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Neisseria gonorrhoeae is an urgent threat to public health in the United States and around the world. Many of the current classes of antibiotics to treat N. gonorrhoeae infection are quickly becoming obsolete due to increased rates of resistance. Thus, there is a critical need for alternative antimicrobial targets and new chemical entities. Our team has repurposed the FDA-approved carbonic anhydrase inhibitor scaffold of acetazolamide to target N. gonorrhoeae and the bacteria's essential carbonic anhydrase, NgCA. This study established both structure-activity and structure-property relationships that contribute to both antimicrobial activity and NgCA activity. This ultimately led to molecules 20 and 23, which displayed minimum inhibitory concentration values as low as 0.25 μg/mL equating to an 8- to 16-fold improvement in antigonococcal activity compared to acetazolamide. These analogues were determined to be bacteriostatic against the pathogen and likely on-target against NgCA. Additionally, they did not exhibit any detrimental effects in cellular toxicity assays against both a human endocervical (End1/E6E7) cell line or colorectal adenocarcinoma cell line (Caco-2) at concentrations up to 128 μg/mL. Taken together, this study presents a class of antigonococcal agents with the potential to be advanced for further evaluation in N. gonorrhoeae infection models.

Ubiquitin (Ub) is a highly conserved protein that is covalently attached to substrate proteins as a post-translational modification to regulate signaling pathways such as proteasomal degradation and cell cycle/transcriptional regulation in the eukaryotic cellular environment. Ub signaling is regulated by the homeostasis of substrate protein ubiquitination/deubiquitination by E3 ligases and deubiquitinating enzymes (DUBs) in healthy eukaryotic systems. One such DUB, ubiquitin C-terminal hydrolase L1 (UCHL1), is endogenously expressed in the central nervous system under normal physiological conditions, but overexpression and/or mutation has been linked to various cancers and neurodegenerative diseases. The lack of UCHL1 probing strategies suggests development of a selective Ub variant (UbV) for probing UCHL1's role in these disease states would be beneficial. We describe a computational design approach to investigate UbVs that lend selectivity, both binding and inhibition, to UCHL1 over the close structural homologue UCHL3 and members of other DUB families. A number of UbVs, mainly those containing Thr9 mutations, displayed appreciable binding and inhibition selectivity for UCHL1 over UCHL3, compared to wild-type Ub in in vitro assays. By appending reactive electrophiles to the C-terminus of the UbVs, we created the first activity-based probe (ABP) with demonstrated reaction selectivity for UCH family DUBs over other families in cell lysates. Further kinetic analysis of covalent inhibition by the UbV-ABP with UCHL1 and UCHL3 offers insight into the future design of UCHL1 selective UbV-ABP. These studies serve as a proof of concept of the viability of the in silico design of ubiquitin variants for UCH family DUBs as a step toward the development of macromolecular UCHL1 inhibitors.

The deubiquitinase (DUB) ubiquitin C-terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small-molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine-based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.

Protein tyrosine phosphatases (PTPs) counteract the enzymatic activity of protein tyrosine kinases to modulate levels of both normal and disease-associated protein tyrosine phosphorylation. Aberrant activity of PTPs has been linked to the progression of many disease states, yet no PTP inhibitors are currently clinically available. PTPs are without a doubt a difficult drug target. Despite this, many selective, potent, and bioavailable PTP inhibitors have been described, suggesting PTPs should once again be looked at as viable therapeutic targets. Herein, we summarize recently discovered PTP inhibitors and their use in the functional interrogation of PTPs in disease states. In addition, an overview of the therapeutic targeting of PTPs is described using SHP2 as a representative target.

Neisseria gonorrhoeae is an urgent threat to public health in the United States and around the world. Many of the current classes of antibiotics to treat N. gonorrhoeae infection are quickly becoming obsolete due to increased rates of resistance. Thus, there is a critical need for alternative antimicrobial targets and new chemical entities. Our team has repurposed the FDA-approved carbonic anhydrase inhibitor scaffold of acetazolamide to target N. gonorrhoeae and the bacteria’s essential carbonic anhydrase, NgCA. This study established both structure-activity and structure-property relationships that contribute to both antimicrobial activity and NgCA activity. This ultimately led to molecules 20 and 23, which displayed minimum inhibitory concentration values as low as 0.25 μg/mL equating to an 8- to 16-fold improvement in anti-gonococcal activity compared to acetazolamide. These analogs were determined to be bacteriostatic against the pathogen and likely on-target against NgCA. Additionally, they did not exhibit any detrimental effects in cellular toxicity assays against both a human endocervical (End1/E6E7) cell line or colorectal adenocarcinoma cell line (Caco-2) at concentrations up to 128 μg/mL. Taken together, this study presents a class of anti-gonococcal agents with the potential to be advanced for further evaluation in N. gonorrhoeae infection models.

The deubiquitinase (DUB) Ubiquitin C-terminal Hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small-molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine-based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.

Vancomycin-resistant enterococci (VRE) are the second leading cause of hospital-acquired infections (HAIs) attributed to a drug-resistant bacterium in the United States and resistance to the frontline treatments is well documented. To combat VRE, we have repurposed the FDA-approved carbonic anhydrase drug acetazolamide to design potent anti-enterococcal agents. Through structure-activity relationship optimization we have arrived at two leads possessing improved potency against clinical VRE strains from MIC = 2 μg/mL (acetazolamide) to MIC = 0.007 μg/mL (22) and 1 μg/mL (26). Physicochemical properties were modified to design leads that either have high oral bioavailability to treat systemic infections or low intestinal permeability to treat VRE infections in the gastrointestinal tract. Our data suggests the intracellular target for the molecules are putative α-carbonic and γ-carbonic anhydrases and homology modeling and molecular dynamics simulations were performed. Together, this study presents potential anti-VRE therapeutic options to provide alternatives for problematic VRE infections.