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.

07 October 2019

The story of erdafitinib – abridged

In April we celebrated the FDA approval of erdafitinib for certain types of urothelial carcinomas with genetic alterations in the FGFR2 or FGFR3 genes. This marked the third launch of a fragment-derived drug. However, although erdafitinib was mentioned in a 2016 review, the fragment origins have remained obscure. The full story has yet to be published, but Chris Murray (Astex), David Newell (Newcastle University) and Patrick Angibuad (Janssen) have recently published brief highlights in MedChemComm.

The story begins all the way back in 2006, with a collaboration between Astex and the Northern Institute of Cancer Research at Newcastle University. The four fibroblast growth factor receptors (FGFR1-4) are kinases whose aberrant activation was known to be associated with multiple cancers. In those days most FGFR inhibitors also inhibited VEGFR2, which was linked to unacceptable side effects. A fragment screen in 2008 led to a series of potent, selective molecules and enticed Janssen to join the collaboration. This particular lead series was ultimately discontinued, though, as discussed below, it turned out to be important.

Meanwhile, fragment 1 was identified, characterized crystallographically bound to FGFR1, and improved to low micromolar compound 2. Virtual screening and medicinal chemistry led to compound 4, with improved selectivity for FGFR3 over VEGFR2. Superimposing the crystal structure of this compound with the previous series suggested building off the aniline nitrogen to improve potency (and, one might assume, solubility). This ultimately led to erdafitinib.


The researchers highlight the cooperative nature of this project. “Each team brought their specific expertise and most importantly, wisely and collaboratively capitalized on each other’s strengths.”

The publication also illustrates that success can take time – thirteen years in this case. This is not unusual for drug discovery, and is in fact halfway between the remarkably fast six years for vemurafenib, the first fragment-based drug approved, and the two decades required for venetoclax, the second.

But in the end good things are worth waiting for, and the 15-20% of metastatic bladder cancer patients with an FGFR alteration now have a new treatment option. Erdafitinib is being tested in at least a dozen other clinical trials for various cancers. Practical Fragments wishes all the participants the best of luck.