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 Kd
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.
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ReplyDeleteHi Dan, I see this study more as an exercise in scaffold-hopping than fragment-linking given that P218 is a known high potency compound. If it were my project, I’d want to know how important, the carboxylic acid is for activity since it’s a substructural feature that can make it difficult for compounds to be orally absorbed and to get into cells. My view is that some (probably a lot) of the loss of potency going from P218 to L4 is due to a likely change in ionization state and I’d argue that 2,4-diamino-5-methoxypyrimidine (pKa: 7.2 at 20°C | JOC 1969 34 821–836 https://doi.org/10.1021/jo01256a011) is a more appropriate potency reference than L4 (likely to be neutral under physiological conditions). I don’t have access to the article so it is just possible that the authors discussed some of these points.
ReplyDeleteHi Pete,
ReplyDeleteI agree this could be characterized as scaffold-hopping or fragment replacement but the authors themselves referred to it as fragment-linking, and they did computationally rebuild the linker.
I also agree that the hydroxyl on L4 is problematic; your suggestion or a methoxy would have been better choices.
Unfortunately the carboxylic acid seems to be important as methyl esters were less active in vitro and still not effective against the parasite. That said, P218 also has a carboxylic acid so it is a bit surprising that compound 8 is so much worse.