which is implicated in the formation of the HIV-1 PR dimer and is usually targeted by dimerization inhibitors.

3.2. Molecular signatures of the HIV-1 PR inhibition Figure 3 The equilibrium distribution of the dimeric and monomeric states of the HIV-1 PR obtained from equilibrium simulations with the inhibitors at T ¼ 300 K: Peptide I is the tripeptide inhibitor Glu-Asp-Leu (shown in red); Peptide II is the tetrapeptide inhibitor Ac-Ser-TyrGlu-Leu-OH (shown in green); Peptide III is the folding inhibitor NIIGRNLLTQI (shown in blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

The molecular basis of the HIV-1 PR inhibition has been also analyzed for an acetylated tetrapeptide inhibitor Ac-SYEL-OH which may act as both dimerization inhibitor with a K i value of 8.7 mM and a competitive active site inhibitor with K i value of about 12.6 mM (Fig. 1B). The active site inhibition for the Ac-SYEL-OH tetrapeptide inhibitor may be fulfilled by the binding mode, which closely resembles the crystal structure of the Glu-Asp-Leu peptide and is characterized by an extensive network of the hydrogen bonds formed exclusively with the active site residues of only one of the HIV-1 PR subunits

Figure 4 Connolly surface representation of the HIV-1 PR (shown in light blue) bound to the Ac-SYEL-OH tetrapeptide (shown in CPK model and default color) in the thermodynamically stable complexes formed by the tetrapeptide inhibitor with the HIV-1 PR dimers (A) and HIV-1 PR monomers (C). A close-up of the binding interface and specific interactions formed by the tetrapeptide inhibitor in complexes with the HIV-1 PR dimmers (B) and HIV-1 PR monomers (D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Computational proteomics analysis of biomolecular interactions (Fig. 4 A and B). In this binding mode, the polar Ser residue of the inhibitor forms hydrogen bonding with the carbonyl oxygen of the Gly-27 and backbone NH group of the Asp-29. These interactions are further consolidated by anchoring the inhibitor via additional two hydrogen bonds with the with the flap residue Gly-48 of the same monomer. The dual character of the Ac-SYEL-OH inhibitor is accurately reproduced in the equilibrium distribution of the HIV-1 PR conformational states, which is almost equally populated by the ensembles of the HIV-1 PR dimers and monomers (Fig. 3). Accordingly, the calculated binding free energies with the HIV-1 PR dimeric and monomeric states reflect similar experimental binding affinities of the Ac-SYEL-OH inhibitor (Fig. 5). We have found that the dimerization mode of inhibition may be achieved by forming a thermodynamically stable binding mode with the favorable hydrophobic interactions to the Phe-99 and Trp-6 residues (Fig. 4 C and D). A network of specific hydrogen bonds can be also formed, in which the NH group of the peptide Leu residue interacts with the carbonyl oxygen of Ile-3; carboxyl group of the Glu peptide residue faces the NH group of the Leu-5; and carbonyl oxygen of the peptide Leu residue contacts the NH group of Asn-98 (Fig. 4 C and D).

Figure 5 A comparison of the computed and experimental binding affinities for the studied HIV-1 PR inhibitors in complexes with the HIV-1 PR dimers (active site inhibition mechanism) and HIV-1 PR monomers (dimerization inhibition). Active site binding free energies for the Peptide I (tripeptide inhibitor Glu-Asp-Leu) are displayed in red. Dimerization binding free energies for the Peptide II (Ac-Ser-Tyr-Glu-Leu-OH) are shown in green and the active site binding free energies for the Peptide II are displayed in blue. Dimerization binding free energies for the Peptide III (the folding inhibitor NIIGRNLLTQI) are shown in maroon. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Monte Carlo simulations with the conformational ensembles of the HIV-1 PR dimer and monomer structures provide access to the slower conformational exchanges between the dimer-inhibitor and monomer-inhibitor complexes, which have been detected in the NMR experiments [54,55]. The results of simulations suggest a dynamic equilibrium between the active site and dimerization modes of inhibition. The predicted structural model of dimerization inhibition agrees with the NMR experiments using a selective Trp side chain labeling, which have shown that the Ac-SYEL-OH inhibitor can disrupt the HIV-1 PR dimer and form a stable complex with the monomeric form of the enzyme [54,55]. The binding free energy, computed using the obtained equilibrium distribution of the inhibitor-protease states, provides an estimate of the binding affinity according to the observed experimental activity of the inhibitor (Fig. 5).

3.3. Structural mimicry mechanism of the HIV-1 PR folding inhibition We have also analyzed the atomic details of the binding mechanism of the folding HIV-1 PR inhibitor (Fig. 1C), a peptide with a sequence NIIGRNLLTQI of the structurally conserved HIV-1 PR unit 83—93 (Fig. 1D), which can inhibit the HIV-1 PR folding and dimerization [41—44]. In this work, we have complemented the recent peptide docking and molecular dynamics simulations of the folding inhibitor with explicit solvent [43,44], by conducting Monte Carlo binding simulations with the ensembles of HIV-1 PR conformational states, which confirm a primary mechanism of the peptide folding inhibition (Fig. 6). A consistent folding of the flexible peptide NIIGRNLLTQI to the stable conformation of the structurally conserved segment 83—93 of the HIV-1 PR may be partly facilitated by the binding requirements to provide a sufficient degree of structural mimicry with the key elements of the HIV-1 PR dimer interface. In the thermodynamically stable binding mode with the HIV-1 PR monomers, the folded peptide is favorably packed against segment 24—34 of the folding core by forming a network specific hydrogen bonds with Asp-25, Asp-29 and the side-chain of the Thr-26. In addition, hydrogen bonds are formed between the inhibitor and the backbone of the conserved Gly-49 and Ile-50 residues, which are unlikely to be mutated in the active protease variants (Fig. 6 A and B). Strikingly, the predicted interactions of the folding inhibitor with the HIV-1 PR monomers bear a considerable degree of structural mimicry with the ‘‘fireman’s grip’’ network of hydrogen bonds between monomers of the active

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Figure 6 Structural characterization of the peptide binding modes in complexes with the HIV-1 monomers. Connolly surface representation of the HIV-1 PR (shown in light blue) bound to the folding inhibitor NIIGRNLLTQI (shown in CPK model and default color) in the thermodynamically stable complexes which may be formed with the HIV-1 PR monomers (A and C). A close-up of the binding interfaces and specific interactions which may be formed by the folding inhibitor (atom-based representation shown in default colors) in complexes with the HIV-1 PR monomers, shown in green ribbons (B and D). A conventional dimerization mode of binding for the folding inhibitor mimics the anti-parallel a b-sheet region of the dimer interface (C and D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

HIV-1 PR dimer. The binding interface of folding HIV1 PR inhibitors may be formed through interactions with the evolutionary and structurally conserved elements of the HIV-1 PR interface. As a result, this mechanism may inhibit consolidation of the HIV-1 PR folding core, consisting of the interacting conserved segments 24—34 and 83—93. In the second energetically favorable binding mode, the peptide may favorably interact with the residues from the anti-parallel b-sheet comprising of residues 1—4 and 96—99 (Fig. 6 C and D). Indeed, since the active site of HIV-1 PR is formed by two half-enzymes, which are connected by a the anti-parallel b-sheet involving the N- and C-termini of both monomers, enzyme activity can be abolished by therapeutic agents targeting the dimer interface thereby interfering with formation or stability of the functional HIV-1 PR dimer. These results provide an additional support to the proposed structural models of intervening with the HIV-1 PR dimerization and folding. The proposed structural models of binding based on Monte Carlo binding simulations with the ensembles of HIV-1 PR dimer and monomer conformations fully agree with the previous all-atom molecular dynamics simula-

tions in explicit solvent [43,44] and, the folding inhibitor may target the conserved HIV-1 PR regions by mimicking specific interactions in the active HIV1 PR dimer and thereby facilitate the inhibitor intervention with the HIV-1 PR folding and dimerization. Consequently, this class of HIV-1 PR therapeutic agents may reduce the emergence of drug resistant strains due to a reduced probability for the active HIV-1 PR mutants to evolve variations at these sites of the interface. Theoretical and experimental studies of the non-conventional HIV-1 PR inhibitors may ultimately facilitate understanding of the viral life cycle and develop novel therapeutic strategies to combat drug resistance.

4. Conclusions The molecular basis underlying diversity of the HIV-1 PR binding mechanisms is examined for a panel of the peptide inhibitors using Monte Carlo simulations with the conformational ensembles of the HIV-1 PR dimeric and monomeric structures. A comparative microscopic analysis of the HIV-1 PR binding is performed for a tripeptide analog Glu-Asp-Leu of the

Computational proteomics analysis of biomolecular interactions transframe octapeptide, acting as a conventional active site inhibitor, and an acetylated tetrapeptide Ac-SYEL-OH, which can act as both dimerization inhibitor and a competitive active site inhibitor. We have also investigated binding of the peptide NIIGRNLLTQI, a folding HIV-1 PR inhibitor with a sequence of the structurally conserved unit 83—93 from the HIV-1 PR folding core. In agreement with the NMR and biochemical studies, we have found that the tetrapeptide inhibitor Ac-SYEL-OH can maintain both the active site and dimerization modes of inhibition using a dynamic equilibrium between thermodynamically stable complexes formed with the HIV-1 PR dimeric and monomeric states. According to the results of this study, the folding inhibitor may target the conserved HIV-1 PR regions by mimicking the ‘‘fireman’s grip’’ hydrogen bond network of the active dimer and thereby intervening with the HIV-1 PR folding and dimerization. The mechanism of structural mimicry may alleviate drug resistance effects because targeting the conserved HIV-1 PR regions, which are responsible for the protease folding and stability, is unlikely to induce mutations in these crucial for the native function regions. In silico screening of the inhibitors for elements of structural mimicry with the dimer interface may assist experimental techniques in discriminating between competitive and dissociative inhibition mechanisms and identifying dimerization interface HIV-1 PR inhibitors. The presented computational approach may be also useful in complementing proteomics profiling to characterize binding signatures and specificities of small molecules against important protein targets.

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