Fragments vs CYP125 and CYP142 for M. tuberculosis

Although 2020 and 2021 were baleful exceptions, tuberculosis is normally the world’s deadliest infectious disease. The pathogen Mycobacterium tuberculosis (Mtb) makes its home inside macrophages, the very cells that normally destroy microorganisms. Worse, some strains have become resistant to approved drugs. In a recent open-access J. Med. Chem. paper, Madeline Kavanagh, Kirsty McLean, and collaborators at University of Manchester, University of Cambridge, and elsewhere explore a new mechanism to fight this ancient disease.

 

An important nutrient source Mtb exploits inside human cells is cholesterol, which bacteria oxidize with the cytochrome P450 enzyme CYP125. A second enzyme, CYP142, is also present in some strains and is functionally redundant. Thus, the researchers set out to make a dual inhibitor.

 

Mtb has some 20 CYPs, and the Cambridge researchers have been studying them for a long time: we wrote about their work on CYP121 in 2016 and their work on CYP126 in 2014. All these enzymes contain a heme cofactor, and much is known about targeting the bound iron. However, some ligands are promiscuous, hitting human P450 enzymes, or they are rapidly effluxed out of cells. Thus, the researchers built a fragment library of just 80 likely heme binders but excluded particularly promiscuous moieties, such as imidazoles. The library was screened using UV-vis spectroscopy; ligands that bind to the heme group cause a red-shift in the λ_max. Only four hits were found for CYP125, while a dozen were found for CYP142, including three of the four CYP125 hits. Compound 1a had modest affinity for CYP125 and low micromolar affinity for CYP142.

 

Compound 1a was soaked into crystals of CYP142, and interestingly two molecules bound at the active site: one coordinating to the iron atom as expected, the other binding near the entrance of the active site. This suggested a linking or merging strategy, so the researchers made small libraries based on compound 1a and tested these against the two enzymes. Compound 5m was the most potent against both. Crystal structures of this molecule bound to both CYP125 and CYP142 confirmed that the pyridine nitrogen maintained its interaction with the heme iron, while the added bit nicely filled the space previously occupied by the second copy of compound 1a.

 

Functional assays revealed that compound 5m inhibited both enzymes with nanomolar activity, comparable to their affinities. It also inhibited the growth of Mtb grown on media containing cholesterol as the sole source of carbon. More impressively, it even inhibited the growth of Mtb in standard media spiked with just low concentrations of cholesterol. Oddly though, it also inhibited the growth of Mtb grown on media not containing cholesterol, albeit at a higher concentration, suggesting perhaps other targets. But one reason tuberculosis is so hard to treat is that the bacteria persist inside human cells. Encouragingly, compound 5m inhibited the growth of Mtb in human macrophages at low micromolar concentrations, and it  did not show cytotoxicity up to 50 micromolar concentration.

 

Unfortunately, compound 5m did show cytotoxicity to human HepG2 cells, and it also inhibited several human P450 enzymes at high nanomolar concentrations, which could cause drug-drug interactions. Also, selectivity against other MTb P450 enzymes is unclear. Finally, no in vitro ADME data are reported. Nonetheless, this is a nice fragment to lead story, and compound 5m could be used – cautiously – as a chemical probe to study Mtb biology.