Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol: part II. integrase inhibition

We report molecular modeling and functional confirmation of Ole and HT binding to HIV-1 integrase. Docking simulations identified two binding regions for Ole within the integrase active site. Region I encompasses the conserved D64-D116-E152 motif, while region II involves the flexible loop region formed by amino acid residues 140-149. HT, on the other hand, binds to region II. Both Ole and HT exhibit favorable interactions with important amino acid residues through strong H-bonding and van der Waals contacts, predicting integrase inhibition. To test and confirm modeling predictions, we examined the effect of Ole and HT on HIV-1 integrase activities including 3'-processing, strand transfer, and disintegration. Ole and HT exhibit dose-dependent inhibition on all three activities, with EC(50)s in the nanomolar range. These studies demonstrate that molecular modeling of target-ligand interaction coupled with structural-activity analysis should facilitate the design and identification of innovative integrase inhibitors and other therapeutics.     

Computational study of bindings of olive leaf extract (OLE) to HIV-1 fusion protein gp41

Recent experimental study found that OLE (olive leaf extract) has anti-HIV activity by blocking the HIV virus entry to host cells [Lee-Huang, S., Zhang, L., Huang, P.L., Chang, Y. and Huang, P.L. (2003) Anti-HIV activity of olive leaf extract (OLE) and modulation of host cell gene expression by HIV-1 infection and OLE treatment. Biochem. Biophys. Res. Commun. 307, 1029; Lee-Huang, S., Huang, P.L., Zhang, D., Lee, J.W., Bao, J., Sun, Y., Chang, Y.-Tae, Zhang, J.Z.H. and Huang, P.L. (2007) Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol. Biochem. Biophys. Res. Commun. 354, 872-878, 879-884]. As part of a joint experimental and theoretical effort, we report here computational study to help identify and characterize the binding complexes of several main compounds of OLE (olive leaf extract) to HIV-1 envelop protein gp41. A number of possible binding modes are found by docking oleuropein and its metabolites, aglycone, elenolic acid and hydroxytyrosol, onto the hydrophobic pocket on gp41. Detailed OLE-gp41 binding interactions and free energies of binding are obtained through molecular dynamics simulation and MM-PBSA calculation. Specific molecular interactions in our predicted OLE/gp41 complexes are identified and hydroxytyrosol is identified to be the main moiety for binding to gp41. This computational study complements the corresponding experimental investigation and helps establish a good starting point for further refinement of OLE-based gp41 inhibitors.     

Small molecule fusion inhibitors: Design,synthesis and biological evaluation of (Z)-3-(5-(3-benzyl-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrroles and related derivatives targeting HIV-1 gp41

By a scaffold elongation strategy, a series of (Z)-3-(5-(3-benzyl-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrroles and related derivatives with a linear multi-aromatic-ring skeleton were designed, synthesized, and evaluated in HIV-1 gp41 and cellular assays. Among them, the most active compounds, 12e, 12g, and 12k with a one-carbon linker (n = 1) between the rhodanine (C) and phenyl (D) rings, exhibited very promising inhibitory potency with IC₅₀ values of 1.8–2.6 μM and EC₅₀ values of 0.3–1.5 μM against gp41 6-HB formation and HIV-1 replication in MT-2 cells, respectively. Additionally, they were almost equally effective against both T20-sensitive and resistant strains. The related SAR studies and molecular modeling results provided potential for further developing a new class of non-peptide small molecular fusion inhibitors targeting the HIV-1 gp41.     

Prediction of HIV-1 entry inhibitors neomycin-arginine conjugates interaction with the CD4-gp120 binding site by molecular modeling and multistep docking procedure

Developing of multi-target HIV-1 entry inhibitors represents an important avenue of drug therapy. Two such inhibitors are hexa-arginine-neomycin-conjugate (NeoR6) and nona-d-arginine-neomycin-conjugate (Neo-r9). Our findings that NeoR6-resistant mutations appear in the gp120 constant regions; and NeoR6 is not CCR5 antagonist, but inhibits CXCR4 and CCR5 HIV-1 using isolates, led us to suggest that NeoR6 may inhibit HIV-1 entry by interfering with the CD4-gp120 binding. To support this notion, we constructed a homology model of unliganded HIV-1_IIIB gp120 and docked NeoR6 and Neo-r9 to it, using a multistep docking procedure: geometric-electrostatic docking by MolFit; flexible ligand docking by Autodock3 and final refinement of the obtained complexes by Discover3. Binding free energies were calculated by MM-PBSA methodology. The model predicts competitive inhibition of CD4-gp120 binding by NeoR6 and Neo-r9. We determined plausible binding sites between constructed CD4-bound gp120 trimer and homology modeled membranal CXCR4, and tested NeoR6 and Neo-r9 interfering with this interaction. These models support our notion that another mechanism of anti-HIV-1 activity of NeoR6 is inhibition of gp120-CXCR4 binding. These structural models and interaction of NeoR6 and Neo-r9 with gp120 and CXCR4 provide a powerful approach for structural based drug design for selective targeting of HIV-1 entry and/or for inhibition of other retroviruses with similar mechanism of entry.     

DNA sequence-specific ligands: XIII. New dimeric Hoechst 33258 molecules,inhibitors of HIV-1 integrase in vitro

Dimeric Hoechst 33258 molecules [dimeric bisbenzimidazoles (DBBIs)] that, upon binding, occupy one turn of the B form of DNA in the narrow groove were constructed by computer simulation. Three fluorescent DBBIs were synthesized; they consist of two bisbenzimidazole units tail-to-tail linked to phenolic hydroxy groups via penta-or heptamethylene or tri(ethylene glycol) spacers and have terminal positively charged N,N-dimethylaminopropyl carboxamide groups in the molecule. The absorption spectra of the DBBIs in the presence of different DNA concentrations showed a hypochromic effect and a small shift of the absorption band to longer wavelengths, which indicated the formation of a complex with DNA. The presence of an isobestic point in the spectrum indicates the formation of one type of DBBI-DNA complexes. The interaction of DBBIs with DNA was studied by CD using a cholesteric liquid-crystalline dispersion (CLD) of DNA. The appearance of a positive band in the absorption region of ligand chromophores in the CD spectrum of the DNA CLD indicates the formation of a DBBI-DNA complex in which ligand chromophores are arranged at an angle close to 54° relative to the helix axis of DNA, which suggests the localization of the DBBI in the narrow groove of DNA. All the DBBIs were found to be in vitro inhibitors of HIV-1 DNA integrase in the 3′-processing reaction, and, of the three DBBIs, two dimers inhibit HIV-1 integrase even in submicromolar concentrations.     

Fused heterocyclic compounds bearing bridgehead nitrogen as potent HIV-1 NNRTIs. Part 1: Design,synthesis and biological evaluation of novel 5,7-disubstituted pyrazolo[1,5-a]pyrimidine derivatives

In our continuous efforts to identify novel potent HIV-1 NNRTIs, a novel class of 5,7-disubstituted pyrazolo[1,5-a]pyrimidine derivatives were rationally designed, synthesized and evaluated for their anti-HIV activities in MT4 cell cultures. Biological results showed that most of the tested compounds displayed excellent activity against wild-type HIV-1 with a wide range of EC₅₀ values from 5.98 to 0.07 μM. Among the active compounds, 5a was found to be the most promising analogue with an EC₅₀ of 0.07 μM against wild-type HIV-1 and very high selectivity index (SI, 3999). Compound 5a was more effective than the reference drugs nevirapine (by 2-fold) and delavirdine (by 2-fold). In order to further confirm their binding target, an HIV-1 RT inhibitory assay was also performed. Furthermore, SAR analysis among the newly synthesized compounds was discussed and the binding mode of the active compound 5a was rationalized by molecular modeling studies.     

Computer-based design of novel HIV-1 entry inhibitors: neomycin conjugated to arginine peptides at two specific sites

Aminoglycoside–arginine conjugates (AAC and APAC) are multi-target inhibitors of human immunodeficiency virus type-1 (HIV-1). Here, we predict new conjugates of neomycin with two arginine peptide chains binding at specific sites on neomycin [poly-arginine-neomycin-poly-arginine (PA-Neo-PA)]. The rationale for the design of such compounds is to separate two short arginine peptides with neomycin, which may extend the binding region of the CXC chemokine receptor type 4 (CXCR4). We used homology models of CXCR4 and unliganded envelope glycoprotein 120 (HIV-1_IIIB gp120) and docked PA-Neo-PAs and APACs to these using a multistep docking procedure. The results indicate that PA-Neo-PAs spread over two negatively charged patches of CXCR4. PA-Neo-PA–CXCR4 complexes are energetically more favorable than AACs/APAC–CXCR4 complexes. Notably, our CXCR4 model and docking procedure can be applied to predict new compounds that are either inhibitors of gp120–CXCR4 binding without affecting stromal cell-derived factor 1α (SDF-1α) chemotaxis activity, or inhibitors of SDF-1α–CXCR4 binding resulting in an anti-metastasis effect. We also predict that PA-Neo-PAs and APACs can interfere with CD4–gp120 binding in unliganded conformation. Figure The r5-Neo-r5-CXCR4 complex. CXCR4 is shown in CPK representation. The negatively charged residues are shown in red and positively charged residues in blue. The r5-Neo-r5 is shown in stick representation, neomycin core is colored yellow and arginine moieties are colored magenta. Two negatively charged patches separated by neutral and positively charged residues are visible.     

Design and synthesis of pyrazolo[3,4-d]pyrimidinone derivatives: Discovery of selective phosphodiesterase-5 inhibitors

A novel series of 1,6-disubstituted pyrazolo[3,4-d]pyrimidin-7-one derivatives, 2a-h, 4a-d, 5 and 6, were successfully synthesized, which showed promising, and potent inhibition of phosphodiesterase 5 (PDE5). The inhibitory activities of 5, 4b, 2a, 2d, 2f, 4d and 4a against PDE5 were similar to that of sildenafil (100%). These compounds exhibited potent relaxant effects on isolated rat cavernosum tissue with pEC₅₀ values ranging from 8.31 to 5.16 µM. Pyrazolo[3,4-d]pyrimidin-7-one scaffolds have been rationally designed via consecutive molecular modelling studies prior to their synthesis and biological evaluation. In addition, the results of the pharmacophore-based virtual screening revealed that 1v0p_PVB might have promising activity as a PDE-5 inhibitor.     

Phosphodiesterase inhibitors. Part 2: Design, synthesis, and structure-activity relationships of dual PDE3/4-inhibitory pyrazolo[1,5-a]pyridines with anti-inflammatory and bronchodilatory activity

A structural survey of pyrazolopyridine-pyridazinone phosphodiesterase (PDE) inhibitors was made with a view to optimization of their dual PDE3/4-inhibitory activity for respiratory disease applications. These studies identified (−)-6-(7-methoxy-2-trifluoromethylpyrazolo[1,5-a]pyridine-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone (KCA-1490, compound 2ac) as a compound with potent combined bronchodilatory and anti-inflammatory activity and an improved therapeutic window over roflumilast.