Effective cancer treatment still remains one of the most critical unmet medical needs. Despite many years of research, we do not have effective drugs for this formidable disease that affects almost everyone in one way or another. However, this will soon change. We have learned a lot about the causes of cancer and the proteins that we need to target to discover new and improved cancer drugs. In addition, we have developed the technologies to drug these highly validated targets. Finally, we now have methods to diagnose cancers based on the genetics of an individual's disease and will eventually be able to treat these individuals with drugs tailored for their particular disease. This is exciting times in cancer research. Our mission is to discover drugs to treat cancer that will effectively shrink tumors and lead to cures. We have chosen to target proteins that make outstanding cancer targets but are technically challenging to drug. To tackle such difficult targets, we will rely heavily on state-of-the-art methodologies such as fragment-based methods and structure-based design.
Fragment-Based Drug Discovery
The traditional approach to drug design involves the screening of a library of relatively large (at least on the chemical scale), intact compounds against the desired protein target. These target proteins have “pockets” to which drugs bind and can interfere with their activity. Our chemists try to find a molecule that will fit best into these pockets and affect the protein’s activity.
To find such molecules, we screen for fragments or pieces of a molecule that bind to subpockets of a binding site and then link them together, like Tinkertoys (See Figure on the left). Once our fragment-based screen identifies chemical fragments that bind to “subpockets” on the target protein’s binding surface, the 3-dimensional structure of the protein bound to the drug fragments is determined with NMR spectroscopy or X-ray crystallography. The 3-dimensional structures provide a picture of how the fragments fit into the protein’s binding site – and how they might be linked together. The fragments can then be assembled into a larger molecule that better fills up the target protein’s binding pocket. It’s a modular approach to drug discovery.
The fragment-based approach has several advantages over conventional methods and is ideal for identifying leads for technically challenging protein targets such as the ones we have chosen to target (e.g., K-Ras). Hits can be found in a fragment-based screen that cannot be found by conventional high throughput screening of large libraries of complex molecules. This is due to the difference in the stringencies in the screens.
The stringency in a conventional screen is typically set for detecting micromolar hits; whereas, a fragment-based screen is generally capable of identifying millimolar hits. In addition, the fragments in a screen are not constrained by the rest of a larger molecule and can more easily find the nooks and crannies in a protein binding site. Another advantage of the fragment-based approach is the greater coverage of chemical diversity space in the screen, which often leads to a wide variety of useful starting points for drug discovery. This method is a great way to create molecules that have never been made before and therefore would not have been found in a traditional high-throughput screen.
Ras proteins play essential roles as molecular switches controlling cell proliferation, growth, differentiation, and apoptosis. Deregulation of the Ras signaling pathway by activating mutations, overexpression, or upstream activation is common in human tumors. K-Ras is frequently mutated in human cancers, with the highest incidence of K-Ras mutations found in pancreatic (90%), colon (50%), and lung (40%) carcinomas. In addition, direct inhibition of K-Ras activity in established tumors or tumor cell lines has been shown to result in the reversal of the transformed phenotype and suppression of tumorigenicity in human cancer cells. Thus, K-Ras is a highly validated cancer target. However, K-Ras exerts its function through interactions with other proteins, and these proteins have traditionally been considered to be undruggable. To tackle this challenging problem, we are using fragment-based methods and structure-based design to discover potent and drug-like small molecule K-Ras inhibitors that bind to multiple forms of K-Ras complexes, including the GDP- and GTP-bound form of wild-type and oncogenic mutant forms of K-Ras. If successful, different chemical classes of K-Ras inhibitors will be obtained that could serve as useful lead molecules and potential clinical candidates for the treatment of many different types of cancer.
Mcl-1 (Myeloid cell leukemia 1) is a member of the Bcl-2 family of proteins that when dysregulated prevents cancer cells from undergoing programmed cell death, a hallmark of cancer. By overexpressing the Mcl-1 protein or amplifying the Mcl-1 gene, a cancerous cell can avoid death, the normal fate for cells exhibiting abnormal and deregulated growth. Indeed, amplification of Mcl-1 is one of the most common genetic aberrations observed in human cancer, including lung, breast, prostate, pancreatic, ovarian, and cervical cancers, as well as melanoma and leukemia. Moreover, Mcl-1 overexpression has emerged as a resistance mechanism against a number of anti-cancer therapies including the widely prescribed microtubule-targeted agents paclitaxel and vincristine as well as gemcitabine, a first-line treatment option for pancreatic cancer. Mcl-1 overexpression also confers resistance to ABT-263, a Bcl-2/Bcl-xL inhibitor currently in clinical trials. Not surprisingly, specific down-regulation of Mcl-1 by RNA interference inhibits cell growth, colony formation, and induces apoptosis in pancreatic cancer cells in vitro, and markedly decreases tumorigenicity in mouse xenograft models. Silencing of Mcl-1 also potently kills particular subgroups of non-small-cell lung cancer (NSCLC) cell lines. Together, these data suggest that direct inhibition of Mcl-1 could be an effective therapeutic option for a wide variety of cancers. In an attempt to target Mcl-1 for the treatment of cancer, small-molecule inhibitors as well as stapled helix peptides that bind to Mcl-1 have been reported. However, no Mcl-1 inhibitors have entered clinical trials. Indeed, targeting Mcl-1 is extremely difficult, since Mcl-1 exerts its activity through protein-protein interactions involving a large binding interface. To overcome the challenges associated with targeting Mcl-1, we have applied a combination of fragment-based methods and structure-based design. This approach leads to a more efficient sampling of chemical space and can provide hits with higher ligand efficiencies. Furthermore, the affinity of the initial fragment hits can be markedly increased by growing, linking, or scaffold merging to obtain potent lead molecules. Using this approach, we have discovered potent small-molecule Mcl-1 inhibitors (Ki <100 nM) that inhibit BH3-containing peptides from binding to Mcl-1 and have obtained X-ray structures that reveal how these lead molecules bind to the protein. We are currently using these structures to design improved inhibitors.
Replication Protein A (RPA)
Targeting the cellular response to DNA damage is a clinically validated approach for the treatment of cancer patients. Many cancer cells have defects in DNA repair pathways that cause genetic instability and support the malignant phenotype. Although select alterations in the rate limiting steps of DNA repair pathways can drive malignancy, excessive deficiencies in DNA repair may be harmful to cancer cells and lead to their demise. In addition, oncogene activation often leads to elevated levels of DNA damage and replication stress in cancer cells, making them particularly dependent on (already defective) DNA repair mechanisms. Thus, cancer cells with defects in DNA repair or elevated oncogene-induced DNA damage may be more susceptible to treatment with general DNA damaging agents (such as cisplatin) or targeted inhibition of select DNA repair pathways. For example, PARP inhibitors are under clinical investigation in several clinical trials, either as single agent therapy based on a synthetic lethal interaction with BRCA1 and BRCA2 mutations, or in combination with other therapies as a sensitizer to chemotherapeutics. In addition, inhibitors of ATM and ATR, two kinases heavily involved in DNA damage response, as well as inhibitors of downstream kinases Chk1 and Chk2, have been investigated for their ability to induce hypersensitivity to DNA damaging agents. The discovery of a new therapeutic target that governs DNA damage response and repair mechanisms may lead to a new treatment for cancer that has the potential to make a dramatic impact across a number of different cancer subtypes. RPA is a heterotrimeric ssDNA-binding protein that is essential for eukaryotic DNA replication, damage response, and repair. The RPA complex binds to and protects ssDNA, and also serves as a scaffold to recruit critical checkpoint and DNA-damage response proteins such as p53, Rad9, ATRIP, and Mre11 through the N-terminal region of the 70 kDa subunit of RPA (RPA70N). Selective inhibition of the protein-protein interactions between RPA70N and the proteins involved in the DNA damage response may result in inhibition of the critical RPA-mediated DNA damage response mechanisms. This may cause selective cytotoxicity to cancer cells, either alone or in combination with DNA-damaging chemotherapeutics. To study the function of RPA in this context, we are discovering potent and selective inhibitors of RPA70 using fragment-based methods and structure-based design and are evaluating their action in cells. We have identified hits in a fragment-based screen that bind to nearby pockets, have obtained X-ray structures that indicate how they bind to the proteins and have linked the fragments together using this structural information.
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