Synthesis and Biological Evaluation of Protein Tyrosine Phosphatase Inhibitors
PublisherThe University of Arizona.
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EmbargoRelease after 26-Sep-2019
AbstractProtein tyrosine phosphatases (PTP) are a group of enzymes that regulate signal transduction by removing a phosphate group from tyrosine residues of their substrates. Despite successes in drug discovery and development based on PTP' counterparts, the kinases, there are currently no molecule in the clinic targeting PTP. Like kinases, one of the major challenges with targeting of PTP is isoform selectivity since the structure of the active site of PTP is highly conserved, as is the basic biochemical mechanism. Further confounding PTP drug discovery are their shallow, positively charged active sites which are often targeted with polar molecules. Although this approach can produce molecules with high affinity, specificity is lacking as well as poor pharmacological properties. To look for isoform-selective PTP inhibitors, we used a parallel screening strategy. A panel of the most highly conserved PTP was cloned, expressed, and purified using a high-throughput protein production strategy from the structural genomics literature. Using our panel of PTP and a general PTP assay, a library of natural products and natural product derivatives was screened for isoform selectivity. From this, we found a number of hits, including a protein tyrosine phosphatase 1B (PTP1B) inhibitor. PTP1B is a negative regulator of insulin and leptin signaling and a validated drug target for the treatment of obesity and type II diabetes. Of those active in the preliminary assay, the most promising compounds were 19 and 20, containing a novel pyrrolopyrazoloisoquinolone scaffold derived from treatment of the HSP90 inhibitor, radicicol, with hydrazine, a procedure we call nitrogen atom augmentation. Intriguingly, 19 and 20 were found to be selective for PTP1B when screened against a panel of highly conserved non-receptor PTP. Biochemical evaluation, molecular docking, and mutagenesis revealed 19 and 20 to be allosteric inhibitors of PTP1B with sub-µM Kis. However, Western blot analysis revealed only 19 to be active in cells, so our efforts were focused on 19. The immunoblot studies of insulin-stimulated C2C12 myoblasts indicated that 19 was capable of restoring insulin signaling in a dose-dependent manner. In addition to natural products and their derivatives, there is a continued need for synthetic strategies to generate enantiopure materials with complex, drug-like architectures that can be used in drug discovery campaigns. To facilitate this, we developed a highly enantioselective [4+1] cycloaddition reaction of aryl diazoacetates and aryl propargyl alcohols involving chiral silver-carbene transfer processes using a bifunctional silver catalyst. This reaction provides a new approach to access optically active 2,5-dihydrofurans. Mechanistic studies revealed that the reaction involves second-order kinetics of the chiral silver catalyst, suggesting that two silver catalysts may be involved synergistically in the intramolecular trapping process of a silver-associated ylide with silver-activated alkynes. Using this strategy, we generated a collection of asymmetric 2,5-dihydrofurans and evaluated them against our collection of protein tyrosine phosphatases (PTP). This revealed several members of this class to have good to excellent selectivity for PTP1B relative to the other highly homologous PTP. Molecular docking, mutagenesis, and enzymology indicated an allosteric, non-competitive mechanism with a possibly unique mode of binding to PTP1B. Western blot analysis showed these compounds could restore insulin signaling in C2C12 myotubes. Last but not the least, a diastereoselectively switchable enantioselective synthesis of 3,4-substituted tetrahydro-β-carbolines (THBCs) via Rh(II)/chiral Brønsted acid co-catalysed three-component cascade Pictet-Spengler reaction has been developed. This process allows rapid and efficient access to both cis- and trans- 3,4-substituted THBCs in moderate to good yields with high diastereoselectivity and enantioselectivity. These THBCs were identified as PTP inhibitors with low µM IC50s. The structure and activity relationship was investigated via experimental and computational tools.
Degree ProgramGraduate College