2B4J was selected because of the 2 2 molecules of LEDGF/p75 that we speculated might aid in docking the viral DNA substrate. model.(DOCX) pone.0077448.s001.docx (135K) GUID:?294B5C4E-8519-495E-BD14-D2BBF882C81A File S1: Construction and refinement of molecular models.? (DOCX) pone.0077448.s002.docx (234K) GUID:?34054F5A-27F1-45A9-9601-F8CCB6EB6AAD Abstract Signature HIV-1 integrase mutations associated with clinical raltegravir resistance involve 1 of 3 primary genetic pathways, Y143C/R, Q148H/K/R and N155H, the latter 2 of which confer cross-resistance to elvitegravir. In accord with clinical findings, in vitro drug resistance profiling studies with wild-type and site-directed integrase mutant viruses have shown significant fold increases in raltegravir and elvitegravir resistance for the specified viral mutants relative to wild-type HIV-1. Dolutegravir, in contrast, has demonstrated clinical efficacy in subjects failing JMV 390-1 raltegravir therapy due to integrase mutations at Y143, Q148 or N155, which is consistent with its distinct in vitro resistance profile as dolutegravirs antiviral activity against these viral JMV 390-1 mutants is equivalent to its activity against wild-type HIV-1. Kinetic studies of inhibitor dissociation from wild-type and mutant integrase-viral DNA complexes have shown that dolutegravir also has a distinct off-rate profile with dissociative half-lives substantially longer than those of raltegravir and elvitegravir, suggesting that dolutegravirs prolonged binding may be an important contributing factor to its distinct resistance profile. To provide a structural rationale for these observations, Rabbit polyclonal to VPS26 we constructed several molecular models of wild-type and clinically relevant mutant HIV-1 integrase enzymes in complex with viral DNA and dolutegravir, raltegravir or elvitegravir. Here, we discuss our structural models and the posited effects that the integrase mutations and the structural and electronic properties of the integrase inhibitors may have on the catalytic pocket and inhibitor binding and, consequently, on antiviral potency in vitro JMV 390-1 and in the clinic. Introduction HIV-1 integrase (IN) is required for viral cDNA integration into the host cell genome, an essential step in the HIV life cycle. First, IN catalyzes the cleavage of a GT dinucleotide from the 3 end of each viral long terminal repeat (LTR) that is downstream from a conserved CA dinucleotide (3 processing). Next, the enzyme catalyzes the concerted insertion of the 2 2 processed 3 ends into opposite strands of the host target DNA 5 base pairs apart from each other by a direct trans-esterification reaction (strand transfer). Because of the vital role that IN plays in HIV replication, the enzyme is an attractive therapeutic target. Extensive research efforts have led to the discovery and development of the IN inhibitors, raltegravir (RAL) and elvitegravir (EVG), and the new IN inhibitor, dolutegravir (DTG) (Figure 1), all of which have demonstrated efficacy in clinical studies by preferentially inhibiting the strand transfer activity of IN [1-3]. Open in a separate window Figure 1 2D structures of (A) dolutegravir, (B) raltegravir and (C) elvitegravir.Red ovals encircle the oxygen atoms that chelate the divalent metal cations in the active site; green ovals encircle the halobenzyl groups; and blue boxes encircle the approximate regions of the scaffolds that can accommodate positive charge after chelation of the metals. The purple circles at (B) encircle raltegravirs gem-dimethyl (small circle) and oxadiazole groups, and the purple oval at (C) encircles elvitegravirs 1-hydroxymethyl-2-methylpropyl group. Clinical RAL resistance is associated with 3 primary genetic pathways that involve IN mutations at Y143, Q148 or N155, whereas EVG resistance is associated with mutations at Q148 or N155 as well as T66, E92, T97 or S147 [4-7]. In subjects who have failed RAL therapy with RAL-resistant HIV-1, DTG has demonstrated greatest efficacy in those harboring HIV-1 with Y143 or N155 pathway mutations, and more limited responses when Q148 pathway viruses with additional secondary mutations are present [8]. In accord with in vivo results, in vitro drug resistance profiling studies with wild-type and site-directed IN mutant viruses have shown that DTG has a distinct profile compared with those of RAL and EVG.The metal-bound conformations of HIV-1 IN residues D64 and D116 were also modeled based on their PFV IN counterparts, residues D128 and D185, respectively. IN catalytic core domain. (B) Nucleotide sequence used to model the HIV-1 U5 LTR end. The 2 2 nucleotides highlighted in yellow are not part of the 3 processed DNA model.(DOCX) pone.0077448.s001.docx (135K) GUID:?294B5C4E-8519-495E-BD14-D2BBF882C81A File S1: Construction and refinement of molecular models.? (DOCX) pone.0077448.s002.docx (234K) GUID:?34054F5A-27F1-45A9-9601-F8CCB6EB6AAD Abstract Signature HIV-1 integrase mutations associated with clinical raltegravir resistance involve 1 of 3 primary genetic pathways, Y143C/R, Q148H/K/R and N155H, the latter 2 of which confer cross-resistance to elvitegravir. In accord with clinical findings, in vitro drug resistance profiling studies with wild-type and site-directed integrase mutant viruses have shown significant fold increases in raltegravir and elvitegravir resistance for the specified viral mutants relative to wild-type HIV-1. Dolutegravir, in contrast, has demonstrated clinical efficacy in subjects failing raltegravir therapy due to integrase mutations at Y143, Q148 or N155, which is consistent with its distinct in vitro resistance profile as dolutegravirs antiviral activity against these viral mutants is equivalent to its activity against wild-type HIV-1. Kinetic studies of inhibitor dissociation from wild-type and mutant integrase-viral DNA complexes have shown that dolutegravir also has a distinct off-rate profile with dissociative half-lives substantially longer than those of raltegravir and elvitegravir, suggesting that dolutegravirs prolonged binding may be an important contributing factor to its distinct resistance profile. To provide a structural rationale for these observations, we constructed several molecular models of wild-type and clinically relevant mutant HIV-1 integrase enzymes in complex with viral DNA and dolutegravir, raltegravir or elvitegravir. Here, we discuss our structural models and the posited effects that the integrase mutations and the structural and electronic properties of the integrase inhibitors may have on the catalytic pocket and inhibitor binding and, consequently, on antiviral potency in vitro and in the clinic. Introduction HIV-1 integrase (IN) is required for viral cDNA integration into the host cell genome, an essential step in JMV 390-1 the HIV existence cycle. First, IN catalyzes the cleavage of a GT dinucleotide from your 3 end of each viral long terminal repeat (LTR) that is downstream from a conserved CA dinucleotide (3 processing). Next, the enzyme catalyzes the concerted insertion of the 2 2 processed 3 ends into reverse strands of the sponsor target DNA 5 foundation pairs apart from each other by a direct trans-esterification reaction (strand transfer). Because of the vital part that IN takes on in HIV replication, the enzyme is an attractive therapeutic target. Considerable research efforts possess led to the finding and development of the IN inhibitors, raltegravir (RAL) and elvitegravir (EVG), and the new IN inhibitor, dolutegravir (DTG) (Number 1), all of which have demonstrated effectiveness in medical studies by preferentially inhibiting the strand transfer activity of IN [1-3]. Open in a separate window Number 1 2D constructions of (A) dolutegravir, (B) raltegravir and (C) elvitegravir.Red ovals encircle the oxygen atoms that chelate the divalent metal cations in the active site; green ovals encircle the halobenzyl organizations; and blue boxes encircle the approximate regions of the scaffolds that can accommodate positive charge after chelation of the metals. The purple circles at (B) encircle raltegravirs gem-dimethyl (small circle) and oxadiazole organizations, and the purple oval at (C) encircles elvitegravirs 1-hydroxymethyl-2-methylpropyl group. Clinical RAL resistance is associated with 3 main genetic pathways that involve IN mutations at Y143, Q148 or N155, whereas EVG resistance is associated with mutations at Q148 or N155 as well as T66, E92, T97 JMV 390-1 or S147 [4-7]. In subjects who have failed RAL therapy with RAL-resistant HIV-1, DTG offers demonstrated greatest effectiveness in those harboring HIV-1 with Y143 or N155 pathway mutations, and more limited reactions when Q148 pathway viruses with additional secondary mutations are present [8]. In accord with in vivo results, in vitro drug resistance profiling studies with wild-type and site-directed IN mutant viruses have shown that DTG has a unique profile compared with those.