ABSTRACT
HIV-1 integrase (IN) catalyzes the integration of viral DNA into the host genome, involving several interactions with the viral and cellular proteins. We have previously identified peptide IN inhibitors derived from the α-helical regions along the dimeric interface of HIV-1 IN. Herein, we show that appropriate hydrocarbon stapling of these peptides to stabilize their helical structure remarkably improves the cell permeability, thus allowing inhibition of the HIV-1 replication in cell culture. Furthermore, the stabilized peptides inhibit the interaction of IN with the cellular cofactor LEDGF/p75. Cellular uptake of the stapled peptide was confirmed in four different cell lines using a fluorescein-labeled analogue. Given their enhanced potency and cell permeability, these stapled peptides can serve as not only lead IN inhibitors but also prototypical biochemical probes or "nanoneedles" for the elucidation of HIV-1 IN dimerization and host cofactor interactions within their native cellular environment.
Subject(s)
Drug Design , Enzyme Inhibitors/pharmacology , HIV Integrase/metabolism , Peptides/pharmacology , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Biocatalysis/drug effects , Cell Line, Tumor , Cell Membrane Permeability/drug effects , Cell Survival/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , HCT116 Cells , HIV-1/drug effects , HIV-1/enzymology , HIV-1/physiology , Host-Pathogen Interactions/drug effects , Humans , Microscopy, Confocal , Models, Chemical , Molecular Structure , Peptides/chemical synthesis , Peptides/pharmacokinetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/virology , Protein Binding/drug effects , Transcription Factors/metabolism , Virus Replication/drug effectsABSTRACT
Human apurinic/apyrimidinic endonuclease 1 (APE1) is an important enzyme in the base excision repair (BER) pathway that is essential for the repair of abasic sites in the genome. Evidence for APE1 as an attractive therapeutic target in anticancer drug development has been demonstrated by studies that link overexpression of APE1 in many cancers to resistance of tumor cells to radio- and chemotherapy. APE1 also shows a protective effect in several cancer cell models to a variety of DNA damaging agents. This study represents the first rational design of selective small-molecule APE1 inhibitors utilizing a three-dimensional interaction-based pharmacophore perception. All of our most potent molecules show inhibitory activity below 10 muM and are selective for APE1 inhibition.
Subject(s)
DNA-(Apurinic or Apyrimidinic Site) Lyase/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Biocatalysis , Combinatorial Chemistry Techniques , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Drug Design , Drug Evaluation, Preclinical , Humans , Inhibitory Concentration 50 , Models, Molecular , Molecular Structure , Protein Binding , Structure-Activity RelationshipABSTRACT
Two decades of intensive research efforts have led to the discovery of a large number of HIV-1 integrase (IN) inhibitors. Recently, the United States Food and Drug Administration (US FDA) approved MK-0518, or raltegravir ( 1), as the first IN inhibitor for HIV/AIDS treatment. Growing clinical evidence also demonstrates that the emergence of HIV-1 virus strains bearing IN amino acid substitutions that confer resistance to IN inhibitors is inevitable. The discovery of second generation inhibitors with potency against viral strains bearing drug resistant IN substitutions is necessary for ongoing effective treatment of viral infections. We generated common feature pharmacophore hypotheses using a training set of quinolone 3-carboxylic acid IN inhibitors, including the clinical candidate GS-9137 ( 2). A database search of small molecules using the quinolone 3-carboxylic acid pharmacophore model, followed by in vitro evaluation of selected hits in an assay specific to IN, resulted in the discovery of potential leads with diverse structural scaffolds useful for the design of second generation IN inhibitors.
Subject(s)
HIV Integrase Inhibitors/chemistry , HIV-1/drug effects , Models, Molecular , Quinolones/chemistry , Animals , Carboxylic Acids/chemistry , Carboxylic Acids/pharmacology , Cell Line , Databases, Factual , Drug Design , HIV Integrase/chemistry , HIV Integrase Inhibitors/pharmacology , HIV-1/enzymology , Humans , Mice , Molecular Conformation , Quinolones/pharmacology , Structure-Activity RelationshipABSTRACT
Peptides deriving from the HIV-1 HXB2 Pol gene sequence were evaluated for inhibitory activity against wild-type (WT) and mutant HIV-1 integrase (IN). The most potent peptide corresponding to a region on the reverse transcriptase (RT) subunit of the Pol polyprotein showed IC(50) value of 5 and 2 microM for 3'-processing and strand transfer, respectively. These peptides, and their analogs, may potentially be used in the elucidation of structural and functional epitopes of IN involved in protein-protein and protein-small molecule interactions.
Subject(s)
Gene Products, pol/chemistry , HIV Integrase Inhibitors/chemical synthesis , HIV Integrase/drug effects , Peptide Fragments/chemical synthesis , Peptide Fragments/pharmacology , Amino Acid Sequence , HIV Integrase/chemistry , HIV Integrase/genetics , HIV Integrase Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Mutation , Structure-Activity RelationshipABSTRACT
Integration of viral DNA into the host chromosome is an essential step in the HIV life cycle. This process is mediated by integrase (IN), a 32 kDa viral enzyme that has no mammalian counterpart, rendering it an attractive target for antiviral drug design. Herein, we present a novel approach toward elucidating "hot spots" of protein-protein or protein-nucleic acid interactions of IN through the design of peptides that encompass conserved amino acids and residues known to be important for enzymatic activity. We designed small peptides (7-17 residues) containing at least one amino acid residue that is important for IN catalytic activities (3'-processing and strand transfer) or viral replication. All these peptides were synthesized on solid phase by fluorenylmethoxycarbonyl (Fmoc) chemistry and evaluated for their inhibition of IN catalytic activities. Such specific sites of interest (i.e., protein-DNA or protein-drug interactions) could potentially be used as drug targets. This novel "sequence walk" strategy across the entire 288 residues of IN has allowed the identification of two peptides NL-6 and NL-9 with 50% inhibitory concentration (IC50) values of 2.7 and 56 microM for strand transfer activity, respectively. Amino acid substitution analysis on these peptides revealed essential residues for activity, and the rational truncation of NL-6 produced a novel hexapeptide (peptide NL6-5) with inhibitory potency equal to that of the parent dodecapeptide (peptide NL-6). More significantly, the retroinverso analogue of NL-6 (peptide RDNL-6) in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted displayed improved inhibitory potency against 3'-processing of HIV-1 IN by 6-fold relative to the parent NL-6, serving as a metabolically stable derivative for further in vitro and in vivo analyses.
Subject(s)
HIV Integrase Inhibitors/chemistry , HIV Integrase/chemistry , Peptides/chemistry , Amino Acid Sequence , Catalysis , Circular Dichroism , Conserved Sequence , DNA, Viral/chemistry , HIV Integrase Inhibitors/chemical synthesis , Molecular Sequence Data , Mutation , Peptides/chemical synthesis , Protein Conformation , Virus ReplicationABSTRACT
HIV-1 integrase (IN) is an attractive and validated target for the development of novel therapeutics against AIDS. Significant efforts have been devoted to the identification of IN inhibitors using various methods. In this context, through virtual screening of the NCI database and structure-based drug design strategies, we identified several pharmacophoric fragments and incorporated them on various aromatic or heteroaromatic rings. In addition, we designed and synthesized a series of 5-aryl(heteroaryl)-isoxazole-3-carboxylic acids as biological isosteric analogues of beta-diketo acid containing inhibitors of HIV-1 IN and their derivatives. Further computational docking studies were performed to investigate the mode of interactions of the most active ligands with the IN active site. Results suggested that some of the tested compounds could be considered as lead compounds and suitable for further optimization.
