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.
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