From noncovalent fragment to reversible covalent CatS inhibitor

We noted just a couple weeks ago that covalent fragment-based approaches have been on a tear. Much of the recent focus has been on irreversible inhibitors, but as we discussed back in 2013 there is much to be said for reversible covalent molecules too. These are the subject of a new paper in J. Med. Chem. by Markus Schade and colleagues at Grünenthal GmbH.

 

The researchers were interested in cathepsin S (CatS), one of 11 members of a family of cysteine proteases. The enzyme has been implicated in a laundry list of diseases, from arthritis to neuropathic pain to Sjögren’s Syndrome, and indeed a few inhibitors entered clinical trials in the early twenty-first century. However, selectivity turns out to be essential: inhibiting the related cathepsin K can lead to cardiovascular problems and stroke. Molecules that appear selective often  contain a basic nitrogen and so can accumulate in lysosomes, achieving sufficiently high local concentrations to inhibit CatK.

 

CatS is a small (24 kDa) enzyme, ideal for protein-observed NMR. An ¹⁵N-HSQC screen of 1858 noncovalent fragments yielded 18 hits, all of which showed similar chemical shift perturbations (CSPs) suggesting binding in the S2 pocket. X-ray crystallography was successful for three fragments, confirming that they do indeed bind in the S2 pocket. Appealingly, this region of the protein is structurally different from the other cathepsins, suggesting a route to selectivity.

 

The sulfonamide moiety of compound 1 (blue) binds in a very similar fashion to the sulfone of a previously reported reversible covalent inhibitor, compound 16 (red). Growing compound 1 to compound 37 led to a significant boost in potency, and crystallography revealed that the binding mode remained the same.

At this point the researchers sought to remove a few heteroatoms as well as introduce the nitrile warhead from compound 16, yielding compound 39b. Surprisingly, this molecule was no more potent than the non-covalent precursor. However, fragment growing into the S3 pocket yielded a massive boost in potency in the form of compound 44. Further SAR and crystallography revealed that much of the increased affinity is due not to specific interactions in the S3 pocket but rather to a hydrogen bond between the newly introduced amide proton and a main chain carbonyl of CatS. Compound 44 is also highly selective against CatK and CatB, showing negligible inhibition of either at 10 µM. Unfortunately, cellular potencies of representative compounds were down by more than three orders of magnitude, likely due to low permeability.

 

While there is still some way to go to establish whether these molecules will succeed where others have failed, this is nonetheless a nice case of fragment-assisted lead discovery And while one can certainly argue that it would have been possible to derive compound 44 from compound 16 through classical medicinal chemistry, fragments clearly helped.