Covalent fragments provide an opportunity to both drug difficult targets and to more completely shut down targets. Success has spurred interest, and the literature is exploding. It has been just over a month since our last post on the topic, and already three new papers are worth highlighting.
The first, in Eur. J. Med. Chem. by György Keserű and collaborators at the Hungarian Research Centre for Natural Sciences and University of Szeged, describes a library of 24 covalent fragments. All of these contain the same relatively simple core but vary in their covalent warheads or how the warhead is attached.
The idea is to explore warhead reactivity in the context of a “vanilla” fragment that could provide modest but nonspecific hydrophobic interactions with proteins. The 14-atom 3,5-bis(trifluoromethyl)phenyl core was chosen because it is commonly used in medicinal chemistry and lacks polar atoms likely to make specific interactions to proteins. Also, the electron withdrawing trifluoromethyl groups make the warheads more reactive. The UV absorbance and lipophilicity also make derivatives synthetically easy to work with, and the fluorine atoms are useful for 19F NMR.
The warheads themselves span a vast range of reactivity as assessed both computationally and experimentally (by reactivity with glutathione). Some, such as maleimides and isothiocyanates, are so highly reactive that they are often used for nonspecific protein labeling, while others, such as styrene and acetylene, are quite unreactive. In the middle are moieties like acrylamides, chloroacetamides, and epoxides.
The researchers screened the library (at 100 µM) against four unrelated kinases: BTK, ERK2, RSK2, and MAP2K6. Unsurprisingly, four of the hottest fragments inhibited all the kinases, while the seven weakest warheads were inactive. Things got interesting in the middle though, with different inhibition profiles seen for different kinases.
Next, the researchers tested their fragment sets against two new kinases, JAK3 and MELK. Both kinases yielded several hits. Replacing the vanilla fragment with small hinge-binding elements for the relevant warheads rapidly yielded nanomolar inhibitors. Covalent inhibitors had already been reported for JAK3 but not for MELK. The researchers suggest using their library as a rapid tool for assessing cysteine accessibility. If you are interested in trying this at home, the authors have offered to send the library upon request.
The second paper, in ChemBioChem by György Keserű, Stanislav Gobec, and a large multinational group of collaborators, describes a slightly expanded covalent library consisting of 28 compounds representing 20 different warhead chemotypes, all with the same 3,5-bis(trifluoromethyl)phenyl core. Usefully, glutathione reactivity kinetics are provided for all the fragments. The fragments were screened against six different (non-kinase) targets, providing hits against all of them. 19F NMR as well as mass spectrometry was used to confirm binding.
It is always nice to see new types of covalent warhead chemistries, but medicinal chemistry tends to be somewhat conservative: if something works clinically and isn’t (too) toxic, we’ll stick with it. Thus the continuing interesting in acrylamides, which are found in five of the six approved covalent kinase inhibitors. Enter the third paper, in J. Med. Chem., by Adam Birkholz and colleagues at Amgen, which systematically explores the glutathione reactivity of substituted N-phenyl acrylamides.
The researchers first examine 11 α-substituted N-phenyl acrylamides. For the most part electron-withdrawing substituents increase the reactivity of the warhead, though fluorine has the opposite effect, attributed to its mesomeric electron-donating ability.
Next, the researchers turn to 21 β-substituted N-phenyl acrylamides. Again, electron withdrawing substituents increase the reactivity of the acrylamides. For aminomethyl substituents, the reactivity is lower than the parent unsubstituted acrylamide for amines with pKa < 6, while the more basic amines show increased reactivity. All experiments were conducted at pH 7.4, and computational modeling suggests that the protonated amine inductively withdraws electron density from the acrylamide, thereby increasing its reactivity.
While the general trends reported in the paper are expected, the actual numbers provide a valuable resource. One of the challenges of covalent drugs is ensuring the warhead is reactive enough to bind to the target but not so reactive that it binds to other targets or is cleared too rapidly. By knowing how much a given substituent is likely to increase – or decrease – reactivity, chemists can more precisely tune their molecules.
Our medicinal chemistry toolkit is expanding, and covalent molecules are playing a growing role.