Execution of a High Throughput HIV-1 Replication Screen and the Identification of a Novel Small Molecule Inhibitor that Targets HIV-1 Envelope Maturation
Wade Blair*, J Cao, L Jackson, Q Peng, J Isaacson, S Butler, H Wu, A Chu, and A Patick
Pfizer Global Research and Development, La Jolla, CA,
Background: All drugs currently approved to treat HIV-1
infection target 1 of 3 steps in the HIV-1 replication cycle. Given that
viruses resistant to one drug of a particular class often exhibit
cross-resistance to other drugs in the same class, therapeutic options for
treatment-experienced patients are often limited. One way to address this
problem is to identify HIV-1 inhibitors directed against new targets in the
HIV-1 replication cycle. As part of an effort to search for inhibitors
targeting new mechanisms, a high throughput HIV-1 full-replication screen (HIV
Rep) was executed resulting in the identification of a novel HIV-1 envelope
(Env) maturation inhibitor.
Methods: An antiviral screen was conducted using the
HIV-1 NL4-3 strain, MT-2 T-cells, and HeLa CD4 LTR/beta-Gal indicator cells.
The antiviral activity of UK-201844 was determined after infection of MT-2
cells or HeLa CD4 LTR/beta-Gal indicator cells with HIV-1 NL4-3 or NL4-3
derivatives. The effect of UK-201844 on infectious virus production was
analyzed after transfection of HEK293 cells with HIV-1 NL4-3 infectious cDNAs.
Results: More than 106 compounds were
evaluated in the HIV Rep screen resulting in the identification of a novel
small molecule inhibitor (UK-201844) that targets HIV-1 Env maturation.
UK-201844 exhibited antiviral activity against HIV-1 NL4-3 in full replication
assays but was not active in single-cycle infection assays using HIV-1 NL4-3
derived reporter viruses, suggesting that the compound targets a late event in
the HIV-1 replication cycle. Consistent with these observations, UK-201844
specifically inhibited the production of infectious virions in an HIV-1 Env
dependent manner. These results confirm that UK201844 acts during the late
stages of HIV replication and suggest that HIV-1 Env is the target of the
compound. In vitro resistant virus studies showed that HIV-1 Env
sequences are critical determinants of compound susceptibility, thus confirming
that UK-201844 targets HIV-1 Env function. Additional mechanism-of-action
studies demonstrated that UK-201844 interferes with HIV-1 gp160 processing in
infected cells, resulting in the production of non-infectious virions.
Conclusions: UK-201844 represents
the prototype for a unique HIV-1 inhibitor class that directly or indirectly
inhibits HIV-1 gp160 processing, resulting in the production of virions with
non-functional Env proteins.