Partnering
Partnering Opportunities
The focus of our research is on drug discovery using fragment-based methods and structurebased design. (Fesik Lab homepage).
Dr. Fesik pioneered the use of fragment-based methods for discovering high affinity ligands for proteins over 25 years ago (1) and has applied this method to several protein targets. After 26 years in drug discovery at Abbott/Abbvie with the last nine-years serving as Divisional Vice President of Cancer Research, Dr. Fesik was recruited by Vanderbilt University in 2009 to be a professor in Biochemistry (primary appointment), Pharmacology, and Chemistry. In addition, many of the Fesik lab members have also worked in the Pharmaceutical Industry, and we apply the same principles, approaches, thought processes, and techniques used in drug companies with an emphasis on targeting “undruggable” targets. As shown in the project descriptions, we have been quite successful over the last several years that has led to projects at various stages ranging from hit identification to clinical candidates. Although KRAS is partnered with Boehringer Ingelheim, the other projects described below are available for partnering.
If you are interested in working with us on any of the projects or are interested in hearing more about these and other ongoing projects in the lab, don’t hesitate to contact Stephen Fesik via email at stephen.fesik@vanderbilt.edu.
Cancer
KRAS (partnered with BI)
KRAS is a GTPase that is heavily mutated in many cancers, including 86-96% of pancreatic cancers, 40-54% of colorectal cancers, and 27-39% of lung cancers. Activating mutations in KRAS increase signaling in several important cellular pathways and are the most common oncogenic drivers in human cancer. Thus, KRAS is a highly validated cancer target. However, despite many attempts to target KRAS over decades, it was long thought impossible to drug. Recent efforts, including the approval of G12C KRAS inhibitors, have changed this way of thinking. Our lab has a 15-year track record of targeting KRAS. Using NMR-based fragment screens, we identified small molecules that bind to KRAS in the active GTP- and inactive GDP-bound forms (2). Subsequent optimization of the fragment hits that bind to KRAS-GTP using structure-based design led to a compound that binds to the switch I/II site with nanomolar affinity, inhibits all GEF, GAP, and effector interactions with KRAS, and displays an antiproliferative effect in KRAS mutant cells (3). We attempted to optimize fragments that bind to KRAS-GDP by using a second site screen to identify compounds that could be linked or merged to the first site hits. However, the molecules simply displaced the first site ligand in the screen. We therefore developed a strategy to hold the first site ligand in place by introducing cysteine mutants near the switch I/II site and identifying a molecule that could covalently attach to these cysteines (4). Using S39C KRAS with a covalently bound switch I/II blocker, we performed a second site screen and identified 20 hits that bound to KRAS at a second site. We obtained X-ray crystal structures that showed these hits unexpectedly bind to the switch II site. Optimization using structure-based design led to BI 1823911, a G12C inhibitor in phase I (5), a pan KRAS inhibitor (BI 3706674) which has recently entered phase I (6), a pan-RAS degrader (7), and mutant selective KRAS inhibitors that are currently in preclinical studies. Some of this work was performed under a collaborative licensing agreement with Boehringer Ingelheim. Interestingly, the reported KRAS inhibitors/degraders under development from BI all contain the fragment that we identified from the second site screen (Fig. 1).
MYC
MYC is a transcription factor that is overexpressed in most cancers. MYC is composed of an N-terminal transactivation domain, a C-terminal basic-helix-loop-helix leucine zipper DNA binding domain, and a central region (Fig. 2). It forms a heterodimer with MAX, binds to Ebox DNA, and drives the expression of genes required for cell growth, proliferation, metabolism, genome instability, and apoptosis. Like KRAS, MYC is a highly validated target but is thought to be difficult or impossible to drug. Unlike KRAS, however, no molecules that directly target MYC have been approved to date.
Our first attempt to drug MYC used an NMR-based fragment screen of the transactivation domain and the DNA binding domain of MYC – two regions of the protein required for activity. No confirmed hits were found for either of these proteins. This outcome was not surprising, as MYC, in the absence of MAX, is intrinsically disordered in solution as evidenced by the poor chemical shift dispersion observed in our NMR spectra. Thus, due to the lack of confirmed hits, we turned to other approaches for targeting MYC.
MYC/WDR5 (available for partnering)
MYC/WDR5 (available for partnering) Using a two-hybrid and proteomic screen, Professor Bill Tansey (Vanderbilt) discovered that the central portion of MYC (MYC box IIIb) binds to WDR5, and this interaction is required for MYCdriven tumorigenesis (8). These data led us to our second approach for targeting MYC using a cofactor of MYC, WDR5, which is much more druggable. From a fragment-based screen of WDR5, we identified hits that bound to the site where MYC interacts with WDR5 (WBM site) (9) and hits that bind to the opposite end of the protein at the site where MLL1 binds to WDR5 (WIN site) (10) (Fig. 3). From these fragments, we discovered potent chemical probes at both the WBM (9,11) and the WIN sites (10,12) that allowed us to study the biology of WDR5 and evaluate WDR5 as a cancer drug target. We found that WDR5 facilitates the recruitment of MYC to chromatin to control the expression of genes linked to ribosome biogenesis—a critical tumor-sustaining function of MYC. Importantly, disrupting the MYC-WDR5 interaction promotes tumor regression in vivo, validating the importance of WDR5 for tumor maintenance by MYC (13). WIN site inhibitors act by displacing WDR5 from chromatin at ribosomal protein genes (14) and not by changes in histone methylation as previously thought (15). These findings indicate that a WDR5 inhibitor may be useful for treating a wide range of cancers and encouraged us to further optimize the chemical probes into drugs. Upon extensive optimization, we discovered single digit picomolar WDR5 inhibitors that are orally bioavailable, efficacious in vivo, and safe (16- 18) (Fig. 4). We have selected a candidate for IND-enabling studies and are currently working with the NCI to advance this molecule into the clinic.
MYC/MAX (available for partnering)
Inhibiting the binding of MYC to DNA represents another method of targeting MYC. OmoMYC, a protein-based inhibitor of DNA-binding to MYC, has been shown to promote tumor regression in a host of cancer types in vivo with only limited and reversible toxicities (19). However, protein-based inhibitors typically face development challenges and often do not make ideal drugs. Small, drug-like molecules that bind to the MYC/MAX dimer and prevent it from recognizing its cognate DNA binding sites would disable all or most of the transcriptional functions of MYC in cancer cells, promote tumor regression, and have superior drug-like properties compared to a protein-based agent like OmoMYC. Therefore, we set out to identify compounds that block the binding of MYC to DNA.
Unlike MYC alone, the MYC/MAX dimer adopts a folded structure and therefore may be more druggable. The MYC/MAX dimer can be labeled and expressed in E. coli, and its 1H and 15N NMR resonances have recently been assigned. Using an NMR-based fragment screen of the MYC/MAX dimer, we identified multiple hits. X-ray crystal structures of two of these hits when bound to the MYC/MAX dimer revealed that these hits bind at the interface of the MYC/MAX dimer with DNA (Fig. 5). Furthermore, these molecules and their analogs were found to weakly disrupt DNA binding by MYC (Fig. 5). We have now obtained multiple X-ray structures of the MYC/MAX dimer when bound to additional hits and their analogs and have used structure-based design to improve binding by 1000-fold. Current work involves exploring the ability of these compounds to inhibit the functions of MYC using assays set up by Bill Tansey and testing our inhibitors for treating MYC-driven cancers in vivo.
WNT pathway: b-catenin (available for partnering)
The Wnt pathway is a highly validated drug discovery target for colorectal cancer (CRC) and other malignancies (20). Mutations in APC, AXIN, or b-catenin occur in 90-95 % of all CRCs, leading to excessive cytosolic b-catenin levels. Dysregulation of Wnt signaling is recognized as the initial, causative event in most CRC cases and has been shown to support tumor growth. However, despite decades of research, direct inhibition of the Wnt pathway downstream of activating mutations has been difficult to achieve. Our project aims to discover potent and selective inhibitors of the Wnt pathway via targeted protein degradation of b-catenin, using heterobifunctional molecules that bind directly to b-catenin.
We have used an NMR-based fragment screen to identify small molecules that bind bcatenin and identified fragments that bind to a site not shared by its known endogenous binding partners. More than 100 high-resolution crystal structures of analogs of these fragments bound to b-catenin have been determined and used to inform structure-based optimization of ligands for b-catenin. We have discovered compounds that bind tightly (KD <20 nM) to b-catenin and contain solvent-facing functional groups suitable for use in heterobifunctional Proteolysis Targeting Chimeras (PROTACs). Using these b-catenin ligands, we have created PROTACs that recruit the E3 ligase CRBN into a ternary complex with b-catenin, induce its proteasome-mediated degradation (DC50 <10 nM), inhibit cancer cell proliferation (EC50 <10 nM), have high in vivo exposure, and display a PD response in vivo (Fig. 6). Optimized b-catenin degraders may thus become a first-in-class therapeutic to address an unmet need in colon and other WNT-driven cancers.
MCL-1 (available for partnering)
MCL-1 is a member of the Bcl-2 family of proteins that binds to pro-death members of the same family and inhibits apoptosis. It is overexpressed in many cancers, allowing cancer cells to avoid apoptosis. Indeed, preventing programmed cell death is one of the hallmarks of cancer. Thus, MCL-1 is a well validated cancer target. However, like other anti-apoptotic members of the Bcl-2 family, MCL-1 is considered difficult to drug. Dr. Fesik has a long history of working with Abbott/Abbvie on the Bcl-2 proteins. Prior work has included solving the first structure of Bcl-xL (21) as well as Bcl-xL when bound to a peptide derived from the pro-death protein BAK (22), discovery of the first potent inhibitor of Bcl-2, Bcl-xL, and Bcl-w (23) that entered clinical trials (navitoclax), and discovery of a Bcl-2 selective inhibitor (venetoclax, VENCLEXTA) that is now being used to treat patients with CLL and AML. At Vanderbilt, the Fesik lab has targeted MCL-1 which causes resistance to Bcl-2 and Bcl-xL inhibitors as well as other anticancer therapies such as gemcitabine, vincristine, and paclitaxel.
To identify starting points for discovering MCL-1 inhibitors, we used a fragment-based screen, which identified two hits that bound to different sites on the protein. We then used a fragment merging strategy to obtain a much more potent MCL-1 inhibitor (24) (Fig. 7). From extensive optimization, we discovered highly selective picomolar MCL-1 inhibitors with enhanced cellular potency, drug-like PK properties, and in vivo efficacy by IV and PO dosing (25-30) (Fig. 7). Under a licensing agreement with our then partner, Boehringer Ingelheim, the MCL-1 inhibitors were extensively profiled. Potent antitumor efficacy in heme malignancies and lung xenograft models was achieved without significant toxicity. Our leads exhibit a different PK profile (lower Vss, shorter t1/2) compared to other MCL-1 inhibitors which may be advantageous to manage the potential cardiovascular issues seen by MCL-1 inhibitors in clinical trials.
E3 ligase ligand discovery (available for partnering)
More than 600 E3 ligases are known, but only a handful (primarily cereblon and VHL) have been used for targeted protein degradation. Novel ligands for different E3 ligases may be of value for several reasons: 1) overcoming resistance caused by mutations in an E3 ligase that block the ability of PROTACS to function, 2) expanding the applicability of PROTACS to target proteins that cannot be effectively degraded with known E3 ligases, and 3) reducing the toxicities associated with non-specific inhibition or degradation of the target protein. One particularly exciting application for new E3 ligases is the prospect of inducing degradation only in certain locations or tissues. PROTACs built from ligands for E3 ligases with expression in cancer but not in normal tissues might be used to degrade target proteins with known tissue-specific toxicities and thus dramatically improve the therapeutic window.
To demonstrate the utility of the approach, we identified E3 ligases with limited protein expression in normal tissues (Fig. 8). One example is KLHL12. PROTACs that recruit this E3 ligase would be ideal to degrade proteins with toxicities that would otherwise limit their use.
Using a fragment-based screen, we first identified hits that bound weakly to KLHL12, optimized binding to~50 nM using iterative structure-based design from >140 X-ray structures of co-complexes, and prepared PROTACS that degrade BRD4 and Bcl-xL (Fig. 8). Degraders that recruit ligases with a restricted expression profile could reduce the toxicity of BRD4 inhibitors, Bcl-xL inhibitors, and inhibitors of other proteins that are highly efficacious but exhibit do
se-limiting toxicities. We have identified hits in fragment-based screens of other E3 ligases (VHL, FEM1B, CBL-c, and TRAF4) using state-of-the-art NMR experiments (Fig. 9) and have determined the X-ray structures of hits bound to the proteins to enable structurebased affinity optimization. We believe that fragment-based methods are ideal for discovering small molecule ligands for E3 ligases and would be open to screening our fragment library against other E3 ligases.
Viral
SARS CoV2 (available for partnering)
The Covid-19 pandemic caused by SARS-CoV-2, the present and future variants of this virus that could emerge, and the potential for other coronaviruses to cause future outbreaks highlight the continuing need for new antiviral drugs targeting critical proteins in the coronavirus life cycle. Following host infection, SARS-CoV-2 translates its genome into two polyproteins, which require subsequent cleavage by two cysteine proteases (main protease and papain-like protease (PLPro )) to generate functionally active proteins. Although inhibitors of the main protease have been developed, no inhibitors of PLPro have reached the clinic. The high homology of PLPro across the coronavirus family makes PLPro an attractive drug target to overcome drug-resistant variants or new emerging coronaviruses in future.
Using an NMR-based screen of an in-house 13,824-molecule fragment library, we identified hits that bind to two sites on PLPro and characterized the binding modes at each site using X-ray crystallography (31) (Fig. 10). Guided by X-ray structures of co-complexes, we have improved the binding affinity > 3-orders of magnitude from the initial hit using fragment growing to exhibit cellular antiviral activity. Unlike other reported PLPro inhibitors that are analogs of GRL-0617 (an inhibitor of SARS-CoV), our inhibitors are structurally distinct and novel. Current work is focused on further optimizing the cellular potency, pharmaceutical properties, and in vivo activity with the goal of discovering a PLPro inhibitor that is suitable for clinical development.
a-viruses (available for partnering)
The Chikungunya virus (CHIKV), Venezuelan equine encephalitis virus (VEEV), and Eastern equine encephalitis virus (EEEV) are alphaviruses that cause diseases ranging from debilitating arthritis to lethal encephalitis in humans. CHIKV has caused several major outbreaks over the past 20 years, is highly infectious via the aerosol route, and is considered a potential bioterror threats. However, there are no FDA-approved antivirals for treating any alphavirus infection. We have selected the CHIKV nsP2 cysteine protease to target due to its essential role in alphavirus replication and its ability to convert the viral nonstructural polyprotein into functional components. We have identified hits in a fragment-based screen that bind to CHIKV nsp2 and are now in the process of determining the X-ray structures of these hits when bound to the protein.
Longevity (available for partnering)
Another field of interest in our lab is longevity. We are at a very early stage identifying potential drug targets that are suitable for fragment-based screens using NMR. We welcome the collaboration with those that may have the same interest in this exciting area.
References
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