A fragment based competitive 19F LB-NMR 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.

Chemical Validation of Mycobacterium tuberculosis Phosphopantetheine Adenylyltransferase Using Fragment Linking and CRISPR Interference.

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 KD <20 µM and on-target anti-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.

Chemical Validation of Mycobacterium tuberculosis Phosphopantetheine Adenylyltransferase Using Fragment Linking and CRISPR Interference.

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 <20 µM and on-target anti-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.

Recognition of abasic sites and single base bulges in DNA by a metalloinsertor.

Abasic sites and single base bulges are thermodynamically destabilizing DNA defects that can lead to cancerous transformations if left unrepaired by the cell. Here we discuss the binding properties with abasic sites and single base bulges of Rh(bpy)(2)(chrysi)(3+), a complex previously shown to bind thermodynamically destabilized mismatch sites via metalloinsertion. Photocleavage experiments show that Rh(bpy)(2)(chrysi)(3+) selectively binds abasic sites with affinities of 1-4 x 10(6) M(-1); specific binding is independent of unpaired base identity but is somewhat contingent on sequence context. Single base bulges are also selectively bound and cleaved, but in this case, the association constants are significantly lower ( approximately 10(5) M(-1)), and the binding is dependent on both unpaired base identity and bulge sequence context. A wide variety of evidence, including strand scission asymmetry, binding enantiospecificity, and MALDI-TOF cleavage fragment analysis, suggests that Rh(bpy)(2)(chrysi)(3+) binds abasic sites, like mismatches, through insertion of the bulky chrysi ligand into the base pair stack from the minor groove side and ejection of the unpaired base. At single base bulge sites, a similar, though not identical, metalloinsertion mode is suggested. The recognition of abasic sites and single base bulges with bulky metalloinsertors holds promise for diagnostic and therapeutic applications.

Targeting abasic sites and single base bulges in DNA with metalloinsertors.

The site-specific recognition of abasic sites and single base bulges in duplex DNA by sterically expansive rhodium metalloinsertors has been investigated. Through DNA photocleavage experiments, Rh(bpy)2(chrysi)3+ is shown to bind both abasic sites and single base bulges site-specifically and, upon irradiation, to cleave the backbone of the defect-containing DNA. Photocleavage titrations reveal that the metal complex binds DNA containing an abasic site with high affinity (2.6(5) x 106 M-1), comparably to the metalloinsertor and a CC mismatch. The complex binds single base bulge sites with lower affinity (approximately 105 M-1). Analysis of cleavage products and the correlation of affinities with helix destabilization suggest that Rh(bpy)2(chrysi)3+ binds both lesions via metalloinsertion, as observed for Rh binding at mismatched sites, a binding mode in which the mismatched or unpaired bases are extruded from the helix and replaced in the base stack by the sterically expansive ligand of the metalloinsertor.