A Noncompetitive Inhibitor forMycobacterium tuberculosis’s Class IIa Fructose 1,6-Bisphosphate Aldolase

Abstract

Class II fructose 1,6-bisphosphate aldolase (FBA) is an enzyme critical for bacterial, fungal, and protozoan glycolysis/gluconeogenesis. Importantly, humans lack this type of aldolase, having instead a class I FBA that is structurally and mechanistically distinct from class II FBAs. As such, class II FBA is considered a putative pharmacological target for the development of novel antibiotics against pathogenic bacteria such as Mycobacterium tuberculosis, the causative agent for tuberculosis (TB). To date, several competitive class II FBA substrate mimic-styled inhibitors have been developed; however, they lack either specificity, potency, or properties that limit their potential as possible therapeutics. Recently, through the use of enzymatic and structure-based assisted screening, we identified 8-hydroxyquinoline carboxylic acid (HCA) that has an IC50 of 10 ± 1 μM for the class II FBA present in M. tuberculosis (MtFBA). As opposed to previous inhibitors, HCA behaves in a noncompetitive manner, shows no inhibitory properties toward human and rabbit class I FBAs, and possesses anti-TB properties. Furthermore, we were able to determine the crystal structure of HCA bound to MtFBA to 2.1 Å. HCA also demonstrates inhibitory effects for other class II FBAs, including pathogenic bacteria such as methicillin-resistant Staphylococcus aureus. With its broad-spectrum potential, unique inhibitory characteristics, and flexibility of functionalization, the HCA scaffold likely represents an important advancement in the development of class II FBA inhibitors that can serve as viable preclinical candidates.

8-Hydroxyquinolines Are Boosting Agents of Copper-Related Toxicity in Mycobacterium tuberculosis

“…S1D in the supplemental material). HCA was previously reported to inhibit the zinc-dependent fructose 1,6-bisphosphate aldolase of M. tuberculosis in enzymatic assays (9,46). In accordance with previous reports (9), we confirmed that the compound is a relatively poor inhibitor of whole cells (see Fig.…”

“…2C). HCA displayed no detectable metal-related activity on whole cells over the concentration range tested, despite being previously shown to inhibit Zn-dependent enzymes (50% inhibitory concentration, 10 M) (9).…”

“…The only existing experimental evidence for a metal-related mode of action for this type of compound against M. tuberculosis therefore probably comes from in vitro enzymatic screens on mycobacterial metalloenzymes. The most recent targets were the two methionine aminopeptidases metA (rv0734) and metB (rv2861c) and fructose 1,6-bisphosphate aldolase (fba, rv0363c) (9,46,58). These enzymes are inhibited by the 8HQ derivative HCA through documented interactions between the inhibitor and a catalytically essential zinc or cobalt ion (9,46,58).…”

“…The most recent targets were the two methionine aminopeptidases metA (rv0734) and metB (rv2861c) and fructose 1,6-bisphosphate aldolase (fba, rv0363c) (9,46,58). These enzymes are inhibited by the 8HQ derivative HCA through documented interactions between the inhibitor and a catalytically essential zinc or cobalt ion (9,46,58). However, an often observed shortcoming of enzymatic inhibitors from such target-based approaches is the lack of significant activity on respective model organisms (59), and unfortunately, HCA is no exception (9).…”

“…Other desirable properties of halogenated or nonhalogenated 8HQ derivatives include a bactericidal mode of action, activity against nonreplicating M. tuberculosis cells, and potency against monoresistant and multidrug-resistant clinical M. tuberculosis isolates (8). In addition, 8-hydroxyquinoline-2-carboxylic acid (HCA) has been identified to be a potent noncompetitive inhibitor of the M. tuberculosis class IIa fructose 1,6-bisphosphate aldolase, which is a well-characterized pathogen-specific drug target (9).…”