Our Platform for Rapid Target Identification
The challenge. To date it has proven challenging to efficiently find the molecular target of orphan cytotoxins. Small molecules bind multiple proteins (polypharmacology), presenting a challenge for unambiguous target identification that is not easily overcome by contemporary approaches based on affinity and crosslinking. Alternatively, an unbiased forward genetic strategy can lead to target identification by pinpointing compound-resistant mutations in binding sites, but until recently, was limited to compounds with activity in classical genetic systems such as yeast. We successfully used HCT116, a diploid human cancer cell line with high mutation rates, as a forward genetic tool to identify the target of indisulam as a molecular glue (the first one discovered after thalidomide). However, the number of successful cases using this system has been limited by the inability to reveal mutations with low penetrance (a lack of sensitivity) and by high background mutation rates (a lack of specificity).
Our solution. We developed a discovery pipeline that (1) uses phenotypic screening to identify bioactive compounds, (2) enriches for novelty by triaging known targets, and (3) exploits creative forward genetic and chemical strategies to identify and validate the precise molecular target for the observed phenotype. To overcome limitations in current forward genetic approaches, we developed an inducible mutagenesis system, which allows us to titrate mutations and compare resistance rates between cells grown in mutation-on or mutation-off conditions. Finding an increased frequency of resistance in the mutator population provides direct evidence that mutations, rather than epigenetic changes, are driving resistance. This system also enables the identification of low penetrance mutations and reduces the number of background mutations by nearly 100-fold, improving both the sensitivity and specificity. To overcome challenges associated with small molecule polypharmacology, we developed a strategy to correlate compound/protein binding (probe displacement EC50 from competitive crosslinking experiments) and cellular toxicity (IC50) across a range of synthetic analogs.
Our platform. To identify and validate the precise molecular targets of biologically active small molecules efficiently, unambiguously, and on scale, we have developed a discovery pipeline that implements several crucial steps detailed below.
Step 1. Phenotypic screening to discover small molecules that block the growth of cancer cells. To maximize the number of potential new targets, we screen large and diverse HTS compound collections and include published orphan cytotoxins and natural products. To expand the range of biological targets, HTS screens are performed in cell lines representing four different lineages.
Step 2. We have implemented a knowledge-based triage of hits to eliminate compounds that engage common targets and increase the likelihood of reaching novel protein space. In so doing, we reserve the more intensive target ID efforts for compounds that modulate novel proteins or pathways. These efforts become more efficient with time since the tools we generate during target ID efforts (crosslinkers, compound-resistant clones) allow us to quickly annotate whether new hits engage the same target. Published in J Med Chem. and Publishein ACS Chem Bio.
Step 3A. Our platform is anchored by an inducible forward genetics strategy, and we evaluate all new hits first with this system. To this end, we developed isogenic cancer cell lines in which we temporally control mutagenesis by regulating the levels of DNA mismatch repair and can compare the frequency of clonal resistance between cells that are either mutagenized (Mut-on) or un-mutagenized (Mut-off). An increased frequency of resistance in the Mut-on condition reflects genetic resistance, which strongly predicts that compounds will operate through direct engagement of a protein target. In addition, by comparing isolated clones to a closely paired reference genome (the parental, Mut-off population), we can refine the list of potential causal mutations and reduce background mutations by 100-fold compared to prior methods. Published in Cell Chem Biol.
Step 3B. When forward genetics does not reveal compound-resistant clones, we use chemical proteomics as an alternative. To overcome the challenge of polypharmacology, we implement medicinal chemistry to reveal embedded structure activity relationships (SAR) related to cellular toxicity (IC50) with a sizable collection of analogs. We then evaluate the same analogs for their ability to dose-responsively displace an alkyne-tagged crosslinking probe in live cells, providing a displacement EC50 for each individual competitor. Correlation of the structure binding relationships (SBR) with the cytotoxicity IC50 SAR pinpoints which, if any, crosslinked protein is on-target. Given the importance of photo-crosslinking probes, we also explore the use of new photoactivatable groups that we design or serendipitously discover to embed in bioactive molecules.
Step 4. New small compound-protein pair candidates are rigorously validated using a variety of chemical, biochemical, genetic, and structural biology techniques. To date, we discovered and validated the molecular targets of > 20 orphan cytotoxins that affect diverse cellular pathways, including the second example of a molecular glue (after thalidomide), tumor activated inhibitors of enzymes in fatty acid metabolism, inhibitors of an endonuclease involved in transcriptional termination, novel inhibitors of the integrated stress response, highly selective inhibitors of the cholesterol biosynthetic pathway, inhibitors of DNA replication, and the first known inhibitors of mammalian ribosome assembly.
Step 4. New small compound-protein pair candidates are rigorously validated using a variety of chemical, biochemical, genetic, and structural biology techniques. To date, we discovered and validated the molecular targets of > 20 orphan cytotoxins that affect diverse cellular pathways, including the second example of a molecular glue (after thalidomide), tumor activated inhibitors of enzymes in fatty acid metabolism, inhibitors of an endonuclease involved in transcriptional termination, novel inhibitors of the integrated stress response, highly selective inhibitors of the cholesterol biosynthetic pathway, inhibitors of DNA replication, and the first known inhibitors of mammalian ribosome assembly.
Related publications:
A Medicinal Chemistry-Driven Approach Identified the Sterol Isomerase EBP as the Molecular Target of TASIN Colorectal Cancer Toxins
Anticancer benzoxaboroles block pre-mRNA processing by directly inhibiting CPSF3
Selective and brain-penetrant lanosterol synthase inhibitors target glioma stem-like cells by inducing 24(S),25-epoxycholesterol production
Translational Opportunities
While novel small molecule – protein pairs will provide tools to advance biological knowledge, occasionally they can also lead to translational opportunities. When additional studies indicate a potential therapeutic rationale, our team is set up to combine medicinal chemistry, and in vitro and in vivo pharmacology to identify potent, selective and bioavailable analogs for in vivo proof-of-concept studies in mouse models of cancer. Some of our current translational programs are highlighted below.
Lanosterol synthase inhibitors
Glioblastoma (GBM) is an aggressive adult brain cancer with few treatment options due in part to the challenges of identifying brain-penetrant drugs. GBM cells are dependent on acquiring cholesterol from neighboring cells or synthesizing cholesterol de novo because circulating LDL cannot cross the blood brain barrier. In support of targeting cholesterol levels in GBM, LXR agonists lead to robust anti-tumor responses in orthotopic patient derived xenografts by promoting cholesterol efflux from the cell. Unfortunately, synthetic LXR agonists also promote fatty acid synthesis, which results in elevated triglycerides and fatty liver in patients.
Endogenous 24,25 epoxycholesterol (EPC) can activate LXRs without stimulating triglyceride synthesis. Therefore, an alternative strategy to target the cholesterol homeostasis in GBM is to develop drugs that upregulate EPC. We identified the mechanism of MM0299, a tetracyclic dicarboximide with anti-glioblastoma activity. MM0299 inhibits lanosterol synthase (LSS) and diverts sterol flux away from cholesterol into a "shunt" pathway that culminates in EPC. EPC synthesis following MM0299 treatment is both necessary and sufficient to block the growth of mouse and human glioma stem-like cells by depleting cellular cholesterol. MM0299 exhibits superior selectivity for LSS over other sterol biosynthetic enzymes. These findings nominate the development of an MM0299 derivative to treat GBM or other neurologic indications.
Relevant publications:
Thiophenyl derivatives of nicotinamide for the treatment of GBM
Inosine monophosphate dehydrogenase (IMPDH), the rate limiting enzyme in guanylate biosynthesis, is upregulated in GBM and provides high levels of guanine nucleotides that allow the tumor cells to overcome DNA damage induced by the combination of radiation and the alkylating agent temozolomide (TMZ), which has been the standard of care for GBM nearly the last two decades. Through an unbiased drug screen and subsequent medicinal chemistry studies, we have identified a novel IMPDH inhibitor, termed compound 9. Genetic screens using compound 9 found that both NAMPT and NMNAT1, enzymes in the NAD salvage pathway, are necessary for activity. Compound 9 is metabolized by NAMPT and NMNAT1 into an adenine dinucleotide (AD) derivative in a cell-free system, cultured cells, and mice, and inhibition of this metabolism blocked compound activity. AD analogues derived from compound 9 inhibit IMPDH in vitro and cause cell death by inhibiting IMPDH in cells. These findings nominate these compounds as preclinical candidates for the development of tumor-activated IMPDH inhibitors to treat neuronal cancers like GBM.
Relevant publication: