A chemical genetics-based approach to biological discovery
Our research group uses the traditional tools of drug discovery--high throughput small molecule screens--coupled with modern target identification techniques to identify new mechanisms and molecules to study and to intervene in the processes causative of human disease. Most projects in the lab start by asking the question whether a drug-like small molecule can be identified to solve a preexisting biological problem by way of interrogating the chemical diversity present in HTS screening libraries (>10^6 molecules). We use chemical proteomics and traditional cell biology-based experiments to retroactively identify and validate the cellular targets and downstream mechanism of action of identified lead molecules (chemical genetics). Our experience suggests this approach frequently results in pharmacologically privileged insight, yielding both the chemical starting points for new drugs and mechanistic information unattainable via classical forward genetics.
Small molecules for regenerative medicine
Despite perceived organismal complexity, mammals possess relatively poor regenerative potential compared to certain lesser species of Animalia (e.g., amphibians). Indeed, there are many human disease states where an insufficient regenerative response limits organ repair and is determining of disease etiology (e.g., heart failure, T1D, IPF). To overcome these limitations, we have ongoing programs aimed at identifying novel small molecules, drug targets, signaling pathways, and repurposed drugs which control the context-specific cell growth and differentiation pathways limiting regenerative potential in a given tissue or cell type. To this end, we have completed a number of pathway-centric discovery programs including the identification of small molecules which activate YAP for organ repair and compounds which promote TERT transcription in human cells for longevity-based applications. We have additionally used high content imaging-based screens involving primary cell preparations to identify including molecules which promote regenerative expansion of tissue specific stem cells from alveolar (AEC2s) and intestinal (ISCs) compartments.
Representative work in regenerative medicine: A pharmacological tool kit targeting the Hippo pathway
The number of signaling pathways and transcriptional programs known to promote endogenous tissue repair and regeneration in mammals is limited. As such, we decided to first focus on a highly promising, albeit ‘under-drugged’ transcriptional program, the Hippo-YAP pathway. Originally identified from genetic screens in Drosophila for organ overgrowth, the highly conserved Hippo-YAP controls stem, progenitor, and parenchymal cell growth to regulate organ size and regeneration. When activated, the transcriptional effector of this pathway, Yes-associated protein (YAP), promotes the positive expression of a few dozen anti-apoptotic and pro-proliferative gene products through interactions with TEAD transcription factors. The net result of YAP activation is cellular proliferation and loss of programmed cell death at the organ level. The transcriptional activities of YAP are restrained by the Hippo pathway, which at its core consists of a kinase cascade nucleated by Hippo (human MST1/2) and Warts (human LATS1/2). Phosphoryl transfer through this cascade results in the phosphorylation and inactivation of YAP resulting in cytoplasmic sequestration. To date, attempts to activate YAP pharmacologically have focused on targeting the traditionally druggable targets in the Hippo pathway, namely in the development of active site inhibitors to MST1/2 and LATS1/2 kinases. However, MST1/2 and LATS1/2 take part in many cellular roles apart from Hippo signaling, including cell cycle control, stress signaling, and transcription, implying that even selective inhibitors of these kinases may possess undesirable on-target effects. An alternative approach towards selective YAP activation might involve chemical targeting of a key scaffolding or sensor protein which is contextually essential in relaying inhibitory phosphorylation to YAP.
To address this gap in pharmacological tools and potential therapeutic agents, we undertook a phenotypic, cell-based high throughput screen to identify agents that activate YAP. We adapted a stable TEAD-LUC 293A reporter assay to 1536-well format and then screened a library of ~738k compounds for YAP-inducing activity. We ultimately identified PY-60, a molecule that robustly activates YAP in reporter assays and in qRT-PCR experiments with suitable properties for in vitro work (EC50 ~ 1.0 µM; CC50 > 20 µM). Likewise, PY-60 much more efficaciously activated YAP-driven transcription (~10-fold) than did the reported MST1/2 inhibitor XMU-MP-1. To identify the relevant target of PY-60, we synthesized a photo-activatable affinity probe molecule--containing an alkyne handle and diazirine photo-crosslinking moiety--informed by our SAR studies of the PY-60 scaffold termed PY-PAP, which retained activity in reporter assays. In situ treatment of probe, subsequent photo-crosslinking followed by streptavidin-based enrichment, and LC-MS/MS-based proteomic identification suggested that the most likely target for this series was the protein Annexin A2 (ANXA2). Loss of ANXA2 by shRNA or siRNA resulted in YAP transcriptional activation, its dephosphorylation (activation), and its nuclear entry. Further, PY-60 was found to bind ANXA in vitro via ITC and BLI with a similar Kd as its EC50 (~1 µM). We ultimately used docking and site directed mutagenesis to uncover the binding site of PY-60, a cleft on the first annexin repeat of ANXA2, a site proposed to bind phospholipids. Although ANXA2 is a known membrane binding protein, its relevance to the Hippo pathway had not been established. From a series of targeted co-IP experiments, we found ANXA2 directly binds to YAP, but does so only with increased cell density, suggesting a cell density sensing-based mechanism for inactivating YAP. We found PY-60 to inhibit the ability of ANXA2 to associate with the plasma membrane. Loss of membrane association of ANXA2 promotes recruitment of the PP-2A phosphatase subunit PPP2CA, which promotes YAP dephosphorylation and activation. This recently published work identified ANXA2 as a new ligandable component of the Hippo pathway and suggests a privileged mechanism by which one might develop specific YAP activators for promoting repair in disease.
Drugging the transcriptional responses to cellular stress
The mammalian cell is continually exposed to stresses arising from its environment and its metabolic activities. Nature has therefore selected for a repertoire of sensors and protective transcriptional programs to curb cell death and to restore homeostasis in response to these stimuli. Pharmacological or genetic augmentation of these responses leads to cellular resiliency in various disease states, but only a fraction of stress related transcriptional programs have been drugged or have annotated drug targets. Our current research program aims to identify novel non-toxic molecules and mechanisms for activating stress related transcriptional responses for which pharmacological probes are lacking (e.g., MTF-1 [heavy metal stress], NRF1 [proteasome stress]). Using this approach, we are revealing key information regarding the regulation and ligandability of these pathways and establishing new therapeutic insight into how specific activation of cell stress responses might be used to intervene in a spectrum of human diseases, including chronic kidney disease, neurodegeneration, and autoimmune disorders.
Representative work in transcriptional stress responses: Connecting central carbon metabolism to the oxidative stress sensing machinery of cells via non-enzymatic PTMs from reactive metabolites
One key example in this area is our work in identifying new chemical matter for activating the master regulator of oxidative stress resistance, NRF2 (NFE2L2). NRF2 is a transcription factor that under normal conditions is continually sent for proteasomal degradation by its repressor protein, KEAP1. KEAP1 acts as a sensor for oxidative stress and electrophilic xenobiotics, as each of its 27 cysteines are tuned to activate NRF2 in response to oxidation or alkylation.15 Upon activation, NRF2 enacts a protective transcriptional program that promotes cellular survival. We have undertaken multiple efforts to identify novel modulators of this pathway, culminating in interesting insight into how flux through central carbon metabolism is integrated into to the oxidative stress sensing machinery of the cell (Figure 2A). In our first effort,16 we identified the non-electrophilic small molecule CBR-470-1 as an efficacious NRF2 activator that does not act by direct modification of KEAP1, unlike many reported NRF2 activating compounds in the literature. Instead, a mechanistic campaign uncovered CBR-470-1 inhibits glycolytic enzyme phosphoglycerate kinase 1 (PGK1), which results in the buildup of triose phosphate degradation product methyl glyoxal (MGx). MGO accumulation results in covalent modification of two KEAP1 molecules through a novel crosslinking PTM, termed MICA/ A follow-up screening effort identified another novel modulator of glycolysis, sAZK692, as a non-electrophilic NRF2-activating compound, which we found to inhibit the more downstream enzyme pyruvate kinase (PKM2). Instead of resulting in MGx buildup as we had anticipated, this molecule promotes accumulation of glyceraldehyde-3-phosphate, which we found modifies a different censor cysteine (C273 and others) than that of MGx (C151). We are continuing to further expand this repertoire of reactive metabolites that communicate with KEAP1 through reactive metabolite derived modifications, an effort which is uncovering a rich interconnectedness of several key metabolic pathways and is defining a host of new non-enzymatically derived PTMs.
Chemical tools for biological discovery
We complement our drug discovery efforts with an additional focus on developing new high throughput- and molecular diversity-based methods to accelerate the rate and scope of potential biological discovery. To this end, we have ongoing projects related to identifying the cellular binding partners of short ORF encoded peptides (SEPs) and the proteinaceous substrates of various classes of enzymes using photo-crosslinking-based methodologies. We are additionally developing multiplexed technology to profile enzyme-inhibitor interactions en masse to more efficiently screen chemical libraries for new lead inhibitor series. Lastly, we have developed miniaturized assays for interrogating the electrophilicity present in existing chemical libraries to discover new warheads for the development of novel covalent inhibitor templates.
Representative chemical biological work: Identifying a new cysteine-selective covalent reactive group
We have additionally sought other high throughput methods for identifying bioactive chemical matter, hoping to leverage the diversity present in chemical libraries to identify novel chemical reactive groups and chemotypes with interesting cellular activity. In one example, we leveraged our knowledge of the KEAP1-NRF2 pathway–namely that KEAP1 is the mammalian cell’s electrophile sensor—to identify novel cysteine reactive groups. From a screen of curated chemical building blocks with potential reactivity, we identified the 2-sulfonyl pyridine group as a surprisingly robust cysteine-reactive molecule in cell-based assays for NRF2 activation. In this work, explored the reactivity of this group, demonstrating the electrophilicity could be tuned by altering the electron withdrawing group in the 3 position and appending either a sulfone or sulfoxide as the leaving group. We further demonstrated the utility of this chemotype by developing compound 21, which covalently inhibits adenosine deaminase (ADA) a key enzyme involved in immune cell activation. Interestingly this molecule does not target an active site but targets a cysteine distal to it, which lies between two residues (G74, R76) that are mutated in severe combined immunodeficiency based on an ADA mutation (ADA-SCID).