Alfred L. Williams

Novel Inhibitors of E. coli RecA ATPase Activity

The bacterial RecA protein has been implicated as a bacterial drug target not as an antimicrobial target, but as an adjuvant target with the potential to suppress the mechanism by which bacteria gain drug resistance. In order to identify small molecules that inhibit RecA/ssDNA nucleoprotein filament formation, we have adapted the phosphomolybdate-blue ATPase assay for high throughput screening to determine RecA ATPase activity against a library of 33,600 compounds, which is a selected representation of diverse structure of 350,000. Four distinct chemotypes were represented among the 40 validated hits. SAR and further chemical synthesis is underway to optimize this set of inhibitors to be used as antimicrobial adjuvant agents.

Old drug new use - Amoxapine and its metabolites as potent bacterial β-glucuronidase inhibitors for alleviating cancer drug toxicity

Purpose: Irinotecan (CPT-11) induced diarrhea occurs frequently in patients with cancer and limits its usage. Bacteria β-glucuronidase (GUS) enzymes in intestines convert the nontoxic metabolite of CPT-11, SN-38G, to toxic SN-38, and finally lead to damage of intestinal epithelial cells and diarrhea. We previously reported amoxapine as a potent GUS inhibitor in vitro. To further understand the molecular mechanism of amoxapine and its potential for treatment of CPT-11–induced diarrhea, we studied the binding modes of amoxapine and its metabolites by docking and molecular dynamics simulation, and tested the in vivo efficacy on mice in combination with CPT-11. Experimental Design: The binding of amoxapine, its metabolites, 7-hydroxyamoxapine and 8-hydroxyamoxapine, and a control drug loxapine with GUS was explored by computational protocols. The in vitro potencies of metabolites were measured by Escherichia coli GUS enzyme and cell-based assay. Low-dosage daily oral administration was designed to use along with CPT-11 to treat tumor-bearing mice. Results: Computational modeling results indicated that amoxapine and its metabolites bound in the active site of GUS and satisfied critical pharmacophore features: aromatic features near bacterial loop residue F365′ and hydrogen bond toward E413. Amoxapine and its metabolites were demonstrated as potent in vitro. Administration of low dosages of amoxapine with CPT-11 in mice achieved significant suppression of diarrhea and reduced tumor growth. Conclusions: Amoxapine has great clinical potential to be rapidly translated to human subjects for irinotecan-induced diarrhea. Clin Cancer Res; 20(13); 3521–30. ©2014 AACR.

A synthetic analog of verbenachalcone potentiates NGF-induced neurite outgrowth and enhances cell survival in neuronal cell models

This study uses NeuroScreen-1 (NS-1) cells, a derivative of pheochromocytoma (PC12) cells, to examine neurite outgrowth induced by a novel synthetic verbenachalcone derivative, DSRB20-022 (C22). We treated NS-1 cells with varying concentrations of C22 in the presence of 2ng/mL nerve growth factor (NGF). A dose-dependent effect of C22 was observed at concentrations of 2microM and above, resulting in significant enhancement of NGF-dependent neurite outgrowth in NS-1 cells. C22 did not exhibit neuritogenic activity in the absence of NGF, but promoted a concentration-dependent increase in neurite-bearing cells without inducing cytotoxicity. Cell viability assays showed that C22 and the parent compound verbenachalcone (VC) are neuroprotective and enhanced survival of NS-1, PC12, and the murine neuro-2A (N2a) cell lines under conditions of serum deprivation. The results show that augmentation of NGF-induced neurite outgrowth by C22 in NS-1 was dependent on MAP kinase. Furthermore, the neuroprotective function of C22 and VC was accompanied by suppression of caspase-3/7 activation. However, C22 and VC exerted their antagonistic effects on caspase-3/7 activation through potentially different mechanisms of action.

A Trifluoromethyl Analog of Verbenachalcone Promotes Neurite Outgrowth and Cell Proliferation of NeuroScreen-1 Cells

Past research has shown that natural products of plant and marine origins and their congeners enhance the actions of neuritogenic factors of the central nervous system (CNS) such as nerve growth factor (NGF). However, the role of fluorine substitutions in their structure–activity relationship (SAR) has not been explored. We have synthesized a trifluoromethyl analog of verbenachalcone (VC), a pharmacologically active natural compound previously shown to potentiate NGF activity. This analog, designated C278, enhances neurite outgrowth and proliferation of NeuroScreen-1™ (NS-1) cells, a subclone of PC12 pheochromocytoma cells. C278 increases the percentage of neurite bearing cells in the presence of suboptimal doses of NGF in comparison with controls treated with NGF alone. In addition, C278 stimulates cell growth in reduced serum and serum-free cell culture conditions based on our observation of increases in cell number and metabolic assessment with MTT reduction and resazurin assays. The addition of C278 partially restored inhibition of NGF-induced neurite outgrowth by the mitogen-activated protein kinase kinase (MEK) inhibitors PD98059 and U0126. Short-term sequential exposure of cells to U0126, C278, and NGF enhanced phosphorylation of extracellular signal-regulated kinase (ERK) in comparison with cells treated with only the MEK inhibitor and NGF. C278 also attenuated cell growth arrest caused by exposure to PD98059, U0126 and the phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002 but did not alter phosphorylation of Akt, a classic downstream target of PI3K during cell survival. These data suggest that C278 promotes NGF-dependent neurite outgrowth in NS-1 cells through a MEK signaling pathway by a mechanism that alters short-term activation of ERK. In contrast, C278 promotes PI3K-mediated survival independently of Akt phosphorylation.

Discovery and characterization of an iminocoumarin scaffold as an inhibitor of MEKK2 (MAP3K2)

The kinase MEKK2 (MAP3K2) activates the MEK5/ERK5 cell signaling pathway and may play an important role in tumor growth and metastasis. Thus, MEKK2 may represent a novel kinase target for cancer. In order to identify inhibitors of MEKK2, we screened a library of compounds using a high throughput MEKK2 intrinsic ATPase enzyme assay. We identified two hits with validated structures and confirmed activity in the primary assay (IC50 values = 322 nM and 7.7 μM) and two orthogonal MEKK2 biochemical assays. Compound 1, the more potent hit, was the subject of further investigation. Limited structure-activity relationship (SAR) studies were performed on this iminocoumarin hit which resulted in ≥20-fold more potent analogs (e.g. 8 and 16 nM IC50). Two analogs had improved selectivity in a 50-member kinase profiling panel compared to the hit. These studies suggested that substitutions around the phenoxy ring of this scaffold can impart improved potency and selectivity for MEKK2. Analog Compound 1s (16 nM IC50) was further verified by external testing to inhibit MEKK2 and MEKK3 with similar potencies. Compound 1s displayed activity in cell-based assays in which it inhibited ERK5 pathway activation in cells and inhibited cell migration in a scratch assay. Thus, we have identified a scaffold that has promising potential to be developed into a highly selective and potent inhibitor of MEKK2. Information from these SAR studies provides specific guidance for the future design of MEKK2 inhibitor probes.