In the Meroueh Lab, we develop small molecules to inhibit the cellular function of well-established oncogenes such as members of the RAS and Rho GTPase superfamilies, transcription factors such as the TEAD-YAP complex, and protein-protein interactions such as HPV E6 oncogenes and urokinase receptor. We are also interested in targets that prevent recovery following spinal cord and traumatic brain injury, such as the Rho GTPases. To accomplish this, we use innovative strategies such developing small molecules that (i) engage hot spots at protein-protein interfaces, (ii) form a covalent bond with target nucleophiles such as cysteine and tyrosine to irreversibly inhibit the target protein, or (iii) degrade proteins using proteolysis targeting chimeras (PROTACs). Small molecules are developed primarily through a fragment-based drug design approach using covalent and non-covalent fragment libraries available in the Meroueh laboratory. Fragments are improved by structure-guided drug design strategies based on co-crystal structures that we solve in my laboratory. We use a range of biochemical and cell biological approaches to explore the potency and selectivity of small molecules that includes fluorescence-based methods, bioanalytical tools such as surface plasmon resonance, and cell-based assays using standard molecular biology methods, or more sophisticated methods like luciferase-based methods.
RAS Oncogene
KRAS is the most frequently mutated oncogene with mutation rates of 95% in pancreatic ductal adenocarcinoma (PDAC), 45% in colorectal cancer, and 30% in lung adenocarcinomas. The most common K-RAS mutations occur at codon 12, namely G12D, G12V, G12C, and G12R. K-RAS was considered undruggable due to lack of well-defined drug-binding pockets. But a recent breakthrough was achieved with covalent inhibitors that form a bond with K-RAS G12C cysteine. Several of these compounds are in clinical trials, and one was given FastTrack status by the FDA. Unfortunately, only a small fraction of K-RAS oncogene mutants harbor the G12C mutation, and G12D, G12V and other mutations do not provide an accessible cysteine nucleophile. In recent work with the RAS GTPase Ral we showed through high-resolution structures and extensive biochemical studies that covalent bond formation with Tyr-82 created a well-defined novel binding site located between the Switch II and the Switch I/II pockets. Additional fragment screening carried out more recently identified a fragment that forms a covalent bond at K-RAS Switch II Tyr-64 to inhibit activation of the GTPase by the Son-of-Sevenless (SOS) guanine exchange factor. Here, we hypothesize that covalent bond formation with tyrosine or other amino acids on K-RAS will inhibit activation or effector binding and block K-RAS signaling in cancer cells. Our preliminary data and extensive experience in the field puts us in a strong position to accomplish our objectives. In the Meroueh lab, we employ ligand- and structure-based methods to generate fragment electrophile libraries from large commercial collections, and we follow a structure-based method to grow hit fragments into neighboring pockets to enhance their binding affinity and reaction rates. We also carry out well-established intact protein mass spectrometry, nucleotide exchange, and effector binding studies to screen fragment libraries for hit compounds, and to characterize small molecules that emerge from fragment growing strategies. We use X-ray crystallography to solve the structure of hit fragments and derivatives that emerge from fragment growing efforts. We also carry out cell biological studies to confirm direct engagement of K-RAS, inhibition of K-RAS signaling, and inhibition of cancer cell proliferation. We expect to identify high quality fragments and small molecules that form a covalent bond at wild-type and mutant K-RAS oncogenes, inhibit K-RAS wild-type or oncogene mutant activity in cancer cell lines, and inhibit PDAC and lung adenocarcinoma cancer cell viability. These compounds will serve as starting points to pursue in lead optimization efforts towards the development of therapeutic agents for the treatment of K-RAS-driven tumors.
TEAD-YAP Transcription Factor Oncogene
Yes-associated protein (YAP) and its paralog TAZ are transcription factor co-activator oncogenes that are sequestered in the cytoplasm through phosphorylation by the tumor suppressor Hippo signaling pathway kinases LATS1 and LAST2, as well as other Hippo-independent kinases. Phosphorylated YAP is degraded in the proteasome or kept in the cytoplasm by 14-3-3 proteins. Cancer cells suppress YAP/TAZ phosphorylation, enabling YAP to enter the nucleus and act as co-activators of the TEAD/TEF transcription factors. The binding of YAP or TAZ to TEAD1-4 is essential to mediate their proliferative and oncogene roles. Activated YAP and TAZ have been shown to be essential for aberrant cell proliferation, survival, and tumor cell spreading in several types of cancers, including breast, pediatric sarcoma, pancreatic ductal adenocarcinoma (PDAC), and lung cancer. To date, despite intense interest in YAP and TAZ, there is no small organic molecule that antagonizes the TEAD•YAP/TAZ interaction in vivo. Thus, there is an unmet need for small-molecule TEAD•YAP/TAZ antagonists to serve as in vivo chemical probes that will lead to the development of new cancer therapeutics. Our short-term objective is to develop small-molecule TEAD•YAP/TAZ antagonists that possess suitable pharmacokinetics and suppress TEAD•YAP/TAZ transcriptional activity in vivo. Our long-term objective is to develop small-molecule therapeutic regimens that include TEAD•YAP/TAZ antagonists either alone or in combination therapies. Our central hypothesis is that small-molecule covalent TEAD•YAP/TAZ antagonists will inhibit tumor growth and metastasis. We pioneered the development of small-molecule TEAD•YAP inhibitors as we reported recently in Cell Chemical Biology (PMID: 30581134). Our innovative approach consisted of taking advantage of a cysteine residue within the deep and well-defined palmitate pocket of TEAD4 to form a covalent bond with the transcription factor and irreversibly inhibit its binding to YAP1. In mammalian cells, the compound, 2 (TED-347), inhibited TEAD4 transcription factor activity, blocked the TEAD4•YAP1 interaction, and suppressed expression of TEAD-dependent CTGF transcript. Based on this discovery, we embarked on an effort to develop small molecules to probe TEAD•YAP in vivo and to develop therapeutic agents targeting this pathway. As a result, we developed a derivative that replaces the reactive group of 2 (TED-347) with an acrylamide, which is found in all recently FDA-approved covalent drugs. Another covalent library screen led to the discovery of an acrylamide with a novel core structure. Both compounds form a covalent bond with TEAD4 at conserved Cys-367 and inhibit TEAD4 binding to YAP1. Both inhibit TEAD4•YAP1 transcription factor activity and suppress TEAD•YAP-dependent CTGF and Cyr61 transcripts in MDA-MB-468 breast and TT2 sarcoma cell lines.
HPV E6 Oncogene
The 8 kilobase pair DNA genome of human papillomavirus (HPV) encodes five early proteins, each of which has multiple activities. The HPV E6 protein is necessary for viral genome replication and maintenance as an episome. The E6 protein of “high-risk” (HR) types is always expressed in HPV-associated pre-malignant, dysplastic, in situ cancers, and in malignant tumors including metastases. HR E6 is best known for its ability to mediate p53 degradation by binding to the ubiquitin ligase E6AP. This complex undergoes allosteric changes that expose the interaction surface for p53 binding on E6 and stimulates the ubiquitin ligase activity of E6AP, which result in destruction of the p53 tumor suppressor protein. X-ray structures of E6•E6AP reveal a pocket in E6 that binds to a charged helical peptide of E6AP, disclosing a druggable protein-protein interaction site. Small molecules that inhibit E6 activity would eliminate HPV infection and the risk of neoplastic progression. Our antiviral strategy is to identify drug-like antiviral molecules that covalently bind and block this pocket of HPV-16 E6, which causes ~50% of all cervical and the vast majority of HPV induced anal and oropharyngeal cancers. There is no medication to treat HPV. A small molecule that covalently and irreversibly binds to the E6•E6AP pocket will act in part by restoring wild-type p53 levels and activity and thereby promote apoptosis or induce senescence of HPV expressing epithelial cells, thereby killing the cells or preventing oncogenic transformation. Extensive preliminary data support this hypothesis. We are following an iterative process of design, synthesis, biochemical screening, complex structure determination, and cell biological studies to isolate drug-like small molecules that would be suitable for pre-clinical evaluation. The concept of treating dysplastic HPV-16 infections with a topically applied E6 antagonist is particularly appealing for prevention of oncogenic progression that will develop in millions of HPV infected people.
