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.

The Sequel is Never as Good as the Original

We are living in a target-driven environment in Pharma, for both good and bad.  The low-hanging fruit have been plucked and the high-hangers are tough.  But, fragments have proven to be highly utile in liganding these targets.  One drawback with target-based screening is the problem with cellular activity, while it may be easy to generate good activity against the isolated target, in the end you need activity in the cell/animal.  Back in the good ole days, people just skipped the target and went straight into cells: compounds are put on bacterial plates and the microbes die if the compound is anti-microbial.  This is the simplest example of phenotypic screening, the phenotype here being "dead cells". [For a discussion of the history of phenotypic screening, go here.]  Fragments could be the worst case scenario for phenotypic screening as fragment-target interactions are very weak, and very commonly do not exert a biological effect. 

In this paper from Rob Leurs and colleagues, including Iota, the describe a fragment-based phenotypic screen process.  This work is a follow on to previous work from this group discussed here, which I quite liked  So, they have a target (PDEB1) but immediately follow their screening with the phenotypic part.  For the phenotypic screen, they used several different parasitic PDE and MRC5 cell-line as a counter-screen. I won't bore you with any of the experimental details. The compounds are recapitulating known molecules, like benadryl.  Now, I really wanted to like this paper, at least from a process approach.  It appears to my eyes, that all the compounds are pretty much equipotent and cytotoxic.  This is a really disappointing paper in that it doesn't really do anything.  They had shown previously that you could get non-cytotoxic compounds with good inhibition of PDEB1.  They didn't repeat that here.  There is no X-ray, they did before.  The compounds are wholly uninteresting and stretch the imagination to be seen as compounds "with a lot of potential to grow into antiparasitic compounds".