Large-Scale Chemical-Genetic Interaction Profiling Identifies a Small-Molecule Inhibitor of Mycobacterium...

Area of Science:

• Microbiology
• Drug Discovery
• Chemical Biology

Background:

• The PROSPECT platform screens compounds against Mycobacterium tuberculosis (Mtb) hypomorphs to identify antimicrobial drug candidates.
• Understanding the mechanism of action (MOA) is crucial for prioritizing drug leads and predicting efficacy.

Purpose of the Study:

• To identify novel antimicrobial compounds targeting Mtb using the PROSPECT platform.
• To elucidate the specific molecular target and MOA of a newly identified inhibitor, BRD1554.
• To demonstrate the utility of PROSPECT in high-resolution MOA assignment and synergy prediction.

Main Methods:

• Chemical-genetic interaction profiling using the PROSPECT platform against Mtb hypomorphs.
• Synthesis and characterization of a novel pyrazole carboxamide inhibitor (BRD1554) and its analogues.
• Mechanism of action assignment using PCL analysis, gene expression profiling, and resistance mutation mapping.
• Biochemical assays using recombinant Pks13 thioesterase domain and computational docking.

Main Results:

• BRD1554 selectively targeted strains depleted of polyketide synthase 13 (Pks13) and Rv2581c.
• PCL analysis predicted Pks13 thioesterase domain as the primary target.
• A potent analogue, 1554-06, demonstrated stereospecific inhibition of Pks13 thioesterase domain (MIC90 = 3.0 μM).
• Resistance mutations and docking studies confirmed Pks13 thioesterase domain as the target.
• Unique chemical-genetic interactions were observed with Mtb detoxifying enzymes.

Conclusions:

• PROSPECT effectively identified a novel Mtb inhibitor (BRD1554) and precisely determined its MOA, targeting the Pks13 thioesterase domain.
• The study confirms Pks13 as a viable target for novel antimicrobials.
• PROSPECT's ability to resolve MOA and identify genetic interactions can guide combination therapy strategies.