A High Throughput Substrate Binding Assay Reveals Hexachlorophene as an Inhibitor of the ER-resident HSP70 Chaperone GRP78

Glucose-regulated protein 78 (GRP78) is the ER resident 70 kDa heat shock protein 70 (HSP70) and has been hypothesized to be a therapeutic target for various forms of cancer due to its role in mitigating proteotoxic stress in the ER, its elevated expression in some cancers, and the correlation between high levels for GRP78 and a poor prognosis. Herein we report the development and use of a high throughput fluorescence polarization-based peptide binding assay as an initial step toward the discovery and development of GRP78 inhibitors. This assay was used in a pilot screen to discover the anti-infective agent, hexachlorophene, as an inhibitor of GRP78. Through biochemical characterization we show that hexachlorophene is a competitive inhibitor of the GRP78-peptide interaction. Biological investigations showed that this molecule induces the unfolded protein response, induces autophagy, and leads to apoptosis in a colon carcinoma cell model, which is known to be sensitive to GRP78 inhibition

Synthesis and Biological Evaluation of Protein Tyrosine Phosphatase Inhibitors

Protein 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.Release after 26-Sep-201

Carbenoid-involved reactions integrated with scaffold-based screening generates a Nav1.7 inhibitor

Abstract The discovery of selective Nav1.7 inhibitors is a promising approach for developing anti-nociceptive drugs. In this study, we present a novel oxindole-based readily accessible library (OREAL), which is characterized by readily accessibility, unique chemical space, ideal drug-like properties, and structural diversity. We used a scaffold-based approach to screen the OREAL and discovered compound C4 as a potent Nav1.7 inhibitor. The bioactivity characterization of C4 reveals that it is a selective Nav1.7 inhibitor and effectively reverses Paclitaxel-induced neuropathic pain (PINP) in rodent models. Preliminary toxicology study shows C4 is negative to hERG. The consistent results of molecular docking and molecular simulations further support the reasonability of the in-silico screening and show the insight of the binding mode of C4. Our discovery of C4 paves the way for pushing the Nav1.7-based anti-nociceptive drugs forward to the clinic

Rh₂(Ph₃COO)₃(OAc)/Chiral Phosphoric Acid Cocatalyzed <i>N</i>‑Alkyl Imines-Involved Multicomponent Reactions Yielding <i>N</i>‑(Anthrancen-9-ylmethyl) Isoserines as Drug Intermediates

N-(Anthrancen-9-ylmethyl) isoserines are useful drug intermediates but short for efficient synthesis. We herein report the synthesis of N-(anthrancen-9-ylmethyl) isoserines via a Rh2(Ph3COO)3(OAc) and chiral phosphoric acid (CPA) synergistically catalyzed multicomponent reaction (MCR) of N-alkyl imines, alcohols, and diazoesters. The method representing the first example of N-alkyl imines-involved MCR is featured by high atom-economy, high diastereo- and enantioselectivities, and broad substrate scope. DFT calculations on the mechanism of the MCR reveals that the hydrophobic interactions and π–π stackings between N-(anthrancen-9-ylmethyl) imines and Rh2(Ph3COO)3(OAc)/CPA cocatalyst is essential to the reactivity and stereocontrol. The synthetic applications of the MCR products include the semisynthesis of paclitaxel, its alkyne-tagged derivative, and β-lactam as an anticancer agent overcoming paclitaxel-resistance. We expect this work to shed light on the development of new N-alkyl imines-involved reactions and on the synthesis of drugs with isoserines as intermediates

A One-Step, Atom Economical Synthesis of Thieno[2,3-d]pyrimidin-4-amine Derivatives by a Four-Component Reaction

A Na2HPO4-catalyzed four-component reaction between a ketone, malononitrile, S-8 and formamide has been realized for the first time. This reaction provides a concise approach to thieno[2,3-d]pyrimidin-4-amines, previously requiring 5 steps. The utility of this reaction was validated by preparing a multi-targeted kinase inhibitor and an inhibitor of the NRF2 pathway with excellent atom- and step-economy.NIH [ES023758]12 month embargo; first published: 30 April 2019This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Non-enzymatic Lysine Lactoylation of Glycolytic Enzymes

Post-translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular metabolites and serve as feedback and feedforward mechanisms of regulation. We have identified a PTM that is derived from the glycolytic by-product, methylglyoxal. This reactive metabolite is rapidly conjugated to glutathione via glyoxalase 1, generating lactoylglutathione (LGSH). LGSH is hydrolyzed by glyoxalase 2 (GLO2), cycling glutathione and generating D-lactate. We have identified the non-enzymatic acyl transfer of the lactate moiety from LGSH to protein Lys residues, generating a "LactoylLys'' modification on proteins. GLO2 knockout cells have elevated LGSH and a consequent marked increase in LactoylLys. Using an alkyne-tagged methylglyoxal analog, we show that these modifications are enriched on glycolytic enzymes and regulate glycolysis. Collectively, these data suggest a previously unexplored feedback mechanism that may serve to regulate glycolytic flux under hyperglycemic or Warburg-like conditions.12 month embargo; published online: 22 November 2019This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Discovery of an eIF4A Inhibitor with a Novel Mechanism of Action

Increased protein synthesis is a requirement for malignant growth, and as a result, translation has become a pharmaceutical target for cancer. The initiation of cap-dependent translation is enzymatically driven by the eukaryotic initiation factor (eIF)­4A, an ATP-powered DEAD-box RNA-helicase that unwinds the messenger RNA secondary structure upstream of the start codon, enabling translation of downstream genes. A screen for inhibitors of eIF4A ATPase activity produced an intriguing hit that, surprisingly, was not ATP-competitive. A medicinal chemistry campaign produced the novel eIF4A inhibitor 28, which decreased BJAB Burkitt lymphoma cell viability. Biochemical and cellular studies, molecular docking, and functional assays uncovered that 28 is an RNA-competitive, ATP-uncompetitive inhibitor that engages a novel pocket in the RNA groove of eIF4A and inhibits unwinding activity by interfering with proper RNA binding and suppressing ATP hydrolysis. Inhibition of eIF4A through this unique mechanism may offer new strategies for targeting this promising intersection point of many oncogenic pathways