Nature Reviewes Microbiology, 2017
A recent study in collaboration between the CDIPD and Elizabeth Winzeler laboratory used a modified strain of yeast that lacks 16 ABC transporter export pumps (ABC16-monster) and is thus more sensitive to cytotoxic drugs, to perform directed evolution. Random mutagenesis was induced in this strain by sublethal doses of MMV001239, a compound from the malaria box from the Medicines for Malaria Venture, known to be effective against the intracellular stages of Trypanosoma cruzi. The yeast clones that survived treatment with the selected compounds were subjected to whole-genome sequencing to identify the mutations responsible for resistance, and single nucleotide variants were detected following comparison with the parental reference genome. A surprisingly low number of SNVs were detected in the evolved clones and only four of these consisted of non-synonymous substitutions, which affected either ERG11 or ERG25. These genes encode enzymes involved in the biosynthesis of ergosterol, which is an essential structural component of the plasma membrane of protozoans that has a role similar to that of cholesterol in mammalian cells. We showed that MMV001239 binds directly to the highly conserved region of CYP51 and inhibits the sterol pathway. Finally, the mutations observed in ERG11 are in close proximity to the active site of the enzyme and predicted to inhibit binding of the drug without affecting substrate binding.
Twenty five years of drug discovery at the University of California targeting Kinetoplastid parasites.
McKerrow JH, Siqueira-Neto JL, McCall L-I and Otrubova K
Journal of Pharmaceutics & Drug Development, 2016, Vol. 4, Issue 1
This review summarizes 25 years of screening compounds against three major kinetoplastid parasites, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. The work was carried out at two University of California campuses by a consortium of scientists. The history of this effort is summarized beginning with DARPA and NIAID TDRU projects. The compound collections that were screened came from both academic and industry sources. To facilitate screening, high throughput or high content microtiter plate-based assays were developed. Three approaches to discovery of new drugs for kinetoplastid diseases are presented. These include structure-based drug design against specific parasite molecular targets, repurposing of already approved drugs, and screening of marine natural products. As similar screening efforts against other molecular targets or with other compound libraries are ongoing, one conclusion is that the current bottleneck in drug development for neglected tropical diseases is downstream of compound screening and hit to lead. More medicinal chemistry efforts for lead optimization and more preclinical package work needs to be done.
X-ray structures of thioredoxin and thioredoxin reductase from Entamoeba histolytica and prevailing hypothesis of the mechanism of Auranofin action.
Parsonage D, Sheng F, Hirata K, Debnath A, McKerrow JH, Reed SL, Abagyan R, Poole LB, Podust LM.
J. Struct. Biol. 2016 May;194(2):180-90.
The anti-arthritic gold-containing drug Auranofin is lethal to the protozoan intestinal parasite Entamoeba histolytica, the causative agent of human amebiasis, in both culture and animal models of the disease. A putative mechanism of Auranofin action proposes that monovalent gold, Au(I), released from the drug, can bind to the redox-active dithiol group of thioredoxin reductase (TrxR). Au(I) binding in the active site is expected to prevent electron transfer to the downstream substrate thioredoxin (Trx), thus interfering with redox homeostasis in the parasite. To clarify the molecular mechanism of Auranofin action in more detail, we determined a series of atomic resolution X-ray structures for E. histolytica thioredoxin (EhTrx) and thioredoxin reductase (EhTrxR), the latter with and without Auranofin. Only the disulfide-bonded form of the active site dithiol (Cys(140)-Cys(143)) was invariably observed in crystals of EhTrxR in spite of the addition of reductants in various crystallization trials, and no gold was found associated with these cysteines. Non-catalytic Cys(286) was identified as the only site of modification, but further mutagenesis studies using the C286Q mutant demonstrated that this site was not responsible for inhibition of EhTrxR by Auranofin. Interestingly, we obtained both of the catalytically-relevant conformations of this bacterial-like, low molecular weight TrxR in crystals without requiring an engineered disulfide linkage between Cys mutants of TrxR and Trx (as was originally done with Escherichia coli TrxR and Trx). We note that the -CXXC- catalytic motif, even if reduced, would likely not provide space sufficient to bind Au(I) by both cysteines of the dithiol group.