14 October 2019

Fragments vs Mycobacterium TrmD – and native ESI-MS revisited

The Mycobacterium genus of bacteria contains more than 190 species, including the pathogens that cause leprosy and tuberculosis. Mycobacterium abscessus (Mab) is not as famous, though it is a growing concern for people living with cystic fibrosis. The enzyme tRNA (m1G37) methyltransferase (TrmD) is essential for Mycobacterium growth. In a recent open-access paper in J. Med. Chem., Chris Abell, Tom Blundell, Anthony Coyne, and collaborators at University of Cambridge, Royal Papworth Hospital, and the US NIH describe potent inhibitors of this target.

The researchers screened 960 fragments using differential scanning fluorimetry (DSF). The 53 hits were soaked into crystals of TrmD, and 27 yielded electron density – all in the cofactor (S-adenosyl methionine, or SAM) binding site. One weak but particularly ligand-efficient fragment was advanced through fragment growing to low micromolar affinity, but further improvements proved challenging.

A powerful feature of FBLD is that screens often yield multiple starting points, and this proved useful here. Specifically, compound 16 bound in the pocket where the adenine of SAM normally sits, while compound 20 bound nearby in the ribose pocket. Merging these led to compound 23,  and a crystal structure suggested that the indole nitrogen would be a good vector for fragment growing, resulting in compound 24c. Addition of a positively charged moiety to engage a couple glutamic acid residues led to compound 29a, a mid-nanomolar compound as assessed by isothermal titration calorimetry (ITC).

Several of the molecules were characterized by native electrospray ionization mass spectrometry (ESI-MS) – an interesting but somewhat controversial technique in which protein-ligand complexes are gently ionized and detected in the gas phase. For large molecules such as proteins, even the gentlest ionization will generate highly charged species; in this case the dimeric TrmD protein had between 13 and 17 positive charges. Each of these charge states represents a different species, or perhaps several since the positive charges could be on different regions of the protein.

Compound 24c has modest affinity, but encouragingly, native ESI-MS at 100 µM ligand revealed two bound ligands, as expected for a protein dimer. However, for the much more potent compound 29a, native ESI-MS at 100 µM ligand revealed two bound ligands for a high charge state but largely unbound protein for a lower charge state. The researchers speculate this could be due to dissociation of the positively charged ligand in the gas phase.

Overall this paper is a nice exercise in fragment merging and growing, guided by crystallographic data. Some of the molecules elaborated from 24c (including 29a) inhibited growth of Mycobacteria species, though further improvements in affinity will be needed, and no DMPK properties are provided. Still, it's good to see solid work being done on new antibacterial agents.

The disconnect between observed affinity by native ESI-MS and that observed by ITC does make one cautious about the utility of the former technique. We’d welcome comments on your experiences with it.

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