Stuart L. Schreiber
The Schreiber laboratory is developing chemical methods to understand and control the cellular function of proteins. To understand a protein's function requires the ability to alter it, for example, by inactivation or activation. This is most often accomplished by genetic manipulation, i.e. mutating the gene encoding a protein of interest. Research in this laboratory has demonstrated ways to alter protein function directly, using cell permeable, small molecules that bind to the target protein and cause either inactivation (equivalent to a "loss of function" mutation) or activation ("gain of function"). A loss of function results when these ligands bind to a site on the target protein that is critical for its function, whereas a gain of function can result when synthetic "dimerizers" bring two target proteins together within the cell. A particularly useful feature is that the gain or loss of function can be switched on and off at will. Since this approach was inspired by the ways in which mutations have been used to study protein function, it has been termed the "chemical genetic" approach. It has emphasized the equivalency of ligands and mutations, and has resulted from the melding of synthetic organic chemistry with cell biology. In order to extend chemical genetics as an approach to the study of all proteins, the principles of genetics used to discover mutations are being emulated in chemical efforts to discover new cell permeable ligands. Split-pool syntheses of natural product-like substances and miniaturization techniques for assaying their intracellular protein-binding properties are being developed in the Howard Hughes Medical Institute laboratory in the Harvard University Department of Chemistry and Chemical Biology and in the Institute of Chemical Biology laboratory in the Harvard Medical School Department of Cell Biology.
This research originated from work that defined the molecular mechanisms of the immunosuppressive agents cyclosporin A, FK506, and rapamycin. The synthetic chemists in the laboratory not only completed total syntheses of the three immunosuppressants, but prepared the reagents used to co-discover (with scientists at Merck) the FKBP family of immunophilin proteins and to characterize them in functional and structural terms. A designed, synthetic ligand named 506BD was used to show that the immunosuppressants cause a gain in the function of an associated immunophilin following receptor binding. The molecular basis for this gain in function was clarified with the discovery in 1991 by a postdoctoral fellow, Jun Liu, that both FKBP12-FK506 and cyclophilin-CsA bind to and inhibit the protein phosphatase calcineurin. This finding led to the discovery that calcineurin is a key molecule in the T cell receptor signaling pathway that activates resting T cells for the cell cycle.
A graduate student, Peter Belshaw, has demonstrated a new strategy that permits structural variants of CsA and FK506 to inhibit calcineurin only in targeted tissues or organs in transgenic animals, and therefore to understand calcineurin's function in these locations. This stratgy involves creating new receptor-ligand pairs using site-directed mutagenesis and synthetic chemistry ("bumps and holes").
The FKBP12-rapamycin complex was shown by two graduate students, Eric Brown and Mark Albers, to bind to a previously unrecognized regulator of the G1 phase of the cell cycle now named FRAP. Using rapamycin as an equivalent of a loss-of-function temperature-sensitive allele of FRAP, the protein's kinase activity was shown to be necessary for the activation of the p70 S6 kinase. A human checkpoint homolog named FRP1 (FRAP-Related Protein) was also discovered, and the lab has begun to shed light on the function of other members of this fascinating family of "PIK-related kinases".
Studies of cell cycle signaling pathways sensitive to natural products have led to the discovery of other new signaling proteins (e.g., histone deacetylase HDAC1, the target of trapoxin; protein palmitoyl transferase, a target of didemnin), and to the identification of valuable probes of known signaling proteins (microtubles, discodermolide; proteasome, lactacystin), and have revealed that the approach has broad generality. The case of trapoxin provides an illustration of the importance of synthetic chemistry in these studies. A total synthesis of trapoxin by a graduate student, Jack Taunton, was adapted to a synthesis of a tritium-labeled analog and, even more importantly, to an immobilized variant that was used as an affinity reagent. This reagent led to the discovery of human histone deacetylase-1 (HDAC1). This previously unkown protein provides a critical link between two active areas of research - transcriptional activation and chromatin remodeling. The most recent work by a graduate student, Christian Hassig, has demonstrated that gene regulation occurs in cells by the targeting of HDAC1 to specific genes through a DNA-protein complex. Histone deacetylase resisted molecular characterization for over 30 years after Allfrey and co-workers first demonstrated its existance in crude nuclear extracts. The laboratory's success illustrates how synthetic organic chemistry can be applied to a problem in cell biology.
Research in the laboratory has also demonstrated that chemical approaches to signal transduction can also be used to control signaling pathways. A key insight came with the recognition that ligand-induced protein dimerization and oligomerization constitute a common means of initiating information transfer, rivaling the role of ligand-induced allosteric change. In collaboration with Dr. Gerald R. Crabtree and members of his laboratory at the Howard Hughes Medical Institute in Stanford, a method has been devised that permits controlled intracellular dimerization or oligomerization of proteins with cell-permeable, dumbbell-shaped, synthetic ligands. Like the immunosuppressive natural products that inspired their design, these molecules have two protein-binding surfaces. This approach has been used in the Harvard-Stanford collaboration to activate proliferative and death pathways involving the T cell, PDGF, insulin, and Fas receptors, and to regulate transcription, protein translocation, and protein degradation. Using this approach, it was demonstrated in 1996 that synthetic dimerizers are able to ablate CD4/CD8 double positive thymocytes in a transgenic mouse expressing a rationally designed, conditional allele of the Fas receptor. This work illustrates for the first time the use of small molecules to achieve spatial and temporal control over a specific signaling pathway in an animal. Ligand-regulated activation and termination of cellular pathways has illustrated the importance of proximity and orientation of proteins in biology and that offers new opportunities in research in biology and medicine.