Designing New Molecular Therapeutics
As we achieve a molecular-level understanding of disease, we are left with the daunting task of developing molecules to intervene. The Kritzer laboratory aims to discover molecules that can be used to probe the mechanisms of human disease and to streamline the development of those molecules as therapeutic agents. We apply methods that integrate genetics, biophysics, and organic synthesis to complement structure-based design and traditional high-throughput screening with innovative techniques.
Figure 1. Large collections of different peptides and peptidomimetics can be synthesized, each attached to an individual bead the size of a grain of sand. A fluorescently-labeled protein can then be used to discover which molecules bind that protein. Beads coated with tight protein binders will accumulate fluorescence (orange glow), while poor binders will remain dark (dark green).
Many of our targets are disease-associated proteins that have been ignored or discarded as "undruggable." By using new classes of molecules as inhibitors, we are reversing long-held assumptions about what proteins can be useful targets for cellular probes and potential therapies. We are currently targeting proteins involved in breast cancer, skin cancer, diabetes, bacterial meningitis, and bacterial pneumonia.
Figure 2. One of our targets is a protease secreted by diverse Gram-positive and Gram-negative pathogens. These proteases are virulence factors that cleave the human mucosal antibody IgA1, and disabling these antibodies is an important step in the infection and invasion of these pathogens. Inhibitors of these IgA1 proteases would be an anti-virulence approach to fighting devastating acute and chronic infections, without promoting resistance.
Getting in shape: Controlling peptide bioactivity and bioavailability using conformational constraints
Molecules can be successfully designed to inhibit a target protein by arraying specific functional groups in three dimensions in order to interact "just so" with the protein surface. Another tenet of drug design is that molecules must have favorable physicochemical properties that ensure it will be stable in biological systems and cell-penetrant. The Kritzer lab focuses on how molecule shape (its 3-D conformation) plays a predominant role in determining a molecule's chemical and biological function.
Figure 3. X-ray crystallography and NMR are used by the Kritzer lab to improve peptide design. In a project designing peptide-based metallocatalysts, we determined the structure of the same cyclic peptide in two states: free (beige) and metal-bound (black). This allows us to visualize the structural transition that occurs upon metal binding.
We are re-examining “exceptions” to physicochemical rules, and discovering surprisingly general scaffolds for highly bioactive and bioavailable molecules. We use novel design and combinatorial screening strategies to identify molecules that inhibit hard-to-target proteins involved in human disease, and then we are use conformation as a tool to improve the molecules' performance in biological systems. In this manner, we are producing not only inhibitors, but useful probes and therapeutic leads for targets that have been overlooked or abandoned as too difficult.
Figure 4. Adding intramolecular covalent constraints, or “staples,” allows us to turn poorly structured peptides into highly structured, cell-pentrant biological probes. In recent work, we used this strategy to identify cell-penetrant peptides that induce autophagy. Molecules that induce autophagy are highly sought after as potential therapeutics, and our stapled peptides are currently being tested in models of infectious disease, metabolic disease and neurodegeneration to measure the therapeutic value of autophagy induction. The figure shows the bioactive peptide, along with human cells in which the autophagy process is visualized as green fluorescent dots.
The hallmarks of our research strategies are efficiency, accessibility, and interdisciplinarity, covering a wide range of relevant science:
- Novel synthetic routes to useful building blocks for controlling peptide conformation
- Synthesis of novel constrained peptide scaffolds with unusual properties and improved cell penetration
- Structural characterization in order to develop design rules for constrained peptides
- Translation from test tube to cell culture to whole-organism models of disease Novel inhibitors of previously "undruggable" proteins