Discovery and characterization of inhibitors of human palmitoyl acyltransferases

Abstract

The covalent attachment of palmitate to specific proteins by the action of palmitoyl acyltransferases (PAT) plays critical roles in the biological activities of several oncoproteins. Two PAT activities are expressed by human cells: type 1 PATs that modify the farnesyl-dependent palmitoylation motif found in H-and N-Ras, and type 2 PATs that modify the myristoyl-dependent palmitoylation motif found in the Src family of tyrosine kinases. We have previously shown that the type 1 PAT HIP14 causes cellular transformation. In the current study, we show that mRNA encoding HIP14 is up-regulated in a number of types of human tumors. To assess the potential of HIP14 and other PATs as targets for new anticancer drugs, we developed three cell-based assays suitable for highthroughput screening to identify inhibitors of these enzymes. Using these screens, five chemotypes, with activity toward either type 1 or type 2 PAT activity, were identified. The activity of the hits were confirmed using assays that quantify the in vitro inhibition of PAT activity, as well as a cell-based assay that determines the abilities of the compounds to prevent the localization of palmitoylated green fluorescent proteins to the plasma membrane. Representative compounds from each chemotype showed broad antiproliferative activity toward a panel of human tumor cell lines and inhibited the growth of tumors in vivo. Together, these data show that PATs, and HIP14 in particular, are interesting new targets for anticancer compounds, and that small molecules with such activity can be identified by high-throughput screening.

Neurotensin receptor-1 inducible palmitoylation is required for efficient receptor-mediated mitogenic-signaling within structured membrane microdomains

“…Recent studies suggest that targeting PATs could lead to the development of novel anti-neoplastic chemotherapeutic agents. 17 Although, characterization of the specific PATs that are responsible for GPCR palmitoylation are still not well defined, there are currently several lipid-based and small molecule based-experimental PAT inhibitors that can serve as lead compounds for the development of more specific inhibitors. 18 We previously published that intact structured membrane micordomains (SMDs) are required for NTSR-1 signaling and interaction with Gα q/11 .…”

“…17,30 In brief, transfected HEK293T cells were seeded at 4.0 x 10 5 cells/ well in 100-mm plates and allowed to grow till they reached 80% confluency. Cells were then treated with 1 μM NTS (15 min) and then washed with ice-cold PBS twice.…”

Palmitoylated proteins: purification and identification

“…Indeed, the mammalian DHHC PAT family is an important set of future drug targets with possible utility in the treatment of cancer and other diseases. Recently, a first generation of compounds with inhibitory effects on palmitoylation has been developed 15 . Comparing the profiles from drug-treated cells with those of mutants deficient in the individual PATs should provide a means of mapping drugs to their target PATs.…”

2-Bromopalmitate and 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one inhibit DHHC-mediated palmitoylation in vitro

“…Human HIP14 is oncogenic, promoting colony formation in soft agar and tumor formation in nude mice (11). HIP14 mRNA is upregulated in numerous tumors (12). DHHC9, a PAT for H-and N-Ras in vitro, is upregulated in microsatellite stable tumors (13), whereas DHHC2 is a putative tumor suppressor (14).…”