Undergraduate, graduate and postdoctoral trainees in the Schreiber laboratory over the past 25 plus years have developed systematic ways to explore biology using small molecules as probes. Their research has uncovered many remarkable small-molecule probes of extremely difficult targets and processes that are at the root of human disease By developing new approaches necessary for these challenging targets, such as next-generation synthesis and niche-based screening, they are changing our view of what can be accomplished in modern drug discovery. By discovering small-molecule probes of undruggable targets such as transcription factors and protein/protein interactions, they have revealed that impossible molecular challenges are actually achievable. Indeed, new drugs have been developed whose therapeutic effects are the direct consequence of proteins and/or cellular mechanisms revealed by the groups research.
The trainees in the Schreiber laboratory have helped to develop the emerging area of chemical biology, for example, through their work in diversity-oriented synthesis (DOS) and small-molecule screening involving complex biology. Using their chemical approach, they have discovered principles that underlie information transfer and storage in cells. For a more detailed account of past efforts read the archived Research Detail.
Several discoveries that emerged from research in this laboratory are highlighted here. These relate to both cell circuitry by signaling proteins calcineurin and mTOR and gene regulation by chromatin-modifying histone deacetylases. Together, they illustrate how chemical biology advances the use of small molecules for exploring biology and medicine.
Calcium-calcineurin-NFAT signaling pathway. Following their co-discovery of the FK506-binding protein FKBP12 in 1988, members of the Schreiber lab reported that the small molecules FK506 and cyclosporin inhibit the activity of the phosphatase calcineurin by forming the ternary complexes FKBP12-FK506-calcineurin and cyclophilin-cyclosporin-calcineurin. This work, together with work by researchers in the Gerald Crabtree lab at Stanford concerning the NFAT proteins, led to the elucidation of the calcium-calcineurin-NFAT signaling pathway. It was an early example of defining an entire cellular signaling pathway from the cell surface to the nucleus, analogous to that of the Ras-Raf-MAPK pathway elucidated the following year. The realization that a successful drug (Novartis Sandimmune/cyclosporin) targeted an intracellular phosphatase, expanded the focus of drug discovery from membrane receptors (e.g., G-protein coupled receptors) to the now commonly targeted intracellular signaling pathways via kinases and phosphatases. In more recent years, the calcium-calcineurin-NFAT pathway has been found to play a critical role in immune function, heart development, and the acquisition of memory in the hippocampus.
Small-molecule dimerizers. The above studies also led the Schreiber and Crabtree labs jointly to develop in 1993 small-molecule dimerizers, which provide small-molecule activation over numerous signaling molecules and pathways (e.g., the Fas, insulin, TGFb and T cell receptors) through proximity effects. Researchers in these labs demonstrated that small molecules could be used to influence signaling pathways in an animal with temporal and spatial control. Dimerizer kits have been distributed to over 1,100 laboratories, and its promise in gene therapy has been highlighted by the stable (over eight years), small molecule-induced production of erythropoeitin (EPO) in a primate (Ariad Pharmaceuticals).
mTOR and the nutrient response-signaling network. In 1994, members of the Schreiber lab discovered that the small molecule rapamycin simultaneously binds FKBP12 and mTOR (originally named FKBP12-rapamycin binding protein, FRAP). Using diversity-oriented synthesis and small-molecule screening, the nutrient-response signaling network involving TOR proteins in yeast and mammalian cells has been illuminated. For example, in their most recent work, trainees discovered that mTOR inhibition induces the metabolic state of aerobic glycolysis, a hallmark of cancer cells, in human cell lines. Small molecules such as uretupamine, SMIRS and SMERs (inhibitors and activators of autophagy), and rapamycin were used to reveal new pathways relevant to cancer.
HDACs and chromatin. In 1996 members of the Schreiber lab used a synthetic version of the small molecule trapoxin to characterize molecularly for the first time the histone deacetylases (HDACs). Prior to this work, the HDAC proteins had not been isolated despite attempts by others in the field who had been inspired by Allfrey's detection of the enzymatic activity in cell extracts over 30 years earlier. Co-incident with the HDAC discovery, David Allis and colleagues reported their discovery of the histone acetyltransferases (HATs). These two contributions catalyzed research in this area, eventually leading to the characterization of numerous histone-modifying enzymes, their resulting histone marks, and numerous proteins that bind to these marks. By taking a global approach to understanding chromatin function, Schreiber and Bernstein proposed a signaling network model of chromatin and compared it to an alternative view, the histone-code hypothesis presented by Strahl and Allis. This approach also led Schreiber and collaborators to discover novel chromatin methylation patterns (bivalent domains) at the promoters of master regulatory genes in embryonic stem cells. The work of many researchers in this area has shined a bright light on chromatin as a key regulatory element central to epigenetics, rather than simply a structural element, and has opened up new avenues in medicine.
During the past 10 years, especially following the founding of the first academic and open-data sharing, small-molecule screening center (Harvard's ICCB, now the Broad Institute's Chemical Biology Program), trainees have focused on systematizing the discovery of small-molecule probes. These include probes that accomplish seemingly impossible tasks for a small molecule; for example, disrupting protein/protein and protein/DNA interactions, targeting transcription factors and even regulatory RNAs, and reprogramming human cells in organ culture. The capabilities that underlie these successes include:
- The development of diversity-oriented synthesis of structurally complex and stereochemically and skeletally diverse small molecules that facilitate all three phases of probe discovery in which synthetic chemistry plays a role. These include the discovery of starting compounds against difficult targets, the optimization and biological characterization of them, and the efficient scaled synthesis of the optimized variants. Diversity synthesis is key to the goal of creating transformative small-molecule screening collections.
- The development of screens and screening methods for the discovery of small molecules that achieve challenging tasks by, for example, using co-cultures to preserve the physiological properties of primary cells, targeting RNAs and proteins in cells, and using signature-based state-switching screens.
- The development of methods to identify comprehensively the proteins to which small molecules bind in cells and cell lysates. These methods include: 1) the first public database (ChemBank) of small-molecule screening data with an underlying information model that enables comparisons of outcomes involving different types of screens and connectivities of compounds using signatures based on compound performance across many screens, 2) quantitative proteomics and small molecule-based affinity reagents, and 3) genetic approaches using microarrays and quantitative trait mapping with overexpression and genotyped yeast strains, respectively.
These advances have resulted in an increase in the pace and impact of small-molecule-based discoveries. Trainees in the Schreiber lab showed for the first time that a small molecule (lactacystin) could selectively target the proteasome, which encouraged the development of the proteasome inhibitor bortezomib, approved for the treatment of multiple myeloma in 2003. Discodermolide was shown to be the second known stabilizer of microtubules (following taxol), and synthetic compounds having this same property such as synstabs A and B were discovered using the cytoblot screening method. Myriocin was shown to bind selectively to a component of the sphingosine biosynthetic pathway the serine palmitoyl transferase. The first sirtuin (the family of NAD-dependent HDACs) inhibitor was discovered using a phenotypic, cell-based screen. (The first sirtuin activator, the red wine component resveratrol, which jump-started the anti-aging biotechnology industry, was discovered in the ICCB.) A cytoblot screen was used to discover monastrol, the first small-molecule inhibitor of mitosis that does not target tubulin. Monastrol was shown to inhibit the kinesin motor protein kinesin-5 and was used to gain new insights into the functions of kinesin-5. This work led several pharmaceutical companies to pursue anti-cancer drugs that target this protein. Methods for multidimensional screening were developed and provided insights into tumorigenesis, cell polarity, nanomaterials, maturity onset diabetes of the young (MODY), and chemical space, among others.
Diversity syntheses have been guided by a powerful synthesis strategy named build/couple/pair that has yielded small-molecule probes of autophagy, histone and tubulin (HDAC6) deacetylases, histone demethylases, transcription factors, cytoplasmic anchoring proteins, developmental signaling proteins, sonic hedgehog, ErbB4 signaling, hepatitis C replication (e.g., histacin, tubacin, haptamide, uretupamine, concentramide, calmodulophilin, robotnikinin, BLG2621, bicyclic epoxides), among many others. More than 200 laboratories from over 50 institutions have collaborated in the open data-sharing environment at the Broad Institute Chemical Biology Program, leading to many additional small-molecule probes and insights into biology.
Impact on human physiology and medicine. Over 100 published papers by trainees in the Schreiber lab have received 100 citations or more (h-index = 109), resulting in one of the top rankings among chemistry labs in terms of citations. In 2007, two new anti-cancer drugs that target proteins discovered in the Schreiber laboratory using the small-molecule approach were approved by the U.S. FDA: torisel (Wyeth; treatment of renal cell carcinoma), which targets mTOR (discovered in 1994) and vorinostat (Merck; treatment of cutaneous T-cell lymphoma), which targets HDACs (HDAC1 discovered in 1996). Chemical biology principles developed in the Schreiber lab have been extended to medicine additionally through the founding of biopharmaceutical companies, including Vertex Pharmaceuticals (1989), ARIAD Pharmaceuticals (1991) and Infinity Pharmaceuticals (2001), each of which has devised new therapeutic agents that are being tested in human clinical trials or used as FDA-approved drugs.
"Broadening Perspectives" C&E News, December 2008
- "Chemical biology at the Broad Institute" Nature Chemical Biology, April 2007