A Nucleophilic Activity-Based Probe Enables Profiling of PLP-Dependent Enzymes.

PLP-dependent enzymes represent an important class of highly "druggable" enzymes that perform a wide array of critical reactions to support all organisms. Inhibition of individual members of this family of enzymes has been validated as a therapeutic target for pathologies ranging from infection with Mycobacterium tuberculosis to epilepsy. Given the broad nature of the activities within this family of enzymes, we envisioned a universally acting probe to characterize existing and putative members of the family that also includes the necessary chemical moieties to enable activity-based protein profiling experiments. Hence, we developed a probe that contains an N-hydroxyalanine warhead that acts as a covalent inhibitor of PLP-dependent enzymes, a linear diazirine for UV crosslinking, and an alkyne moiety to enable enrichment of crosslinked proteins. Our molecule was used to study PLP-dependent enzymes in vitro as well as look at whole-cell lysates of M. tuberculosis and assess inhibitory activity. The probe was able to enrich and identify LysA, a PLP-dependent enzyme crucial for lysine biosynthesis, through mass spectrometry. Overall, our study shows the utility of this trifunctional first-generation probe. We anticipate further optimization of probes for PLP-dependent enzymes will enable the characterization of rationally designed covalent inhibitors of PLP-dependent enzymes, which will expedite the preclinical characterization of these important therapeutic targets.

Cephem-Pyrazinoic Acid Conjugates: Circumventing Resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) is a leading source of infectious disease mortality globally. Antibiotic-resistant strains comprise an estimated 10 % of new TB cases and present an urgent need for novel therapeutics. ß-lactam antibiotics have traditionally been ineffective against M. tuberculosis (Mtb), the causative agent of TB, due to the organism's inherent expression of ß-lactamases that destroy the electrophilic ß-lactam warhead. We have developed novel ß-lactam conjugates, which exploit this inherent ß-lactamase activity to achieve selective release of pyrazinoic acid (POA), the active form of a first-line TB drug. These conjugates are selectively active against M. tuberculosis and related mycobacteria, and activity is retained or even potentiated in multiple resistant strains and models. Preliminary mechanistic investigations suggest that both the POA "warhead" as well as the ß-lactam "promoiety" contribute to the observed activity, demonstrating a codrug strategy with important implications for future TB therapy.

A Versatile Chemoenzymatic Synthesis for the Discovery of Potent Cryptophycin Analogs.

The cryptophycins are a family of macrocyclic depsipeptide natural products that display exceptionally potent antiproliferative activity against drug-resistant cancers. Unique challenges facing the synthesis and derivatization of this complex group of molecules motivated us to investigate a chemoenzymatic synthesis designed to access new analogs for biological evaluation. The cryptophycin thioesterase (CrpTE) and the cryptophycin epoxidase (CrpE) are a versatile set of enzymes that catalyze macrocyclization and epoxidation of over 20 natural cryptophycin metabolites. Thus, we envisioned a drug development strategy involving their use as standalone biocatalysts for production of unnatural derivatives. Herein, we developed a scalable synthesis of 12 new unit A-B-C-D linear chain elongation intermediates containing heterocyclic aromatic groups as alternatives to the native unit A benzyl group. N-Acetyl cysteamine activated forms of each intermediate were assessed for conversion to macrocyclic products using wild type CrpTE, which demonstrated the exceptional flexibility of this enzyme. Semipreparative scale reactions were conducted for isolation and structural characterization of new cryptophycins. Each was then evaluated as a substrate for CrpE P450 and its ability to generate the epoxidized products from these substrates that possess altered electronics at the unit A styrenyl double bond position. Finally, biological evaluation of the new cryptophycins revealed a des-ß-epoxy analog with low picomolar potency, previously limited to cryptophycins bearing epoxide functionality.