An unexpected lysine-reactive covalent fragment against a Pleckstrin Homology domain

Last week we highlighted a study in which an attempt to optimize a covalent fragment led to a non-covalent fragment that bound in a different location. A new open-access paper in J. Med. Chem. by David Spring, Marko Hyvönen and colleagues at University of Cambridge is almost the inverse – a surprisingly covalent binder from a conventional fragment screen.

 

The researchers were interested in Pleckstrin Homology (PH) domains. The 250 or so PH domains in humans bind to the intracellular side of the plasma membrane by interacting with phosphoinositides such as PIP3. Blocking membrane recruitment could be useful for multiple diseases, but the highly charged nature of the interaction (the “P3” in PIP3 means three phosphate groups) makes drugging PH domains difficult, a perfect challenge for fragments.

 

The study focuses on the PH domain from Bruton’s Tyrosine Kinase (BTK), an important oncology and immunology target with several approved drugs, all of which bind to BTK’s kinase domain. Its PH domain was screened against 720 fragments using differential scanning fluorimetry (DSF). Seven fragments stabilized the melting temperature by a whopping 5 degrees C or more. Crystallography was successful for compound 1, which surprisingly revealed that the ketone reacts with the terminal amine of lysine 12, which normally makes electrostatic interactions with two phosphates in phosphoinositides.

 

The crystal structure showed unoccupied space nearby; subsequent fragment growing and optimization led to compound 24, with low micromolar affinity as assessed by DSF. The researchers obtained some two dozen crystal structures, which they were able to correlate with structure-activity relationships (SAR). All of the molecules formed the imine with K12; reducing the ketone to an alcohol abolished stabilization in DSF.

 

Intact protein mass spectrometry was used for assessing SAR and confirming that the covalent bond formation is reversible: adding two fragments with different affinities led to the same distribution of products regardless of the order of addition of the fragments. Not surprisingly, the reaction was faster at higher pH, but nearly complete covalent modification still took place within an hour at pH 7.4.

 

Even at high compound to protein ratios the molecules largely gave single protein modification, and two separate computational studies of the pK_a values of the 15 lysine residues in the BTK PH domain revealed that K12 is an outlier, with a calculated pK_a of 7.1 or 8.8 compared to an average of 10.5 or 10.8 for the others. This means that K12 is largely unprotonated at physiological pH, increasing its reactivity. A similar analysis of four related PH domains suggested that the equivalent lysine residues also have anomalously low pK_a values.

 

The fact that K12 is conserved across related PH domains does raise the question as to whether selectivity will be possible with this type of ligand. Also, no in vitro ADME properties are provided, so it is not clear whether this warhead is advanceable. I wish the researchers had explored other heterocycle alternatives to the furan moiety to assess the tunability of the reactivity; perhaps those will come later. Overall though, this paper is a nice case study where following up on unexpected observations identified a new approach for covalently targeting lysine residues. As Isaac Asimov famously said, “The most exciting phrase to hear in science … is not ‘Eureka!’ but ‘That’s funny.’”