Correlation between shiftide activity and HIV-1 integrase inhibition by a peptide selected from a combinatorial library

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein is an emerging target for the development of anti-HIV drugs. We recently described a new approach for inhibiting IN by “shiftides”—peptides that inhibit the protein by shifting its oligomerization equilibrium from the active dimer to the inactive tetramer. In this study, we used the yeast two-hybrid system with the HIV-1 IN as a bait and a combinatorial peptide aptamer library as a prey to select peptides of 20 amino acids that specifically bind IN. Five non-homologous peptides, designated as IN-1 to IN-5, were selected. ELISA studies confirmed that IN binds the free peptides. All the five peptides interact with IN with comparable affinity (K_d≈10 μM), as was revealed by fluorescence anisotropy studies. Only one peptide, IN-1, inhibited the enzymatic activity of IN in vitro and the HIV-1 replication in cultured cells. In correlation, fluorescence anisotropy binding experiments revealed that of the five peptides, only the inhibitory IN-1 inhibited the DNA binding of IN. Analytical gel filtration experiments revealed that only the IN-1 and not the four other peptides shifted the oligomerization equilibrium of IN towards the tetramer. Thus, the results show a distinct correlation between the ability of the selected peptides to inhibit IN activity and that to shift its oligomerization equilibrium.     

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.     

A comparative study of backbone versus side chain peptide cyclization: application for HIV-1 integrase inhibitors

Peptide cyclization is an important tool for overcoming the limitations of linear peptides as drugs. Backbone cyclization (BC) has advantages over side chain (SC) cyclization because it combines N-alkylation for extra peptide stability. However, the appropriate building blocks for BC are not yet commercially available. This problem can be overcome by preparing SC cyclic peptide analogs of the most active BC peptide using commercially available building blocks. We have recently developed BC peptides that inhibit the HIV-1 integrase enzyme (IN) activity and HIV-1 replication in infected cells. Here we used this system as a model for systematically comparing the BC and SC cyclization modes using biophysical, biochemical and structural methods. The most potent SC cyclic peptide was active almost as the BC peptide and inhibited IN activity in vitro and blocked IN activity in cells even after 6 days. We conclude that both cyclization types have their respective advantages: The BC peptide is more active and stable, probably due to the N-alkylation, while SC cyclic peptides are easier to synthesize. Due to the high costs and efforts involved in preparing BC peptides, SC may be a more approachable method in many cases. We suggest that both methods are interchangeable.     

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.     

A novel short peptide is a specific inhibitor of the human immunodeficiency virus type 1 integrase

The retroviral encoded protein integrase (IN) is required for the insertion of the human immunodeficiency virus type 1 (HIV-1) proviral DNA into the host genome. In spite of the crucial role played by IN in the retroviral life cycle, which makes this enzyme an attractive target for the development of new anti-AIDS agents, very few inhibitors have been described and none seems to have a potential use in anti-HIV therapy. To obtain potent and specific IN inhibitors, we used the two-hybrid system to isolate short peptides. Using HIV-1 IN as a bait and a yeast genomic library as the source of inhibitory peptides (prey), we isolated a 33-mer peptide (I33) that bound tightly to the enzyme. I33 inhibited both in vitro IN activities, i.e. 3' end processing and strand transfer. Further analysis led us to select a shorter peptide, EBR28, corresponding to the N-terminal region of I33. Truncated variants showed that EBR28 interacted with the catalytic domain of IN interfering with the binding of the DNA substrate. Alanine single substitution of each EBR28 residue (alanine scanning) allowed the identification of essential amino acids involved in the inhibition. The EBR28 NMR structure shows that this peptide adopts an alpha-helical conformation with amphipathic properties. Additionally, EBR28 showed a significant antiviral effect when assayed on HIV-1 infected human cells. Thus, this potentially important short lead peptide may not only be helpful to design new anti-HIV agents, but also could prove very useful in further studies of the structural and functional characteristics of HIV-1 IN.     

Conformational changes of peptides at solid/liquid interfaces: a Monte Carlo study

Monte Carlo simulations were performed to study the conformational changes of negatively charged model peptides dissolved in water adsorbed onto charged surfaces. 8-, 16-, and 20-residues peptides were used, each of them consisted of repeating diblock units of aspartic acid (ASP, polar amino acid) and isoleucine (ILE, nonpolar amino acid) residues. We found that a water patch was retained at the charged surface, separating the peptide from it. We believed that these water molecules were primarily responsible for giving a particular orientation to the peptide at the surface. Water did play a role to some extent in the structural stability of the 8-residues peptide. However, for higher chain lengths (16-residues and 20-residues), the intrinsic hydrogen-bonding network (or intrinsic structural stability) showed a predominant effect over hydrophobic dehydration for the stability of the peptide at the surface.     

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.     

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.     

Structure-activity of inhibition of HIV-1 integrase and virus replication by G-quartet oligonucleotides

As novel anti-HIV agents, the G-tetrad-forming oligonucleotides have been explored for their structure-activity relations with regard to inhibition of integrase (IN) (N. Jing, Expert Opin. Investig. Drugs (2000) 9, 1777-1785). We have now developed two families of G-quartet oligonucleotides: T40217-T40222, with potential formation of a tail-to-tail G-quartet dimer, and T40224-T40227, with phosphorothioate (PT) linkages in the guanine loops. The results obtained from biophysical measurements and the assays of the inhibition of HIV-1 IN and virus replication demonstrated that an increase in the length of the G-quartet structure from a monomer (15A) to a tail-to-tail dimer (47A) does not distinctly disrupt the inhibition of HIV-1 IN activity or the inhibition of HIV-1 replication in cell cultures. G-quartet oligonucleotides were observed to induce molecular aggregation of HIV-1 IN and interrupt the binding of viral DNA to HIV-1 IN. Also, PT substitutions did not confer any advantages compared with the regular phosphodiesters for the inhibition of HIV-1 replication by intramolecular G-quartets. The G-quartet motif is the primary requirement for the remarkable nuclease resistance and pronounced biological efficacy of these oligonucleotides.     

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.