Anthony G. Coyne

Tuberculosis is the deadliest infectious disease in history and new drugs are urgently required to combat multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (Mtb). Here, we exploit the relience of Mtb on host-derived cholesterol to develop a novel class of antitubercular compounds that target Mtb CYP125 and CYP142; the enzymes that catalyze the first step of cholesterol metabolism. A combination of fragment screening and structure-based drug design was used to identify a hit compound and guide synthetic optimization of a dual CYP125/142 ligand 5m (K_D 40-160 nM), which potently inhibits enzyme activity in vitro (K_I < 100 nM), and the growth of Mtb in extracellular (MIC₉₉ 0.4-1.5 μM) and intracellular assays (IC₅₀ 1.7 μM). The structural data and lead compounds reported here will help study Mtb cholesterol metabolism and guide the development of novel antibiotics to combat MDR Mtb.

Mycobacterium tuberculosis ( Mtb ) is the world’s most deadly infectious pathogen and new drugs are urgently required to combat the emergence of multi-(MDR) and extensively-(XDR) drug resistant strains. The bacterium specifically upregulates sterol uptake pathways in infected macrophages and the metabolism of host-derived cholesterol is essential for Mtb’s long-term survival in vivo. Here, we report the development of antitubercular small molecules that inhibit the Mtb cholesterol oxidases CYP125 and CYP142, which catalyze the initial step of cholesterol metabolism. An efficient biophysical fragment screen was used to characterize the structure-activity relationships of CYP125 and CYP142, and identify a non-azole small molecule 1a that can bind to the heme cofactor of both enzymes. A structure-guided fragment-linking strategy was used to optimize the binding affinity of 1a , yielding a potent dual CYP125/142 inhibitor 5m (K _D CYP125/CYP142 = 0.04/0.16 µM). Compound 5m potently inhibits the catalytic activity of CYP125 and CYP142 in vitro (K _I values < 0.1 µM), and rapidly depletes Mtb intracellular ATP (IC ₅₀ = 0.15 µM). The compound has antimicrobial activity against both drug susceptible and MDR Mtb ( MIC ₉₉ values 0.4 - 1.5 µM ) in extracellular assays, and inhibits the growth of Mtb in human macrophages (MIC = 1.7 µM) with good selectivity over mammalian cytotoxicity (LD ₅₀ ≥ 50 µM). The combination of small molecule inhibitors and structural data reported here provide useful tools to study the role of cholesterol metabolism in Mtb and are a promising step towards novel antibiotics targeting bioenergetic pathways, which could be used to help combat MDR-TB.

Ligand binding hotspots are regions of protein surfaces that form particularly favourable interactions with small molecule pharmacophores. Targeting interactions with these hotspots maximises the efficiency of ligand binding. Existing methods are capable of identifying hotspots but often lack assays to quantify ligand binding and direct elaboration at these sites. Herein, we describe a fragment‐based competitive ¹⁹F Ligand Based NMR (LB‐NMR) screening platform that enables routine, quantitative ligand profiling focused at ligand‐binding hotspots. As a proof of concept, the method was applied to 4′‐phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium abscessus (Mabs). X‐ray crystallographic characterisation of the hits from a 960‐member fragment screen identified three ligand‐binding hotspots across the PPAT active site. From the fragment hits a collection of ¹⁹F reporter candidates were designed and synthesised. By rigorous prioritisation and use of optimisation workflows, a single ¹⁹F reporter molecule was generated for each hotspot. Profiling the binding of a set of structurally characterised ligands by competitive ¹⁹F LB‐NMR with this suite of ¹⁹F reporters recapitulated the binding affinity and site ID assignments made by ITC and X‐ray crystallography. This quantitative mapping of ligand binding events at hotspot level resolution establishes the utility of the fragment‐based competitive ¹⁹F LB‐NMR screening platform for hotspot‐directed ligand profiling

Ligand binding hotspots are regions of protein surfaces that form particularly favourable interactions with small molecule pharmacophores. Targeting interactions with these hotspots maximises the efficiency of ligand binding. Existing methods are capable of identifying hotspots but often lack assays to quantify ligand binding and direct elaboration at these sites. Herein, we describe a fragment‐based competitive ¹⁹F Ligand Based NMR (LB‐NMR) screening platform that enables routine, quantitative ligand profiling focused at ligand‐binding hotspots. As a proof of concept, the method was applied to 4′‐phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium abscessus (Mabs). X‐ray crystallographic characterisation of the hits from a 960‐member fragment screen identified three ligand‐binding hotspots across the PPAT active site. From the fragment hits a collection of ¹⁹F reporter candidates were designed and synthesised. By rigorous prioritisation and use of optimisation workflows, a single ¹⁹F reporter molecule was generated for each hotspot. Profiling the binding of a set of structurally characterised ligands by competitive ¹⁹F LB‐NMR with this suite of ¹⁹F reporters recapitulated the binding affinity and site ID assignments made by ITC and X‐ray crystallography. This quantitative mapping of ligand binding events at hotspot level resolution establishes the utility of the fragment‐based competitive ¹⁹F LB‐NMR screening platform for hotspot‐directed ligand profiling

Mycobacterium tuberculosis (Mtb) was responsible for approximately 1.6 million deaths in 2021. With the emergence of extensive drug resistance, novel therapeutic agents are urgently needed, and continued drug discovery efforts required. Host‐derived lipids such as cholesterol not only support Mtb growth, but are also suspected to function in immunomodulation, with links to persistence and immune evasion. Mtb cytochrome P450 (CYP) enzymes facilitate key steps in lipid catabolism and thus present potential targets for inhibition. Here we present a series of compounds based on an ethyl 5‐(pyridin‐4‐yl)‐1H‐indole‐2‐carboxylate pharmacophore which bind strongly to both Mtb cholesterol oxidases CYP125 and CYP142. Using a structure‐guided approach, combined with biophysical characterization, compounds with micromolar range in‐cell activity against clinically relevant drug‐resistant isolates were obtained. These will incite further development of much‐needed additional treatment options and provide routes to probe the role of CYP125 and CYP142 in Mtb pathogenesis.

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much‐needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X‐ray crystal structures, we could link weakly‐binding fragments to produce an active site binder with a K_D Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT‐targeting anti‐TB drug.

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much‐needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X‐ray crystal structures, we could link weakly‐binding fragments to produce an active site binder with a K_D Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT‐targeting anti‐TB drug.

Recently reported inhibitors developed against targets from Mycobacterium absecessus (Mab).

Pseudomonas aeruginosa is of major concern for cystic fibrosis patients where this infection can be fatal. With the emergence of drug-resistant strains, there is an urgent need to develop novel antibiotics against P. aeruginosa. MurB is a promising target for novel antibiotic development as it is involved in the cell wall biosynthesis. MurB has been shown to be essential in P. aeruginosa, and importantly, no MurB homologue exists in eukaryotic cells. A fragment-based drug discovery approach was used to target Pa MurB. This led to the identification of a number of fragments, which were shown to bind to MurB. One fragment, a phenylpyrazole scaffold, was shown by ITC to bind with an affinity of K_d = 2.88 mM (LE 0.23). Using a structure guided approach, different substitutions were synthesized and the initial fragment was optimized to obtain a small molecule with K_d = 3.57 μM (LE 0.35).