Peptides derived from HIV-1 Rev inhibit HIV-1 integrase in a shiftide mechanism

The HIV-1 Integrase protein (IN) mediates the integration of the viral cDNA into the host genome. IN is an emerging target for anti-HIV drug design, and the first IN-inhibitor was recently approved by the FDA. We have developed a new approach for inhibiting IN by "shiftides": peptides derived from its cellular binding protein LEDGF/p75 that inhibit IN by shifting its oligomerization equilibrium from the active dimer to an inactive tetramer. In addition, we described two peptides derived from the HIV-1 Rev protein that interact with IN and inhibit its activity in vitro and in cells. In the current study, we show that the Rev-derived peptides also act as shiftides. Analytical gel filtration and cross-linking experiments showed that IN was dimeric when bound to the viral DNA, but tetrameric in the presence of the Rev-derived peptides. Fluorescence anisotropy studies revealed that the Rev-derived peptides inhibited the DNA binding of IN. The Rev-derived peptides inhibited IN catalytic activity in vitro in a concentration-dependent manner. Inhibition was much more significant when the peptides were added to free IN before it bound the viral DNA than when the peptides were added to a preformed IN-DNA complex. This confirms that the inhibition is due to the ability of the peptides to shift the oligomerization equilibrium of the free IN toward a tetramer that binds much weaker to the viral DNA. We conclude that protein-protein interactions of IN may serve as a general valuable source for shiftide design.     

发现靶向蛋白质间相互作用的小分子药物研究进展

蛋白质-蛋白质相互作用在多种细胞功能中具有重要的作用。靶向蛋白质-蛋白质相互作用已经成为新药发现的重要策略,但发现能阻断蛋白质-蛋白质相互作用的小分子药物是一个巨大的挑战。尽管如此,近年来人们还是发现了许多能调控蛋白质-蛋白质相互作用的小分子。该文主要总结了在病毒进入、细胞凋亡通路和神经退行性疾病等方面的蛋白质-蛋白质相互作用小分子抑制剂的研究进展。     

Peptide inhibitors of HIV-1 integrase: From mechanistic studies to improved lead compounds

The HIV-1 integrase enzyme (IN) catalyzes integration of viral DNA into the host genome. We previously developed peptides that inhibit IN in vitro and HIV-1 replication in cells. Here we present the design, synthesis and evaluation of several derivatives of one of these inhibitory peptides, the 20-mer IN1. The peptide corresponding to the N-terminal half of IN1 (IN1 1–10) was easier to synthesize and much more soluble than the 20-mer IN1. IN1 1–10 bound IN with improved affinity and inhibited IN activity as well as HIV replication and integration in infected cells. While IN1 bound the IN tetramer, its shorter derivatives bound dimeric IN. Mapping the peptide binding sites in IN provided a model that explains this difference. We conclude that IN1 1–10 is an improved lead compound for further development of IN inhibitors.     

Identification of old drugs as potential inhibitors of HIV-1 integrase – human LEDGF/p75 interaction via molecular docking

Integration of viral-DNA into host chromosome mediated by the viral protein HIV-1 integrase (IN) is an essential step in the HIV-1 life cycle. In this process, human protein Lens epithelium-derived growth factor (LEDGF/p75) is discovered to function as a cellular co-factor for integration. LEDGF/p75-HIV-1 IN interaction represents an attractive target for anti-HIV therapy. In this study, approved drugs were investigated for the finding of potential inhibitors on this target. Via molecular docking against the LEDGF/p75-binding pocket of HIV-1 IN, 26 old drugs were selected from the DrugBank and purchased for bioassays. Among them, eight, namely Atorvastatin, Bumetanide, Candesartan, Carbidopa, Diclofenac, Diflunisal, Eprosartan, and Sulindac, were identified as potential inhibitors of LEDGF/p75- HIV-1 IN interaction, whose IC₅₀ values ranged from 6.5?μM to 36.8?μM. In addition, Atorvastatin was previously reported to block HIV-1 replication and may have an important implication for the treatment of AIDS. Our results suggested a mechanism of action for the anti-HIV effects of Atorvastatin. This work provides a new example of inhibitors targeting protein-protein interaction and confirmed that old drugs were valuable sources for antiviral drug discovery.     

A time-resolved fluorescence assay to identify small-molecule inhibitors of HIV-1 fusion

Fusion of host cell and human immunodeficiency virus type 1 (HIV-1) membranes is mediated by the 2 "heptad-repeat" regions of the viral gp41 protein. The collapse of the C-terminal heptad-repeat regions into the hydrophobic grooves of a coiled-coil formed by the corresponding homotrimeric N-terminal heptad-repeat regions generates a stable 6-helix bundle. This brings viral and cell membranes together for membrane fusion, facilitating viral entry. The authors developed an assay based on soluble peptides derived from the gp41 N-terminal heptad-repeat region (IQN36) as well as from the C-terminal region (C34). Both peptides were labeled with fluorophores, IQN36 with allophycocyanin (APC) and C34 with the lanthanide europium (Eu3+). Formation of the 6-helix bundle brings both fluorophores in close proximity needed for F?rster resonance energy transfer (FRET). Compounds that interfere with binding of C34-Eu with IQN36-APC suppress the FRET signal. The assay was validated with various peptides and small molecules, and quenching issues were addressed. Evaluation of a diversified compound collection in a high-throughput screening campaign enabled identification of small molecules with different chemical scaffolds that inhibit this crucial intermediate in the HIV-1 entry process. This study's observations substantiate the expediency of time-resolved FRET-based assays to identify small-molecule inhibitors of protein-protein interactions.     

Protein-protein interactions and cancer: small molecules going in for the kill

There has been much progress in the discovery of small, organic molecules that inhibit protein-protein interactions, particularly in the field of cancer. Tubulin polymerization represents a classic target whose function can be allosterically modulated by small molecules. Several protein-protein complexes that regulate apoptosis, or programmed cell death, appear to be particularly amenable to inhibition by small molecules, and recently described compounds have helped to characterize Bcl-2, MDM2 and XIAP as drug targets. Additionally, small-molecule antagonists have recently been described for several new targets, including Rac1-Tiam1, beta-catenin-T cell factor (Tcf), and Sur-2-ESX. Not only is the list of protein-protein inhibitors growing, but the inhibitors themselves are moving closer to treating disease.     

Towards structure-based protein drug design

Structure-based drug design for chemical molecules has been widely used in drug discovery in the last 30 years. Many successful applications have been reported, especially in the field of virtual screening based on molecular docking. Recently, there has been much progress in fragment-based as well as de novo drug discovery. As many protein-protein interactions can be used as key targets for drug design, one of the solutions is to design protein drugs based directly on the protein complexes or the target structure. Compared with protein-ligand interactions, protein-protein interactions are more complicated and present more challenges for design. Over the last decade, both sampling efficiency and scoring accuracy of protein-protein docking have increased significantly. We have developed several strategies for structure-based protein drug design. A grafting strategy for key interaction residues has been developed and successfully applied in designing erythropoietin receptor-binding proteins. Similarly to small-molecule design, we also tested de novo protein-binder design and a virtual screen of protein binders using protein-protein docking calculations. In comparison with the development of structure-based small-molecule drug design, we believe that structure-based protein drug design has come of age.     

Identification of Peptide Inhibitors of Enveloped Viruses Using Support Vector Machine

The peptides derived from envelope proteins have been shown to inhibit the protein-protein interactions in the virus membrane fusion process and thus have a great potential to be developed into effective antiviral therapies. There are three types of envelope proteins each exhibiting distinct structure folds. Although the exact fusion mechanism remains elusive, it was suggested that the three classes of viral fusion proteins share a similar mechanism of membrane fusion. The common mechanism of action makes it possible to correlate the properties of self-derived peptide inhibitors with their activities. Here we developed a support vector machine model using sequence-based statistical scores of self-derived peptide inhibitors as input features to correlate with their activities. The model displayed 92% prediction accuracy with the Matthew’s correlation coefficient of 0.84, obviously superior to those using physicochemical properties and amino acid decomposition as input. The predictive support vector machine model for self- derived peptides of envelope proteins would be useful in development of antiviral peptide inhibitors targeting the virus fusion process.     

A structural model of the HIV-1 Rev-integrase complex: the molecular basis of integrase regulation by Rev

The HIV-1 Rev and integrase (IN) proteins control important functions in the viral life cycle. We have recently discovered that the interaction between these proteins results in inhibition of IN enzymatic activity. Peptides derived from the Rev and IN binding interfaces have a profound effect on IN catalytic activity: Peptides derived from Rev inhibit IN, while peptides derived from IN stimulate IN activity by inhibiting the Rev-IN interaction. This inhibition leads to multi integration, genomic instability and specific death of virus-infected cells. Here we used protein docking combined with refinement and energy function ranking to suggest a structural model for the Rev-IN complex. Our results indicate that a Rev monomer binds IN at two sites that match our experimental binding data: (1) IN residues 66-80 and 118-128; (2) IN residues 174-188. According to our model, IN binds Rev and its cellular cofactor, lens epithelium derived growth factor (LEDGF), through overlapping interfaces. This supports previous observations that IN is regulated by a tight interplay between Rev and LEDGF. Rev may bind either the IN dimer or tetramer. Accordingly, Rev is suggested to inhibit IN by two possible mechanisms: (i) shifting the oligomerization equilibrium of IN from an active dimer to an inactive tetramer; (ii) displacing LEDGF from IN, resulting in inhibition of IN binding to the viral DNA. Our model is expected to contribute to the development of lead compounds that inhibit the Rev-IN interaction and thus lead to multi-integration of viral cDNA and consequently to apoptosis of HIV-1 infected cells.     

Genetic selection for dissociative inhibitors of designated protein-protein interactions

Many biological processes rely on protein-protein interactions. These processes include signal transduction, cell cycle regulation, gene regulation, and viral assembly and replication. Moreover, many proteins and enzymes manifest their function as oligomers. We describe here an efficient means to sift through large combinatorial libraries and identify molecules that block the interaction of target proteins in vivo. The power of this approach is demonstrated by the identification of nine-residue peptides from a combinatorial library that inhibit the intracellular dimerization of HIV-1 protease. Fewer than 1 in 106 peptides do so. In vitro biochemical analyses of one such peptide demonstrate that it acts by dissociating HIV-1 protease into monomers, which are inactive catalysts. Inhibition is enhanced further by dimerizing the peptide. This approach enables the facile identification of new molecules that control cellular processes.     

Computational identification of self-inhibitory peptides from envelope proteins

Fusion process is known to be the initial step of viral infection and hence targeting the entry process is a promising strategy to design antiviral therapy. The self-inhibitory peptides derived from the enveloped (E) proteins function to inhibit the protein-protein interactions in the membrane fusion step mediated by the viral E protein. Thus, they have the potential to be developed into effective antiviral therapy. Herein, we have developed a Monte Carlo-based computational method with the aim to identify and optimize potential peptide hits from the E proteins. The stability of the peptides, which indicates their potential to bind in situ to the E proteins, was evaluated by two different scoring functions, dipolar distance-scaled, finite, ideal-gas reference state and residue-specific all-atom probability discriminatory function. The method was applied to α-helical Class I HIV-1 gp41, β-sheet Class II Dengue virus (DENV) type 2 E proteins, as well as Class III Herpes Simplex virus-1 (HSV-1) glycoprotein, a E protein with a mixture of α-helix and β-sheet structural fold. The peptide hits identified are in line with the druggable regions where the self-inhibitory peptide inhibitors for the three classes of viral fusion proteins were derived. Several novel peptides were identified from either the hydrophobic regions or the functionally important regions on Class II DENV-2 E protein and Class III HSV-1 gB. They have potential to disrupt the protein-protein interaction in the fusion process and may serve as starting points for the development of novel inhibitors for viral E proteins.     

Dynamic modulation of HIV-1 integrase structure and function by cellular lens epithelium-derived growth factor (LEDGF) protein

The mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra- and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target.     

Interaction between HIV-1 Rev and integrase proteins: a basis for the development of anti-HIV peptides

Human immunodeficiency virus 1 (HIV-1) Rev and integrase (IN) proteins are required within the nuclei of infected cells in the late and early phases of the viral replication cycle, respectively. Here we show using various biochemical methods, that these two proteins interact with each other in vitro and in vivo. Peptide mapping and fluorescence anisotropy showed that IN binds residues 1-30 and 49-74 of Rev. Following this observation, we identified two short Rev-derived peptides that inhibit the 3'-end processing and strand-transfer enzymatic activities of IN in vitro. The peptides bound IN in vitro, penetrated into cultured cells, and significantly inhibited HIV-1 in multinuclear activation of a galactosidase indicator (MAGI) and lymphoid cultured cells. Real time PCR analysis revealed that the inhibition of HIV-1 multiplication is due to inhibition of the catalytic activity of the viral IN. The present work describes novel anti-HIV-1 lead peptides that inhibit viral replication in cultured cells by blocking DNA integration in vivo.     

Study on the molecular mechanism of inhibiting HIV-1 integrase by EBR28 peptide via molecular modeling approach

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the HIV-1 lifecycle which aids the integration of viral DNA into the host chromosome. Recently synthesized 12-mer peptide EBR28, which can strongly bind to IN, is one of the most potential small peptide leading compounds inhibiting IN binding with viral DNA. However, the binding mode between EBR28 peptide with HIV-1 IN and the inhibition mechanism remain uncertain. In this paper, the binding modes of EBR28 with HIV-1 IN monomer core domain (IN(1)) and dimmer core domain (IN(2)) were investigated by using molecular docking and molecular dynamics (MD) simulation methods. The results indicated that EBR28 bound to the interfaces of the IN(1) and IN(2) systems mainly through the hydrophobic interactions with the beta3, alpha1 and alpha5 regions of the proteins. The binding free energies for IN(1) with a series of EBR28 mutated peptides were calculated with the MM/GBSA model, and the correlation between the calculated and experimental binding free energies is very good (r=0.88). Thus, the validity of the binding mode of IN(1) with EBR28 was confirmed. Based on the binding modes, the inhibition mechanism of EBR28 was explored by analyzing the essential dynamics (ED), energy decomposition and the mobility of EBR28 in the two docked complexes. The proposed inhibition mechanism is represented that EBR28 binds to the interface of IN(1) to form the IN(1)_EBR28 complex and preventes the formation of IN dimmer, finally leads to the partial loss of binding potency for IN with viral DNA. All of the above simulation results agree well with experimental data, which provide us with some helpful information for designing anti-HIV small peptide drugs.     

Why is Substrate Peptide Binding Unsusceptible to Multidrug-Resistant Mutations in HIV-1 Protease? A Structural and Energetic Analysis

Understanding of the molecular mechanism and biological implication underlying the difference in binding of substrate peptides and small-molecule inhibitors to multidrug-resistant mutants of HIV-1 protease would help to develop new anti-HIV agents combating drug resistance. Here, an integration of rigorous quantum mechanics/molecular mechanics (QM/MM) analysis and empirical Poisson–Boltzmann/surface area (PB/SA) model is described to investigate the structural basis and energetic property of wild-type HIV-1 protease and its mutants in recognizing and binding with a wide variety of ligands, including the peptides derived from its cognate cleavage sites and the cleavage site variants as well as a number of FDA-approved protease inhibitors, attempting to explain why is substrate binding unsusceptible to most observed HIV-1 protease mutations. A preliminary test study demonstrates that the combined QM/MM–PB/SA scheme is able to effectively reproduce the relative ligand binding energy changes upon protease single- and double-mutations, albeit the absolute values appear to be different significantly between the calculated and experimental results. With the QM/MM–PB/SA calculations a complete mutation energy map of HIV-1 protease–ligand interactions is created, which unravels distinct affinity pictures of wild-type substrates, substrate variants and, particularly, the protease inhibitors bound to HIV-1 protease mutants, suggesting that, on the one hand, the evaluation pressure under anti-HIV chemotherapies addresses site-directed protease mutations that impair and undermine the intermolecular interactions specific to inhibitors but not substrates; on the other hand, co-evaluation of protease and its substrate peptides provides a more effective mechanism to avoid therapeutic surveillance. Further, nonbonded interaction analysis and computational alanine scanning reveal 12 key residues that is critical for substrate binding, from which the Asn25, Gly27, Ala28, Asp29 and Pro81 are identified that have not yet been found to cause drug resistance and hence would be the promising sites targeted by new protease inhibitors.     

Structure-based virtual screening toward the discovery of novel inhibitors for impeding the protein-protein interaction between HIV-1 integrase and human lens epithelium-derived growth factor (LEDGF/p75)

HIV-1 integrase is a unique promising component of the viral replication cycle, catalyzing the integration of reverse transcribed viral cDNA into the host cell genome. Generally, IN activity requires both viral as well as a cellular co-factor in the processing replication cycle. Among them, the human lens epithelium-derived growth factor (LEDGF/p75) represented as promising cellular co-factor which supports the viral replication by tethering IN to the chromatin. Due to its major importance in the early steps of HIV replication, the interaction between IN and LEDGF/p75 has become a pleasing target for anti-HIV drug discovery. The present study involves the finding of novel inhibitor based on the information of dimeric CCD of IN in complex with known inhibitor, which were carried out by applying a structure-based virtual screening concept with molecular docking. Additionally, Free binding energy, ADME properties, PAINS analysis, Density Functional Theory, and Enrichment Calculations were performed on selected compounds for getting a best lead molecule. On the basis of these analyses, the current study proposes top 3 compounds: Enamine-Z742267384, Maybridge-HTS02400, and Specs-AE-848/37125099 with acceptable pharmacological properties and enhanced binding affinity to inhibit the interaction between IN and LEDGF/p75. Furthermore, Simulation studies were carried out on these molecules to expose their dynamics behavior and stability. We expect that the findings obtained here could be future therapeutic agents and may provide an outline for the experimental studies to stimulate the innovative strategy for research community.