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Vancomycin-resistant enterococci (VRE), consisting of pathogenic Enterococcus faecalis and E. faecium, is a leading cause of hospital-acquired infections (HAIs). We recently repurposed the FDA-approved human carbonic anhydrase (CA) inhibitor acetazolamide (AZM) against VRE agent with the likely mechanism of action for the molecules being inhibition of one, or both, of the bacterial CA isoforms expressed in VRE. To elucidate how inhibitor binding to the enzymes relates to MIC, we further characterised the inhibition constants (K (i)) against the E. faecium alpha-CA (Ef alpha-CA) and gamma-CA (Ef gamma-CA), as well as against human CA I (hCAI) and human CA II (hCAII) to assess selectivity. We have also utilised homology modelling and molecular dynamics (MD) simulations to gain a better understanding of the potential interactions the molecules are making with the targets. In this paper, we elaborate on the SAR for the AZM analogs as it pertains to MIC and K (i) for each CA. Purdue Institute for Drug Discovery Programmatic; NIH/NIAID [1R01AI148523]; Italian Ministry for University and Research [FISR2019_04819] Published version The research program was partially funded by a Purdue Institute for Drug Discovery Programmatic [Grant (M.N.S and D.P.F.) and NIH/NIAID 1R01AI148523 (M.N.S and D.P.F.)]. This work was also supported by the Italian Ministry for University and Research, [grant FISR2019_04819 BacCAD (C.T.S.)].

Recently, inorganic anions and sulphonamides, two of the main classes of zinc-binding carbonic anhydrase inhibitors (CAIs), were investigated for inhibition of the alpha-class carbonic anhydrase (CA, EC 4.2.1.1) from Neisseria gonorrhoeae, NgCA. As an extension to our previous studies, we report that dithiocarbamates (DTCs) derived from primary or secondary amines constitute a class of efficient inhibitors of NgCA. K(I)s ranging between 83.7 and 827 nM were measured for a series of 31 DTCs that incorporated various aliphatic, aromatic, and heterocyclic scaffolds. A subset of DTCs were selected for antimicrobial testing against N. gonorrhoeae, and three molecules displayed minimum inhibitory concentration (MIC) values less than or equal to 8 mu g/mL. As NgCA was recently validated as an antibacterial drug target, the DTCs may lead to development of novel antigonococcal agents. Italian Ministry for University and Research (MIUR) [FISR2019_04819 BacCAD]; Purdue Institute for Drug Discovery Programmatic Grant; NIH/NIAID [1R01AI148523]; Fondazione Cassa di Risparmio di Firenze [ECR2018.1001]; "Bando di Ateneo per il Finanziamento di Progetti Competitivi per Ricercatori a Tempo Determinato (RTD) dell'Universita di Firenze" [2020-202100] Published version CTS thank the Italian Ministry for University and Research (MIUR), project FISR2019_04819 BacCAD. The research program was partially funded by a Purdue Institute for Drug Discovery Programmatic Grant (to M.N.S. and D.P.F.) and NIH/NIAID 1R01AI148523 (to M.N.S and D.P.F.). Fabrizio Carta (F~C.) is grateful to "Bando di Ateneo per il Finanziamento di Progetti Competitivi per Ricercatori a Tempo Determinato (RTD) dell'Universita di Firenze" 2020-202100, and Fondazione Cassa di Risparmio di Firenze (Grant Number ECR2018.1001) for partially supporting this work.

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.

Neisseria gonorrhoeae is a high-priority pathogen of concern due to the growing prevalence of resistance development against approved antibiotics. Herein, we report the anti-gonococcal activity of ethoxzolamide, the FDA-approved human carbonic anhydrase inhibitor. Ethoxzolamide displayed an MIC50, against a panel of N. gonorrhoeae isolates, of 0.125 µg/mL, 16-fold more potent than acetazolamide, although both molecules exhibited almost similar potency against the gonococcal carbonic anhydrase enzyme (NgCA) in vitro. Acetazolamide displayed an inhibition constant (Ki) versus NgCA of 74 nM, while Ethoxzolamide’s Ki was estimated to 94 nM. Therefore, the increased anti-gonococcal potency of ethoxzolamide was attributed to its increased permeability in N. gonorrhoeae as compared to that of acetazolamide. Both drugs demonstrated bacteriostatic activity against N. gonorrhoeae, exhibited post-antibiotic effects up to 10 hours, and resistance was not observed against both. Taken together, these results indicate that acetazolamide and ethoxzolamide warrant further investigation for translation into effective anti-N. gonorrhoeae agents.

There is currently a lack of reliable methods and strategies to probe the deubiquitinating enzyme UCHL3. Current small molecules reported for this purpose display reduced potency and selectivity in cellular assays. To bridge this gap and provide an alternative approach to probe UCHL3, our group has carried out the rational design of ubiquitin-variant activity-based probes with selectivity for UCHL3 over the closely related UCHL1 and other DUBs. The approach successfully produced a triple-mutant ubiquitin variant activity-based probe, UbVQ40V/T66K/V70F-PRG, that was ultimately 20,000-fold more selective for UCHL3 over UCHL1 when assessed by rate of inactivation assays. This same variant was shown to selectively form covalent adducts with UCHL3 in MDA-MB-231 breast cancer cells and no reactivity toward other DUBs expressed. Overall, this study demonstrates the feasibility of the approach and also provides insight into how this approach may be applied to other DUB targets.

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 deubiquitinating enzyme (DUB) UCHL1 is implicated in various disease states including neurodegenerative disease and cancer. However, there is a lack of quality probe molecules to gain a better understanding on UCHL1 biology. To this end a study was carried out to fully characterize and optimize the irreversible covalent UCHL1 inhibitor VAEFMK. Structure-activity relationship studies identified modifications to improve activity versus the target and a full cellular characterization was carried out for the first time with this scaffold. The studies produced a new inhibitor, 34, with an IC50 value of 7.7 µM against UCHL1 and no observable activity versus the closest related DUB UCHL3. The molecule was also capable of selectively inhibiting UCHL1 in cells and did not demonstrate any discernible off-target toxicity. Finally, the molecule was used for initial probe studies to assess the role of UCHL1 role in proliferation of myeloma cells and migration behavior in small cell lung cancer cells making 34 a new tool to be used in the biological evaluation of UCHL1.

We report the co-crystal structure of the (catalytic Cys)-to-Ala mutant of the deubiquitinase domain of the Legionella pneumophila effector SdeA (SdeADUB) with its ubiquitin (Ub) product. Most of the intermolecular interactions are preserved in this product-bound structure compared to that of the previously characterized complex of SdeADUB with the suicide inhibitor ubiquitin vinylmethyl ester (Ub-VME), whose structure models the acyl-enzyme thioester intermediate. Nuclear magnetic resonance (NMR) titration studies show a chemical shift perturbation pattern that suggests that the same interactions also exist in solution. Isothermal titration calorimetry and NMR titration data reveal that the affinity of wild-type (WT) SdeADUB for Ub is significantly lower than that of the Cys-to-Ala mutant. This is potentially due to repulsive interaction between the thiolate ion of the catalytic Cys residue in WT SdeADUB and the carboxylate group of the C-terminal Gly76 residue in Ub. In the context of SdeADUB catalysis, this electrostatic repulsion arises after the hydrolysis of the scissile isopeptide bond in the acyl-enzyme intermediate and the consequent formation of the C-terminal carboxylic group in the Ub fragment. We hypothesize that this electrostatic repulsion may expedite the release of the Ub product by SdeADUB. We note that similar repulsive interactions may also occur in other deubiquitinases and hydrolases of ubiquitin-like protein modifiers and may constitute a fairly general mechanism of product release within this family. This is a potentially important feature for a family of enzymes that form extensive protein-protein interactions during enzyme-substrate engagement.

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.