In our model of compound 5 with the NNRTI-binding pocket, the thiol group around the imidazole moiety forms a hydrogen bond with the main chain carbonyl oxygen of K101(SHimidazole COK101 = 1.81 ?). positioning of the primer DNA strand in the polymerase active site. This switch in primer grip conformation may alter the position and conformation of the template/primer (T/P) substrate thereby preventing the establishment of a catalytically qualified ternary complex . (3) Kohlstaedt proposed that this NNRTI-BP may normally function as a hinge between IBMX the palm and thumb domains. Since the mobility of the thumb may IBMX be important to facilitate T/P translocation, the binding of NNRTIs may restrict the mobility of the thumb domain name thereby slowing down or preventing T/P translocation and/or elongation of nascent viral DNA . The three mechanisms suggested above are not mutually unique, and NNRTIs may exert multiple inhibitory effects on RT catalyzed DNA synthesis. In fact, several kinetic and thermodynamic studies have illustrated the complexities of the RT-NNRTI conversation Rabbit Polyclonal to TNAP1 [4C7]. Although NNRTI made up of regimens are effective and generally well-tolerated in the majority of patients, treatment durability is limited by drug-related side effects and the development of drug resistance. HIV-1 resistance to NNRTIs correlates directly with mutations of one or more RT residues in the NNRTI-BP. Mutations associated with resistance to NNRTIs include L100I, K101E, K103N, V106A, V108I, V179D, Y181C, Y188C/L/H, G190A/E/S, M230L, P236L and Y318F . These mutations can affect inhibitor binding in a number of ways. (1) They can remove one or more favorable interactions between the inhibitor and NNRTI-binding pocket. For example, the Y181C mutation eliminates -stacking interactions between this residue and the aromatic ring of the NNRTI pharmacophore . (2) They can introduce steric barriers to NNRTI binding. For example, the G190E mutations introduce a bulky side-chain which may prevent NNRTI binding by sterically interfering with functional groups, such as the cyclopropyl ring of nevirapine . (3) The mutations may expose or eliminate inter-residue contacts in the NNRTI-binding pocket which interfere with the ability of other residues in the pocket to fold down over the NNRTI . Accordingly, there is still an urgent need to develop NNRTI with a high-genetic barrier to resistance that facilitate patient adherence and allow durable suppression of viral replication. In this regard, previous studies have exhibited that NNRTI which contain an imidazole nucleus exhibit activity against both wild-type (WT) and drug-resistant strains of HIV-1. For example capravirine (observe Fig. (1)) retains activity against HIV-1 made up of the single key NNRTI mutations K103N, V106A or L100I . The N-aminoimidazoles (NAIMs) have also been reported to inhibit replication of the WT computer virus as well as an HIV-1 strain that contained both the K103N and Y181C mutations . In light of these studies, we have synthesized and characterized the anti-HIV-1 activity of a novel series of NAIMs that contained a thiourea moiety (N-NH-CS-NH) with one aryl ring at the 5th position of the imidazole nucleus and the other aryl ring via an NHCSNH linker. Open in a separate windows Fig. (1) Chemical structures of capravirine, NAIM analogs explained by Lagoja (13) and the novel NAIM analogs synthesized in this study. Methods Materials The wild-type (WT), K103N, and Y181C HIV-1RTs (subtype B, LAI) were purified as explained previously [14, 15]. The protein concentration of the purified enzymes was decided spectrophotometrically at 280 nm using an extinction coefficient (280) of 260450 M?1 cm?1, and by Bradford protein assays (Sigma-Aldrich, St. Louis, MO). The RNA- and DNA-dependent DNA polymerase activities of the purified WT and mutant enzymes were essentially identical (data not shown). [3H]TTP was acquired from PerkinElmer Life Sciences (Boston, MA). RNA and DNA oligonucleotides were synthesized by IDT (Coralville, IA). All the starting anilines were obtained from Central Drug House (CDH), Delhi, India. 85% hydrazine hydrate was procured from S.D. Fine Chemicals. docking studies to investigate the conversation of 5 with the NNRTI-binding pocket of the WT HIV-1 RT (PDB access code 1rt2) using the Extra Precision (XP) mode of Glide software  (observe Fig.(2)). To validate the Glide software, we first modeled the conversation between TNK651 and HIV-1 RT. Superimposition of the experimental bound (co-crystallized) conformation of TNK651  and that predicted by Glide are shown in Fig. (2A). Glide successfully reproduced the experimental binding conformations of TNK 651 in the NNRTI-binding pocket of HIV-1 RT with an acceptable root-mean-square deviation (RMSD) of IBMX 2.4 ?. In our model of compound 5 with the NNRTI-binding pocket, the.