Discovery of arylsulfonamides as a novel class of allosteric integrase inhibitors with antiviral activity.

Lens epithelial-derived growth factor (LEDGF) increases the efficiency of proviral DNA integration into the host genome by interacting with HIV integrase (IN) and directing it to a chromatin environment that favors viral transcription. Allosteric integrase inhibitors (ALLINIs), such as known 2-(tert-butoxy)acetic acid (1), bind to the LEDGF pocket on the catalytic core domain (CCD) of IN, but exert more potent antiviral activities by inhibition of late-stage HIV-1 replication events than through disruption of proviral integration at an earlier phase. A high-throughput screen (HTS) for compounds that disrupt IN-LEDGF interaction led to the identification of a novel arylsulfonamide series, as exemplified by 2, possessing ALLINI-like properties. Further SAR studies led to more potent compound 21 and provided key chemical biology probes revealing that arylsulfonamides are a novel class of ALLINIs with a distinct binding mode than that of 2-(tert-butoxy)acetic acids.

Discovery of a Distinct Chemical and Mechanistic Class of Allosteric HIV-1 Integrase Inhibitors with Antiretroviral Activity.

Allosteric integrase inhibitors (ALLINIs) bind to the lens epithelial-derived growth factor (LEDGF) pocket on HIV-1 integrase (IN) and possess potent antiviral effects. Rather than blocking proviral integration, ALLINIs trigger IN conformational changes that have catastrophic effects on viral maturation, rendering the virions assembled in the presence of ALLINIs noninfectious. A high-throughput screen for compounds that disrupt the IN·LEDGF interaction was executed, and extensive triage led to the identification of a t-butylsulfonamide series, as exemplified by 1. The chemical, biochemical, and virological characterization of this series revealed that 1 and its analogs produce an ALLINI-like phenotype through engagement of IN sites distinct from the LEDGF pocket. Key to demonstrating target engagement and differentiating this new series from the existing ALLINIs was the development of a fluorescence polarization probe of IN (FLIPPIN) based on the t-butylsulfonamide series. These findings further solidify the late antiviral mechanism of ALLINIs and point toward opportunities to develop structurally and mechanistically novel antiretroviral agents with unique resistance patterns.

Specificity of RCN1-mediated protein phosphatase 2A regulation in meristem organization and stress response in roots.

Protein dephosphorylation by the serine/threonine protein phosphatase 2A (PP2A) modulates a broad array of cellular functions. PP2A normally acts as a heterotrimeric holoenzyme complex comprising a catalytic subunit bound by regulatory A and B subunits. Characterization of the regulatory A subunit isoforms (ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 [RCN1], PP2AA2, and PP2AA3) of Arabidopsis thaliana PP2A has shown that RCN1 plays a primary role in controlling root and hypocotyl PP2A activity in seedlings. Here we show that hypocotyl and root growth exhibit different requirements for RCN1-mediated regulation of PP2A activity. Roots of rcn1 mutant seedlings exhibit characteristic abnormalities in cell division patterns at the root apical meristem, as well as reduced growth under ionic, osmotic, and oxidative stress conditions. We constructed chimeric A subunit genes and found that restoration of normal root tip development in rcn1 plants requires both regulatory and coding sequences of RCN1, whereas the hypocotyl elongation defect of rcn1 plants can be complemented by either RCN1 or PP2AA3 transgenes. Furthermore, the RCN1 and PP2AA3 proteins exhibit ubiquitous subcellular localization patterns in seedlings and both associate with membrane compartments. Together, these results show that RCN1-containing PP2A has unique functions that cannot be attributed to isoform-specific expression and localization patterns. Postembryonic RCN1 function is required to maintain normal auxin distribution and stem cell function at the root apex. Our data show that RCN1-regulated phosphatase activity plays a unique role in regulating postembryonic root development and stress response.

A PP2A active site mutant impedes growth and causes misregulation of native catalytic subunit expression.

Activity of protein phosphatase 2A (PP2A) is tightly regulated and performs a diverse repertoire of cellular functions. Previously we isolated a dominant-negative active site mutant of the PP2A catalytic (C) subunit using a yeast complementation assay. We have established stable fibroblastic cell lines expressing epitope-tagged versions of the wild-type and H118N mutant C subunits and have used these cells to investigate mechanisms that regulate PP2A activity. Cells expressing the mutant C subunit exhibit a decreased growth rate and a prolonged G1 cell cycle phase. The mutant protein is enzymatically inactive, but extracts made from cells expressing the H118N C subunit show normal levels of total PP2A activity in vitro. The H118N mutant shows reduced binding to the regulatory A subunit, but binds normally to the alpha4 protein, a non-canonical regulator of PP2A. Expression of the H118N mutant interferes with the normal control of C subunit abundance, causing accumulation of the endogenous wild-type protein as well as the mutant transgene product. Our results indicate that the H118N mutant isoform retards C subunit turnover and suggest that PP2A C subunit turnover may be important for normal cell cycle progression.