the 8-hydroxy-1,6-naphthyridine based INSTI's led to the N-benzyl carboxamide L-870,810 (23), a very potent antiviral agent with an IC95 = 0.019 |xM against wild type virus (53,54). This compound entered phase I clinical trials in 2002 (55). Proof of concept studies with the related naphthyridine amide L-870,812 (24) demonstrated a significant antiviral effect in a group of rhesus macaques infected with the simian-human chimeric virus SHIV 89.6p. Macaques receiving a 10 mg kg"1 dose of L-870,812 twice a day had sustained decreases in viral RNA of 1-3 log copies ml"1 over an 87 day period (56). Structurally related 3-substituted-8-hydroxyquinolines have been reported as integrase strand transfer inhibitors. Although no antiviral activity is reported, compound 25 has an integrase inhibition IC5o = 0.331 |iM (57).
RNase-H Inhibitors - Within the p66 subunit of the p66/p51 heterodimer of HIV reverse transcriptase lies a second catalytic region capable of breaking / making phosphodiester bonds. Known as the ribonuclease H domain (RNase-H), this region is independent of the DNA polymerase site and serves to remove the RNA template from the growing DNA:RNA heteroduplex (58,59). As an independent catalytic site with its own unique enzymic function, RNase-H is a target for antiviral drug design. Structurally there are many similarities between the RNase-H domain of RT and HIV integrase which suggest they use a common alkali metal cation/carboxylate mechanism (60). Indeed, such suggestions led to discovery of RNase-H inhibitory activity with a series of 2,4-diketobutanoic acid integrase inhibitors (61). The N-benzoylaminothiophene diketobutanoic acid 26 has an IC50 = 4.7 nM for RNA cleavage using an isolated RNase-H domain with Mn++ as the catalytic metal. Certain phenylhydrazones inhibit both the polymerase and RNase H activities of RT. Recently the 4-dimethylaminophenylhydrazone 27 was reported to show selectivity for inhibition of RNase-H over the polymerase domain of RT with an IC50 = 4.0 jtM
for RNA cleavage (62). 1,4-Naphthoquinone and integrase inhibitory DNA aptamers also have been shown to selectively inhibit the RNase-H activity of RT (63,64).
Inhibitors of Viral Entry - Entry of HIV virions into host cells involves attachment of viral coat glycoproteins to host cell receptors followed by fusion between virus and host cell membranes. In the initial phase of this process the viral envelope glycoprotein gp120 binds with high affinity to a host cell CD4 receptor. This single protein-protein interaction is insufficient to trigger fusion by itself. A second interaction, one between the gp120/CD4 complex and a host cell "co-receptor" is required. The co-receptors involved the viral entry process belong to a series of transmembrane G-protein coupled receptors for certain chemokines. The most commonly used chemokine receptors in the viral entry process are CCR5 and CXCR4. The interaction of gp120 with CD4 and a chemokine receptor is believed to be the trigger that sets in motion a membrane fusion process involving a second viral glycoprotein, gp41. Gp41, which is situated at the base of gp120, reorganizes in such a manner as to bring the viral and cellular membranes into contact. Fusion of the two membranes then permits entry of the viral nucleoprotein core (65). Inhibitors of the CD4/gp120 interaction include the antibody like tetravalent immunoglobulin molecule containing CD4 binding domains PR0542, and the 11kDa cyanobacterial protein cyanovirin-N (66,67). Cyanovirin contains two high affinity carbohydrate binding sites that interact with mannosyl disaccharide residues on gp120. The nanomolar affinity of these two binding sites is in line with the potent, broad-spectrum anti-HIV activity of cyanovirin in cell culture. While large molecules such as PR0542 and cyanovirin are effective in disrupting the gp120/CD4 interaction, a more surprising observation, that a small molecule can efficiently block this same interaction was recently reported. BMS-806 (structure not disclosed) is a small molecule inhibitor of the gp120/CD4 interaction that has submicromolar antiviral activity in cell culture (68). However, envelope heterogeneity among viruses is problematic for BMS-806, several hundred fold differences in antiviral activity were seen with a panel of diverse viral isolates. Selection of resistant variants to BMS-806 revealed amino acid substitutions near the CD4 binding domains of gp120.
Antagonists of chemokine receptors are antiviral agents that prevent the interaction of the gp120/CD4 complex by blocking the host cells' co-receptor. The CCR5 antagonist SCH-C (SCH 351125) is a potent antiviral agent that inhibits viral replication of NSI viruses with ICso's in the 0.4 to 9 nM range in vitro. Importantly, SCH-C has no effect on SI viruses that employ the CXCR4 co-receptor for viral entry (69). Clinical studies with SCH-C demonstrated a viral load reduction of 0.5-1.0 log in a patient group that excluded those having SI phenotype virus at baseline (70). Other small molecule CCR5 antagonists in preclinical development, notably TAK-220 and AK-602, show similar low nanomolar antiviral activity and SI phenotype selectivity in vitro (70,71).
Inhibitors of the gp41 mediated fusion process are the most clinically advanced viral entry inhibitors. Enfuvirtide (T-20, DP-178, pentafuside) is a 36 amino acid residue peptide that binds to one of the two heptad repeat regions on gp41. The high affinity binding between T-20 and gp41 results in subnanomolar antiviral activity
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