Partnering
Partnering Opportunities
The focus of our research is on drug discovery using fragment-based methods and structure-based
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)
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 dose-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 structure-based 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.
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