Confirmation of the cellular targets of benomyl and rapamycin using next-generation sequencing of resistant mutants in S. cerevisiae.

Investigating the mechanisms of action (MOAs) of bioactive compounds and the deconvolution of their cellular targets is an important and challenging undertaking. Drug resistance in model organisms such as S. cerevisiae has long been a means for discovering drug targets and MOAs. Strains are selected for resistance to a drug of interest, and the resistance mutations can often be mapped to the drug's molecular target using classical genetic techniques. Here we demonstrate the use of next generation sequencing (NGS) to identify mutations that confer resistance to two well-characterized drugs, benomyl and rapamycin. Applying NGS to pools of drug-resistant mutants, we develop a simple system for ranking single nucleotide polymorphisms (SNPs) based on their prevalence in the pool, and for ranking genes based on the number of SNPs that they contain. We clearly identified the known targets of benomyl (TUB2) and rapamycin (FPR1) as the highest-ranking genes under this system. The highest-ranking SNPs corresponded to specific amino acid changes that are known to confer resistance to these drugs. We also found that by screening in a pdr1Δ null background strain that lacks a transcription factor regulating the expression of drug efflux pumps, and by pre-screening mutants in a panel of unrelated anti-fungal agents, we were able to mitigate against the selection of multi-drug resistance (MDR) mutants. We call our approach "Mutagenesis to Uncover Targets by deep Sequencing", or "MUTseq", and show through this proof-of-concept study its potential utility in characterizing MOAs and targets of novel compounds.

Large-scale cytological profiling for functional analysis of bioactive compounds.

Cytological profiling (CP) is an unbiased image-based screening technique that uses automated microscopy and image analysis to profile compounds based on numerous quantifiable phenotypic features. We used CP to evaluate a library of nearly 500 compounds with documented mechanisms of action (MOAs) spanning a wide range of biological pathways. We developed informatics techniques for generating dosage-independent phenotypic "fingerprints" for each compound, and for quantifying the likelihood that a compound's CP fingerprint corresponds to its annotated MOA. We identified groups of features that distinguish classes with closely related phenotypes, such as microtubule poisons vs. HSP90 inhibitors, and DNA synthesis vs. proteasome inhibitors. We tested several cases in which cytological profiles indicated novel mechanisms, including a tyrphostin kinase inhibitor involved in mitochondrial uncoupling, novel microtubule poisons, and a nominal PPAR-gamma ligand that acts as a proteasome inhibitor, using independent biochemical assays to confirm the MOAs predicted by the CP signatures. We also applied maximal-information statistics to identify correlations between cytological features and kinase inhibitory activities by combining the CP fingerprints of 24 kinase inhibitors with published data on their specificities against a diverse panel of kinases. The resulting analysis suggests a strategy for probing the biological functions of specific kinases by compiling cytological data from inhibitors of varying specificities.

Hit-to-lead development of the chamigrane endoperoxide merulin A for the treatment of African sleeping sickness.

BACKGROUND: Human African trypanosomiasis (HAT) is an infectious disease with a large global health burden occurring primarily in Central and Eastern Africa. Most current treatments have poor blood brain barrier (BBB) penetration, which prevent them from targeting the most lethal stage of the infection. In addition, current therapeutics suffer from a variety of limitations ranging from serious side effects to difficulties with treatment administration. Therefore it is of crucial importance to find new treatments that are safe, affordable, and effective against both sub-species of Trypanosoma brucei. METHODS: Semi-synthetic derivatization of the fungally-derived natural product merulin A (1) has led to the discovery of new development candidates for the protozoan parasite T. brucei, the causative agent of HAT. Creation of an initial SAR library based around the merulin scaffold revealed several key features required for activity, including the endoperoxide bridge, as well as one position suitable for further derivatization. Subsequent synthesis of a 20-membered analogue library, guided by the addition of acyl groups that improve the drug-like properties of the merulin A core, resulted in the development of compound 12 with an IC(50) of 60 nM against T. brucei, and a selectivity index greater than 300-fold against HeLa and immortalized glial cells. SIGNIFICANCE: We report the semi-synthetic optimization of the merulin class of endoperoxide natural products as development candidates against T. brucei. We have identified compounds with low nM antiparasitic activities and high selectivity indices against HeLa cells. These compounds can be produced economically in large quantities via a one step derivatization from the microbial fermentation broth isolate, making them encouraging lead candidates for further development.