Fragments vs malarial DHFR

Malaria continues to be a worldwide scourge, with some quarter billion cases last year. A seventy-year-old drug called pyrimethamine targets the dihydrofolate reductase (DHFR) enzyme from Plasmodium falciparum, but resistance mutations have rendered this molecule mostly useless. An analog called P218 was developed to overcome this resistance and completed a handful of phase 1 clinical trials, but unfortunately the human pharmacokinetics were found lacking. In a new RSC Med. Chem. paper, Marie Hoarau and colleagues at the National Center for Genetic Engineering and Biotechnology in Thailand describe their efforts to improve this molecule.

 

The researchers recognized that the phenyl propanoate moiety of P218 was a metabolic liability and sought a replacement. They screened a library of 1163 fragments (from Key Organics) at 1 mM using a thermal shift assay. This resulted in 64 hits, 52 of which confirmed by SPR. Of these, 22 showed some level of inhibition at 0.5 mM against mutant PfDHFR.

 

Among the hits, five were “bi-aromatic carboxylates,” such as compound 136. These were prioritized because, while reminiscent of the phenyl propanoate in P218, they had fewer rotatable bonds. Some of them also showed slow off-rates by SPR, though in my opinion the sensorgrams look suspicious, perhaps due to excessive protein loading on the chip. (For example, the K_d for compound 136 calculated from the on and off rates comes in at 160 nM, unrealistically potent given that it shows only 20% enzymatic inhibition at 0.5 mM. Note – all values here and in the figure are for the mutant form of the enzyme.)

 

SAR by catalog was used to find additional analogs, such as compound AF10, which showed measurable inhibition of the enzyme. Next, the researchers tested hits in the presence of a pyrimidine fragment (L4) derived from P218, known to bind nearby. Compound AF10 showed greater inhibition than would be expected by simple additivity, perhaps suggesting some preorganization of the binding site, as in a different example discussed here.

 

Molecular modeling was used to link the carboxylate fragments with L4, and eight were made and tested. All inhibited both wild type and mutant PfDHFR, and compound 8 showed good selectivity over human DHFR too. A crystal structure confirmed that it bound as predicted. From a fragment-linking perspective, the sub-nanomolar affinity of compound 8 is impressively better than would be expected given the weak affinities of L4 and AF10.

 

Unfortunately, despite similar in vitro potency against the isolated enzymes, compound 8 and the other molecules tested showed “disappointing” activity against Plasmodium falciparum carrying either wild-type or mutant DHFR, roughly 100- to 1000-fold less potent than P218. The researchers suggest solubility may be a factor.

 

This paper is a useful reminder of the dramatic disconnects often seen between enzymatic and cell activity. Nonetheless, it is another good example of using fragment-based methods to replace one portion of an existing molecule.