Discovery and Evaluation of C6-Substituted Pyrazolopyrimidine-Based Bisphosphonate Inhibitors of the Human Geranylgeranyl Pyrophosphate Synthase and Evaluation of Their Antitumor Efficacy in Multiple Myeloma, Pancreatic Ductal Adenocarcinoma, and Colorectal Cancer.

Novel C6-substituted pyrazolo[3,4-d]pyrimidine- and C2-substituted purine-based bisphosphonate (C6-PyraP-BP and C2-Pur-BP, respectively) inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS) were designed and evaluated for their ability to block the proliferation of multiple myeloma (MM), pancreatic ductal adenocarcinoma (PDAC), and colorectal cancer (CRC) cells. Pyrazolo[3,4-d]pyrimidine analogs were identified that induce selective intracellular target engagement leading to apoptosis and downregulate the prenylation of Rap-1A in MM, PDAC, and CRC cells. The C6-PyraP-BP inhibitor RB-07-16 was found to exhibit antitumor efficacy in xenograft mouse models of MM and PDAC, significantly reducing tumor growth without substantially increasing liver enzymes or causing significant histopathologic damage, usually associated with hepatotoxicity. RB-07-16 is a metabolically stable compound in cross-species liver microsomes, does not inhibit key CYP 450 enzymes, and exhibits good systemic circulation in rat. Collectively, the current studies provide encouraging support for further optimization of the pyrazolo[3,4-d]pyrimidine-based GGPPS inhibitors as potential human therapeutics for various cancers.

Synthesis and Evaluation of Structurally Diverse C-2-Substituted Thienopyrimidine-Based Inhibitors of the Human Geranylgeranyl Pyrophosphate Synthase.

Novel analogues of C-2-substituted thienopyrimidine-based bisphosphonates (C2-ThP-BPs) are described that are potent inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS). Members of this class of compounds induce target-selective apoptosis of multiple myeloma (MM) cells and exhibit antimyeloma activity in vivo. A key structural element of these inhibitors is a linker moiety that connects their (((2-phenylthieno[2,3-d]pyrimidin-4-yl)amino)methylene)bisphosphonic acid core to various side chains. The structural diversity of this linker moiety, as well as the side chains attached to it, was investigated and found to significantly impact the toxicity of these compounds in MM cells. The most potent inhibitor identified was evaluated in mouse and rat for liver toxicity and systemic exposure, respectively, providing further optimism for the potential value of such compounds as human therapeutics.

The SMS domain of Trs23p is responsible for the in vitro appearance of the TRAPP I complex in Saccharomyces cerevisiae.

Saccharomyces cerevisiae transport protein particle (TRAPP) is a family of related multisubunit complexes required for endoplasmic reticulum-to-Golgi transport (TRAPP I), endosome-to-Golgi transport (TRAPP II) or cytosol to vacuole targeting (TRAPP III). To gain insight into the relationship between these complexes, we generated random and targeted mutations in the Trs23p core subunit. Remarkably, at physiological salt concentrations only two peaks (TRAPP I and a high molecular weight peak) are detected in wild-type cells. As the salt was raised, the high molecular weight peak resolved into TRAPP II and III peaks. Deletion of a Saccharomycotina-specific domain of Trs23p resulted in destabilization of TRAPP I but had no effect on TRAPP II or III. This mutation had no observable growth phenotype, normal levels of Ypt1p-directed guanine nucleotide exchange factor activity in vivo and did not display any in vivo nor in vitro blocks in membrane traffic. Biochemical analysis indicated that TRAPP I could be produced from the TRAPP II/III peak in vitro by increasing the salt concentration. Our data suggest that the SMS domain of Trs23p is responsible for the in vitro appearance of TRAPP I in S. cerevisiae. The implications of these findings are discussed.