Discovery and validation of a novel inhibitor of HYPE-mediated AMPylation.

Adenosyl monophosphate (AMP)ylation (the covalent transfer of an AMP from Adenosine Triphosphate (ATP) onto a target protein) is catalyzed by the human enzyme Huntingtin Yeast Interacting Partner E (HYPE)/FicD to regulate its substrate, the heat shock chaperone binding immunoglobulin protein (BiP). HYPE-mediated AMPylation of BiP is critical for maintaining proteostasis in the endoplasmic reticulum and mounting a unfolded protein response in times of proteostatic imbalance. Thus, manipulating HYPE's enzymatic activity is a key therapeutic strategy toward the treatment of various protein misfolding diseases, including neuropathy and early-onset diabetes associated with two recently identified clinical mutations of HYPE. Herein, we present an optimized, fluorescence polarization-based, high-throughput screening (HTS) assay to discover activators and inhibitors of HYPE-mediated AMPylation. After challenging our HTS assay with over 30,000 compounds, we discovered a novel AMPylase inhibitor, I2.10. We also determined a low micromolar IC50 for I2.10 and employed biorthogonal counter-screens to validate its efficacy against HYPE's AMPylation of BiP. Further, we report low cytotoxicity of I2.10 on human cell lines. We thus established an optimized, high-quality HTS assay amenable to tracking HYPE's enzymatic activity at scale, and provided the first novel small-molecule inhibitor capable of perturbing HYPE-directed AMPylation of BiP in vitro. Our HTS assay and I2.10 compound serve as a platform for further development of HYPE-specific small-molecule therapeutics.

Biochemical pharmacology of adenylyl cyclases in cancer.

Globally, despite extensive research and pharmacological advancement, cancer remains one of the most common causes of mortality. Understanding the signaling pathways involved in cancer progression is essential for the discovery of new drug targets. The adenylyl cyclase (AC) superfamily comprises glycoproteins that regulate intracellular signaling and convert ATP into cyclic AMP, an important second messenger. The present review highlights the involvement of ACs in cancer progression and suppression, broken down for each specific mammalian AC isoform. The precise mechanisms by which ACs contribute to cancer cell proliferation and invasion are not well understood and are variable among cancer types; however, AC overactivation, along with that of downstream regulators, presents a potential target for novel anticancer therapies. The expression patterns of ACs in numerous cancers are discussed. In addition, we highlight inhibitors of AC-related signaling that are currently under investigation, with a focus on possible anti-cancer strategies. Recent discoveries with small molecules regarding more direct modulation AC activity are also discussed in detail. A more comprehensive understanding of different components in AC-related signaling could potentially lead to the development of novel therapeutic strategies for personalized oncology and might enhance the efficacy of chemoimmunotherapy in the treatment of various cancers.

Covalent Fragment Screening and Optimization Identifies the Chloroacetohydrazide Scaffold as Inhibitors for Ubiquitin C-terminal Hydrolase L1.

Dysregulation of the ubiquitin-proteasome systems is a hallmark of various disease states including neurodegenerative diseases and cancer. Ubiquitin C-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is expressed primarily in the central nervous system under normal physiological conditions, however, is considered an oncogene in various cancers, including melanoma, lung, breast, and lymphoma. Thus, UCHL1 inhibitors could serve as a viable treatment strategy against these aggressive cancers. Herein, we describe a covalent fragment screen that identified the chloroacetohydrazide scaffold as a covalent UCHL1 inhibitor. Subsequent optimization provided an improved fragment with single-digit micromolar potency against UCHL1 and selectivity over the closely related UCHL3. The molecule demonstrated efficacy in cellular assays of metastasis. Additionally, we report a ligand-bound crystal structure of the most potent molecule in complex with UCHL1, providing insight into the binding mode and information for future optimization.

Altered Protein Dynamics and a More Reactive Catalytic Cysteine in a Neurodegeneration-associated UCHL1 Mutant.

A mutant of ubiquitin C-terminal hydrolase L1 (UCHL1) detected in early-onset neurodegenerative patients, UCHL1R178Q, showed higher catalytic activity than wild-type UCHL1 (UCHL1WT). Lying within the active-site pocket, the arginine is part of an interaction network that holds the catalytic histidine in an inactive arrangement. However, the structural basis and mechanism of enzymatic activation upon glutamine substitution was not understood. We combined X-ray crystallography, protein nuclear magnetic resonance (NMR) analysis, enzyme kinetics, covalent inhibition analysis, and biophysical measurements to delineate activating factors in the mutant. While the crystal structure of UCHL1R178Q showed nearly the same arrangement of the catalytic residues and active-site pocket, the mutation caused extensive alteration in the chemical environment and dynamics of more than 30 residues, some as far as 15 Å away from the site of mutation. Significant broadening of backbone amide resonances in the HSQC spectra indicates considerable backbone dynamics changes in several residues, in agreement with solution small-angle X-ray scattering (SAXS) analyses which indicate an overall increase in protein flexibility. Enzyme kinetics show the activation is due to a kcat effect despite a slightly weakened substrate affinity. In line with this, the mutant shows a higher second-order rate constant (kinact/Ki) in a reaction with a substrate-derived irreversible inhibitor, Ub-VME, compared to the wild-type enzyme, an observation indicative of a more reactive catalytic cysteine in the mutant. Together, the observations underscore structural plasticity as a factor contributing to enzyme kinetic behavior which can be modulated through mutational effects.

Inhibition of pathogenic bacterial carbonic anhydrases by monothiocarbamates.

Carbonic anhydrases (CAs) from the pathogenic bacteria Nesseria gonorrhoeae and vancomycin-resistant enterococci (VRE) have recently been validated as antibacterial drug targets. Here we explored the inhibition of the α-CA from N. gonorrhoeae (α-NgCA), of α- and γ-class enzymes from Enterococcus faecium (α-EfCA and γ-EfCA) with a panel of aliphatic, heterocyclic and aryl-alkyl primary/secondary monothiocarbamates (MTCs). α-NgCA was inhibited in vitro with KIs ranging from 0.367 to 0.919 µM. The compounds inhibited the α-EfCA and γ-EfCA with KI ranges of 0.195-0.959 µM and of 0.149-1.90 µM, respectively. Some MTCs were also investigated for their inhibitory effects on the growth of clinically-relevant N. gonorrhoeae and VRE strains. No inhibitory effects on the growth of VRE were noted for all MTCs, whereas one compound (13) inhibited the growth N. gonorrhoeae strains at concentrations ranging from 16 to 64 µg/mL. This suggests that compound 13 may be a potential antibacterial agent against N. gonorrhoeae.

Identification of covalent fragment inhibitors for Plasmodium falciparum UCHL3 with anti-malarial efficacy.

Malaria continues to be a major burden on global health, responsible for 619,000 deaths in 2021. The causative agent of malaria is the eukaryotic parasite Plasmodium. Resistance to artemisinin-based combination therapies (ACTs), the current first-line treatment for malaria, has emerged in Asia, South America, and more recently Africa, where >90% of all malaria-related deaths occur. This has necessitated the identification and investigation of novel parasite proteins and pathways as antimalarial targets, including components of the ubiquitin proteasome system. Here, we investigate Plasmodium falciparum deubiquitinase ubiquitin C-terminal hydrolase L3 (PfUCHL3) as one such target. We carried out a high-throughput screen with covalent fragments and identified seven scaffolds that selectively inhibit the plasmodial UCHL3, but not human UCHL3 or the closely related human UCHL1. After assessing toxicity in human cells, we identified four promising hits and demonstrated their efficacy against asexual P. falciparum blood stages and P. berghei sporozoite stages.

Mechanism-based inhibition of gut microbial tryptophanases reduces serum indoxyl sulfate.

Indoxyl sulfate is a microbially derived uremic toxin that accumulates in late-stage chronic kidney disease and contributes to both renal and cardiovascular toxicity. Indoxyl sulfate is generated by the metabolism of indole, a compound created solely by gut microbial tryptophanases. Here, we characterize the landscape of tryptophanase enzymes in the human gut microbiome and find remarkable structural and functional similarities across diverse taxa. We leverage this homology through a medicinal chemistry campaign to create a potent pan-inhibitor, (3S) ALG-05, and validate its action as a transition-state analog. (3S) ALG-05 successfully reduces indole production in microbial culture and displays minimal toxicity against microbial and mammalian cells. Mice treated with (3S) ALG-05 show reduced cecal indole and serum indoxyl sulfate levels with minimal changes in other tryptophan-metabolizing pathways. These studies present a non-bactericidal pan-inhibitor of gut microbial tryptophanases with potential promise for reducing indoxyl sulfate in chronic kidney disease.

Carbonic Anhydrase Inhibition as a Target for Antibiotic Synergy in Enterococci.

Enterococcus faecalis is a hospital-associated opportunistic pathogen that can cause infections with high mortality, such as infective endocarditis. With an increasing occurrence of multidrug-resistant enterococci, there is a need for alternative strategies to treat enterococcal infections. We isolated a gentamicin-hypersusceptible E. faecalis strain from a patient with infective endocarditis that carried a mutation in the alpha-carbonic anhydrase (α-CA) and investigated how disruption of α-CA sensitized E. faecalis to killing with gentamicin. The gentamicin-hypersusceptible α-CA mutant strain showed increased intracellular gentamicin uptake in comparison to an isogenic strain encoding full-length, wild-type α-CA. We hypothesized that increased gentamicin uptake could be due to increased proton motive force (PMF), increased membrane permeability, or both. We observed increased intracellular ATP production in the α-CA mutant strain, suggesting increased PMF-driven gentamicin uptake contributed to the strain's gentamicin susceptibility. We also analyzed the membrane permeability and fatty acid composition of isogenic wild-type and α-CA mutant strains and found that the mutant displayed a membrane composition that was consistent with increased membrane permeability. Finally, we observed that exposure to the FDA-approved α-CA inhibitor acetazolamide lowered the gentamicin MIC of eight genetically diverse E. faecalis strains with intact α-CA but did not change the MIC of the α-CA mutant strain. These results suggest that α-CA mutation or inhibition increases PMF and alters membrane permeability, leading to increased uptake of gentamicin into E. faecalis. This connection could be exploited clinically to provide new combination therapies for patients with enterococcal infections. IMPORTANCE Enterococcal infections can be difficult to treat, and new therapeutic approaches are needed. In studying an E. faecalis clinical strain from an infected patient, we found that the bacteria were rendered hypersusceptible to aminoglycoside antibiotics through a mutation that disrupted the α-CA. Our follow-on work suggested two different ways that α-CA disruption causes increased gentamicin accumulation in E. faecalis: increased proton motive force-powered uptake and increased membrane permeability. We also found that a mammalian CA inhibitor could sensitize a variety of E. faecalis strains to killing with gentamicin. Given that mammalian CA inhibitors are frequently used to treat conditions such as glaucoma, hypertension, and epilepsy, our findings suggest that these "off-the-shelf" inhibitors could also be useful partner antibiotics for the treatment of E. faecalis infections.

Evaluation of 1,3,4-Thiadiazole Carbonic Anhydrase Inhibitors for Gut Decolonization of Vancomycin-Resistant Enterococci.

Vancomycin-resistant enterococci (VRE), Enterococcus faecium and Enterococcus faecalis, are high-priority drug-resistant pathogens in need of new therapeutic approaches. VRE originate in the gastrointestinal tract of carriers and can lead to more problematic downstream infections in the healthcare setting. Having a carrier of VRE admitted into a healthcare setting increases the risk to other patients for acquiring an infection. One strategy to eliminate the downstream infections is decolonization of VRE from carriers. Here, we report the activity of a set of carbonic anhydrase inhibitors in the in vivo VRE gastrointestinal decolonization mouse model. The molecules encompass a range of antimicrobial potency and intestinal permeability, and these factors were shown to influence the in vivo efficacy for VRE gut decolonization. Overall, carbonic anhydrase inhibitors exhibited superior VRE decolonization efficacy compared to the current drug of choice, linezolid.

Structural Characterization of Thiadiazolesulfonamide Inhibitors Bound to Neisseria gonorrhoeae α-Carbonic Anhydrase.

Drug-resistant Neisseria gonorrhoeae is a critical threat to public health, and bacterial carbonic anhydrases expressed by N. gonorrhoeae are potential new therapeutic targets to combat this pathogen. To further expand upon our recent reports of bacterial carbonic anhydrase inhibitors for the treatment of N. gonorrhoeae, our team has solved ligand-bound crystal structures of the FDA-approved carbonic anhydrase inhibitor acetazolamide, along with three analogs, in complex with the essential α-carbonic anhydrase isoform from N. gonorrhoeae. The structural data for the analogs presented bound to N. gonorrhoeae α-carbonic anhydrase supports the observed structure-activity relationship for in vitro inhibition with this scaffold against the enzyme. Moreover, the ligand-bound structures indicate differences in binding poses compared to those traditionally observed with the close human ortholog carbonic anhydrase II. These results present key differences in inhibitor binding between N. gonorrhoeae α-carbonic anhydrase and the human carbonic anhydrase II isoform.

Protein-protein interaction-based high throughput screening for adenylyl cyclase 1 inhibitors: Design, implementation, and discovery of a novel chemotype.

Genetic and preclinical studies have implicated adenylyl cyclase 1 (AC1) as a potential target for the treatment of chronic inflammatory pain. AC1 activity is increased following inflammatory pain stimuli and AC1 knockout mice show a marked reduction in responses to inflammatory pain. Previous drug discovery efforts have centered around the inhibition of AC1 activity in cell-based assays. In the present study, we used an in vitro approach focused on inhibition of the protein-protein interaction (PPI) between Ca2+/calmodulin (CaM) and AC1, an interaction that is required for activation of AC1. We developed a novel fluorescence polarization (FP) assay focused on the PPI between an AC1 peptide and CaM and used this assay to screen over 23,000 compounds for inhibitors of the AC1-CaM PPI. Next, we used a cellular NanoBiT assay to validate 21 FP hits for inhibition of the AC1-CaM PPI in a cellular context with full-length proteins. Based on efficacy, potency, and selectivity for AC1, hits 12, 13, 15, 18, 20, and 21 were prioritized. We then tested these compounds for inhibition of AC1 activity in cyclic AMP (cAMP) accumulation assays, using HEK293 cells stably expressing AC1. Hit 15 contained a dithiophene scaffold and was of particular interest because it shared structural similarities with our recently reported benzamide series of AC1 inhibitors. We next tested a small set of 13 compounds containing the dithiophene scaffold for structure-activity relationship studies. Although many compounds were non-selective, we observed trends for tuning AC1/AC8 selectivity based on heterocycle type and substituents. Having an ethyl on the central thiophene caused the scaffold to be more selective for AC8. Cyclization of the alkyl substituent fused to the thiophene significantly reduced activity and also shifted selectivity toward AC8. Notably, combining the fused cyclohexane-thiophene ring system with a morpholine heterocycle significantly increased potency at both AC1 and AC8. Through designing a novel FP screen and NanoBiT assay, and evaluating hits in cAMP accumulation assays, we have discovered a novel, potent, dithiophene scaffold for inhibition of the AC1- and AC8-CaM PPI. We also report the most potent fully efficacious inhibitor of AC8 activity known to-date.

Structure-activity relationship studies for inhibitors for vancomycin-resistant Enterococcus and human carbonic anhydrases.

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 (Ki) against the E. faecium α-CA (Efα-CA) and γ-CA (Efγ-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 Ki for each CA.

Optimization of a Pyrimidinone Series for Selective Inhibition of Ca2+/Calmodulin-Stimulated Adenylyl Cyclase 1 Activity for the Treatment of Chronic Pain.

Adenylyl cyclase type 1 (AC1) is involved in signaling for chronic pain sensitization in the central nervous system and is an emerging target for the treatment of chronic pain. AC1 and a closely related isoform AC8 are also implicated to have roles in learning and memory signaling processes. Our team has carried out cellular screening for inhibitors of AC1 yielding a pyrazolyl-pyrimidinone scaffold with low micromolar potency against AC1 and selectivity versus AC8. Structure-activity relationship (SAR) studies led to analogues with cellular IC50 values as low as 0.25 µM, selectivity versus AC8 and other AC isoforms as well as other common neurological targets. A representative analogue displayed modest antiallodynic effects in a mouse model of inflammatory pain. This series represents the most potent and selective inhibitors of Ca2+/calmodulin-stimulated AC1 activity to date with improved drug-like physicochemical properties making them potential lead compounds for the treatment of inflammatory pain.

Inhibition studies of bacterial α-carbonic anhydrases with phenols.

The α-class carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogens Neisseria gonorrhoeae (NgCAα) and Vibrio cholerae (VchCAα) were investigated for their inhibition by a panel of phenols and phenolic acids. Mono-, di- and tri-substituted phenols incorporating additional hydroxyl/hydroxymethyl, amino, acetamido, carboxyl, halogeno and carboxyethenyl moieties were included in the study. The best NgCAα inhibitrs were phenol, 3-aminophenol, 4-hydroxy-benzylalcohol, 3-amino-4-chlorophenol and paracetamol, with KI values of 0.6-1.7 µM. The most effective VchCAα inhibitrs were phenol, 3-amino-4-chlorophenol and 4-hydroxy-benzyl-alcohol, with KI values of 0.7-1.2 µM. Small changes in the phenol scaffold led to drastic effects on the bacterial CA inhibitory activity. This class of underinvestigated bacterial CA inhibitors may thus lead to effective compounds for fighting drug resistant bacteria.

Coumarins effectively inhibit bacterial α-carbonic anhydrases.

Coumarins are known to act as prodrug inhibitors of mammalian α-carbonic anhydrases (CAs, EC 4.2.1.1) but they were not yet investigated for the inhibition of bacterial α-CAs. Here we demonstrate that such enzymes from the bacterial pathogens Neisseria gonorrhoeae (NgCAα) and Vibrio cholerae (VchCAα) are inhibited by a panel of simple coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system. The nature and the position of the substituents in the coumarin ring were the factors which strongly influenced inhibitory efficacy. NgCAα was inhibited with KIs in the range of 28.6-469.5 µM, whereas VchCAα with KIs in the range of 39.8-438.7 µM. The two human (h)CA isoforms included for comparison reason in the study, hCA I and II, were less prone to inhibition by these compounds, with KIs of 137-948.9 µM for hCA I and of 296.5-961.2 µM for hCA II, respectively. These findings are relevant for discovering coumarin bacterial CA inhibitors with selectivity for the bacterial over human isoform, with potential applications as novel antibacterial agents.

Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3.

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.

Dithiocarbamates effectively inhibit the α-carbonic anhydrase from Neisseria gonorrhoeae.

Recently, inorganic anions and sulphonamides, two of the main classes of zinc-binding carbonic anhydrase inhibitors (CAIs), were investigated for inhibition of the α-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. KIs 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 µg/mL. As NgCA was recently validated as an antibacterial drug target, the DTCs may lead to development of novel antigonococcal agents.

Repurposing FDA-approved sulphonamide carbonic anhydrase inhibitors for treatment of Neisseria gonorrhoeae.

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.

Anion inhibition studies of the α-carbonic anhydrases from Neisseria gonorrhoeae.

The bacterial pathogen Neisseria gonorrhoeae encodes for an α-class carbonic anhydrase (CA, EC 4.2.1.1), NgCA, which was investigated for its inhibition with a series of inorganic and organic anions. Perchlorate and hexafluorophosphate did not significantly inhibit NgCA CO2 hydrase activity, whereas the halides, azide, bicarbonate, carbonate, stannate, perosmate, diphosphate, divanadate, perruthenate, and trifluoromethanesulfonate showed inhibition constants in the range of 1.3-9.6 mM. Anions/small molecules such as cyanate, thiocyanate, nitrite, nitrate, bisulphite, sulphate, hydrogensulfide, phenylboronic acid, phenylarsonic acid, selenate, tellurate, tetraborate, perrhenate, peroxydisulfate, selenocyanate, iminodisulfonate, and fluorosulfonate showed KIs in the range of 0.15-1.0 mM. The most effective inhibitors detected in this study were sulfamide, sulfamate, trithiocarbonate and N,N-diethyldithiocarbamate, which had KIs in the range of 5.1-88 µM. These last compounds incorporating the CS2- zinc-binding group may be used as leads for developing even more effective NgCA inhibitors in addition to the aromatic/heterocyclic sulphonamides, as this enzyme was recently validated as an antibacterial drug target for obtaining novel antigonococcal agents.