Internally quenched fluorogenic probe provides selective and rapid detection of cathepsin L activity

Cathepsin L (CTL) is a cysteine protease that demonstrates upregulated activity and/or altered trafficking during disease states such as cancer. The overlapping substrate specificity of cathepsin family members makes selective detection of activity from a single cathepsin difficult, and CTL activity is particularly difficult to parse from its close homologue CTV and the ubiquitous CTB. Despite this, screening campaigns have explored the extended chemical space in the cathepsin binding sites and identified unique substrate structures that offer selectivity for one enzyme over others. In this vein, we present CTLAP, a fluorogenic probe that is rapidly activated by CTL and displays good selectivity over CTB and CTV, the closest competing analytes for CTL activity probes. CTLAP exhibits intrinsically low background fluorescence, which we attribute to possible self-quenching mechanisms. CTLAP demonstrates markedly higher turn-on ratios (24-fold) and moderately improved enzyme selectivity compared to Z-FR-AMC (10-fold turn-on ratio), a commercially available CTL-selective probe commonly used to detect CTL activity in mixed samples. Optimum selectivity for CTL is achieved within 10 min of incubation with the enzyme, suggesting that CTLAP is amenable for rapid detection of CTL, even in the presence of competing cathepsins.

Cofactor Selectivity in Methylmalonyl Coenzyme A Mutase, a Model Cobamide-Dependent Enzyme

ABSTRACT Cobamides, a uniquely diverse family of enzyme cofactors related to vitamin B12, are produced exclusively by bacteria and archaea but used in all domains of life. While it is widely accepted that cobamide-dependent organisms require specific cobamides for their metabolism, the biochemical mechanisms that make cobamides functionally distinct are largely unknown. Here, we examine the effects of cobamide structural variation on a model cobamide-dependent enzyme, methylmalonyl coenzyme A (CoA) mutase (MCM). The in vitro binding affinity of MCM for cobamides can be dramatically influenced by small changes in the structure of the lower ligand of the cobamide, and binding selectivity differs between bacterial orthologs of MCM. In contrast, variations in the lower ligand have minor effects on MCM catalysis. Bacterial growth assays demonstrate that cobamide requirements of MCM in vitro largely correlate with in vivo cobamide dependence. This result underscores the importance of enzyme selectivity in the cobamide-dependent physiology of bacteria. IMPORTANCE Cobamides, including vitamin B12, are enzyme cofactors used by organisms in all domains of life. Cobamides are structurally diverse, and microbial growth and metabolism vary based on cobamide structure. Understanding cobamide preference in microorganisms is important given that cobamides are widely used and appear to mediate microbial interactions in host-associated and aquatic environments. Until now, the biochemical basis for cobamide preferences was largely unknown. In this study, we analyzed the effects of the structural diversity of cobamides on a model cobamide-dependent enzyme, methylmalonyl-CoA mutase (MCM). We found that very small changes in cobamide structure could dramatically affect the binding affinity of cobamides to MCM. Strikingly, cobamide-dependent growth of a model bacterium, Sinorhizobium meliloti, largely correlated with the cofactor binding selectivity of S. meliloti MCM, emphasizing the importance of cobamide-dependent enzyme selectivity in bacterial growth and cobamide-mediated microbial interactions.

Benzimidazole inhibitors of the major cysteine protease of Trypanosoma brucei

Aim: Limitations in available therapies for trypanosomiases indicate the need for improved medicines. Cysteine proteases cruzain and rhodesain are validated targets for treatment of Chagas disease and human African trypanosomiasis. Previous studies reported a benzimidazole series as potent cruzain inhibitors. Results & methodology: Considering the high similarity between these proteases, we evaluated 40 benzimidazoles against rhodesain. We describe their structure-activity relationships (SAR), revealing trends similar to those observed for cruzain and features that lead to enzyme selectivity. This series comprises noncovalent competitive inhibitors (best Ki = 0.21 μM against rhodesain) and micromolar activity against Trypanosoma brucei brucei. A cheminformatics analysis confirms scaffold novelty, and the inhibitors described have favorable predicted physicochemical properties. Conclusion: Our results support this series as a starting point for new human African trypanosomiasis medicines.

Cofactor selectivity in methylmalonyl-CoA mutase, a model cobamide-dependent enzyme

Cobamides, a uniquely diverse family of enzyme cofactors related to vitamin B12, are produced exclusively by bacteria and archaea but used in all domains of life. While it is widely accepted that cobamide-dependent organisms require specific cobamides for their metabolism, the biochemical mechanisms that make cobamides functionally distinct are largely unknown. Here, we examine the effects of cobamide structural variation on a model cobamide-dependent enzyme, methylmalonyl-CoA mutase (MCM). Thein vitrobinding affinity of MCM for cobamides can be dramatically influenced by small changes in the structure of the lower ligand of the cobamide, and binding selectivity differs between bacterial orthologs of MCM. In contrast, variations in the lower ligand have minor effects on MCM catalysis. Bacterial growth assays demonstrate that cobamide requirements of MCMin vitrolargely correlate within vivocobamide dependence. This result underscores the importance of enzyme selectivity in the cobamide-dependent physiology of bacteria.

DHFR Inhibitors: Reading the Past for Discovering Novel Anticancer Agents

Dihydrofolate reductase inhibitors are an important class of drugs, as evidenced by their use as antibacterial, antimalarial, antifungal, and anticancer agents. Progress in understanding the biochemical basis of mechanisms responsible for enzyme selectivity and antiproliferative effects has renewed the interest in antifolates for cancer chemotherapy and prompted the medicinal chemistry community to develop novel and selective human DHFR inhibitors, thus leading to a new generation of DHFR inhibitors. This work summarizes the mechanism of action, chemical, and anticancer profile of the DHFR inhibitors discovered in the last six years. New strategies in DHFR drug discovery are also provided, in order to thoroughly delineate the current landscape for medicinal chemists interested in furthering this study in the anticancer field.

Comparison of selective hydrolysis of α-lactalbumin by acid Protease A and Protease M as alternative to pepsin: potential for β-lactoglobulin purification in whey proteins

The experiments reported in this research paper examine the potential of digestion using acidic enzymes Protease A and Protease M to selectively hydrolyse α-lactalbumin (α-La) whilst leaving β-lactoglobulin (β-Lg) relatively intact. Both enzymes were compared with pepsin hydrolysis since its selectivity to different whey proteins is known. Analysis of the hydrolysis environment showed that the pH and temperature play a significant role in determining the best conditions for achievement of hydrolysis, irrespective of which enzyme was used. Whey protein isolate (WPI) was hydrolysed using pepsin, Acid Protease A and Protease M by randomized hydrolysis conditions. Reversed-phase high performance liquid chromatography was used to analyse residual proteins. Regarding enzyme selectivity under various milieu conditions, all three enzymes showed similarities in the reaction progress and their potential for β-Lg isolation.

Mechanistic insights into a non-heme 2-oxoglutarate-dependent ethylene-forming enzyme: selectivity of ethylene-formation versusl-Arg hydroxylation

QM/MM calculations for a non-heme 2-oxoglutarate-dependent ethylene-forming enzyme reveal that the selectivity of ethylene formation versus succinate formation is determined by the relative energies of transition states for the competing CO2 insertion and O–O bond cleavage of the FeII-peroxysuccinate species.

Inhibition of Pantothenate Synthetase by Analogs of β-Alanine Precursor Ineffective as an Antibacterial Strategy

Background: Pantothenate, the fundamental precursor to coenzyme A, is required for optimal growth and virulence of microbial pathogens. It is synthesized by the enzyme-catalyzed condensation of β-alanine and pantoate, which has shown susceptibility to inhibition by analogs of its molecular constituents. Accordingly, analogs of β-alanine are gaining inquiry as potential antimicrobial chemotherapeutics. Methods: We synthesized and evaluated 35 derivatives of β-alanine, substituted at the α, β, amine, and carboxyl sites, derived from in silico, dynamic molecular modeling to be potential competitive inhibitors of pantothenate synthetase. Employing the Clinical Laboratory Standards M7-A6 broth microdilution method, we tested these for inhibition of growth in Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Results: All compounds proved entirely ineffective in all species tested, with no inhibition of growth being observed up to 200 µM/mL. Conclusions: Upon revision of the literature, we conclude that high enzyme selectivity or external salvage mechanisms may render this strategy futile against most bacteria.