Research laboratory of William DeGrado, PhD

Research & Projects

Our research focuses on structural characterization of membrane proteins and de novo protein design in order to understand biological processes relevant to human disease and develop novel therapeutics.

We approach many diverse biological issues:

  • Protein design: We use de novo protein design to test whether our knowledge has advanced sufficiently to allow generation of structure and function from first principles. Our lab also uses this method to incorporate desired features into biological systems.
  • Integrin inhibitors: We study the mechanisms of signal transduction and conformational change in integrins, and have also designed novel small molecule inhibitors of specific integrins to use as therapeutics for tissue fibrosis and other diseases.
  • Antimicrobials: We study the mechanism of action of and bacterial response to rationally designed small molecule mimics of antimicrobial peptides.
  • Influenza: We study the matrix 2 protein (M2) of influenza A, which is a drug target found in the viral envelope of the flu.
  • HIV: We aim to characterize the membrane proximal external region of HIV-1’s envelope protein to rationally design an HIV vaccine.
  • Alzheimer’s disease: We are interested in structurally characterizing and disrupting the accumulations of Aβ that are associated with Alzheimer’s disease.
  • Bacterial sensing: We are interested in the mechanisms by which bacteria sense their environment and adapt to different environmental stresses.
  • Computational tools: We have taken a nature-inspired approach to protein design.

Protein design

Protein Design

Proteins catalyze countless vital physicochemical reactions. Proteins do this by coordinating the substrates in specific, three-dimensional orientations, so understanding the structure is essential to understanding the function, disease etiology and drug design. Our lab uses a de novo design approach to explore the principals that govern folding, protein-cofactor, and protein-protein interaction, as well as subsequent functions, for both water- and lipid-soluble proteins. We then use various biophysical methods to define the success of our designs. Please, check out our de novo metalloprotein, DF2, featured as the Molecule of the Month in the PDB.

Integrin inhibitors

Integrin inhibitors Figure

Integrins are heterodimeric transmembrane proteins that play a pivotal role in the signaling pathways that regulate processes as diverse as cell proliferation, differentiation, apoptosis, and cell migration.In collaboration with Joel Bennett (UPenn), we study the mechanism of signal transduction of integrins such as integrin αIIbβ3, with a particular focus on the role played by the membrane-spanning regions of this protein. We have also developed small molecule inhibitors of integrin α2β1 in the platelet collagen receptor and now are expanding our small molecule inhibitor research to integrin αvβ1 in collaboration with Dean Sheppard (UCSF).

Pulmonary fibrosis is a currently untreatable condition with a high mortality rate. One central common step in the development and progression of pulmonary fibrosis is the differentiation and expansion of pathologic fibroblasts that are largely responsible for the excess production of collagen and other extracellular matrix components that characterize tissue fibrosis. Transforming growth factor β (TGFβ plays an important role in fibroblast differentiation and expansion and is widely recognized as a key factor in driving pulmonary fibrosis. However, TGFβ is not itself the most attractive drug target, because of the potential toxicity of inhibiting many of the other important roles this growth factor plays in normal tissue homeostasis.

A subset of integrins are important activators of TGFβ. TGFβ is secreted and stored in latent form and needs to be activated extracellularly to bind to its receptors and induce pathologic and homeostatic effects. Because of the diversity of activation mechanisms, targeting specific pathways of TGFβ activation is a strategy to selectively treat TGFβ-mediated pathology without the broad risks of indiscriminately blocking all TGFβ effects. Working with Dean Sheppard (Chief of the Division of Pulmonary, Critical Care, Allergy and Sleep in the Department of Medicine, UCSF) we have designed small molecules that inhibit the integrin avb1, and used these to demonstrate that this integrin is the primary responsible for activation of TGFβ on pulmonary fibroblasts. This molecule is also highly active in mouse models for lung, kidney and liver fibrosis. Current efforts focus on the design of dual-specificity integrin antagonists to inhibit TGFβ activation on multiple cell types, as well as the development of tools to guide clinical studies of integrin antagonists as antifibrotic agents.

Antimicrobials

Antimicrobials

The rise of multidrug resistance is an alarming health concern in both nosocomial and community-acquired bacterial infections. We have designed small molecule mimics of antimicrobial peptides (evolutionarily conserved components of innate immunity in higher organisms), which display potent and selective activity against a broad spectrum of pathogens. The lead compound is currently in phase II clinical trials against multi-drug resistant Staphylococcal infections. We study the mechanism of action of these mimetics and the bacterial response to the presence of sub-inhibitory concentrations of these agents. We are also using these compounds as a tool for studying bacterial signaling systems involved in drug resistance and virulence.

Influenza

Influenza Figure

We are interested in the M2 protein of influenza because it is both a drug target and a model system for proton transport. M2 is a proton channel that is the target of a class of drugs called the adamantanes, though viral mutations have caused adamantane-resistant strains of influenza to become prevalent. M2 is also one of the smallest proton channels found in nature, since it is a homotetramer, with a minimally functional monomer length of only 25 amino acids. We perform structural studies on this system using NMR and crystallography, and we also create new M2 inhibitors targeting M2 then test their effectiveness.

HIV

HIV Figure

A vaccine candidate that elicits broadly neutralizing monoclonal antibodies that confer immunity to HIV is yet to be discovered, but increasing data suggest that developing an effective vaccination is possible. In particular, the membrane proximal external region (MPER) of HIV-1’s gp41 envelope protein is a promising target. The MPER is a highly conserved, dynamic region of gp41, exposed during the transient, pre-fusion state while the virus is binding to host T-cells and initiating fusion. A successful anti-MPER vaccine will elicit antibodies that recognize the MPER in this pre-fusion state, halt fusion, and render the virus incapable of reinitiating fusion. Using existing structural information on the HIV MPER and related viral proteins, we are designing vaccine constructs and working with collaborators to test their immunogenicity in rabbits. These design efforts would greatly benefit from characterization of the pre-fusion structure of the MPER, which is another goal of this project.

Alzheimer’s disease

Alzheimer's Disease Figure

Alzheimer’s disease is a devastating neurodegenerative disease that cruelly strips patients of memory, cognitive ability, and independence. Within the brains of patients, the Aβ peptide accumulates into fibrillar plaques that disrupt neuronal networks and soluble oligomeric complexes that are highly cytotoxic. However, relatively little is known about the protein conformations adopted by Aβ during disease pathogenesis. We are using biophysical techniques such as NMR to characterize protein conformation of oligomers and to identify structural polymorphisms in fibrils, as well as protein design to create novel disruption strategies that prevent these dangerous peptide accumulations.

Bacterial sensing

Bacterial signaling Figure

The two-component system is an essential stimulus-response mechanism in bacteria. It consists of a transmembrane histidine kinase that senses the environment and a response regulator that mediates the cellular response. We study the protein structures and conformational dynamics associated with signal transduction via two-component systems.

Our methodology includes in-cell studies on bacteria, biochemical assays on purified proteins, structural and biophysical studies by x-ray crystallography, H/D exchange mass-spectrometry, NMR and EPR.

This project is synergistic with new antimicrobial development as many bacteria use two-component systems to develop antibiotic resistance.

Computational tools

Computational Tools

We use several computational approaches to help us design proteins with desired properties. By efficiently searching the Protein Data Bank for backbone or amino acid motifs of interest, we can identify interactions frequently occurring in nature which can then be used as foundations for our designs. Often we combine this nature-inspired approach with molecular modeling tools such as Rosetta and molecular dynamics simulations. Some applications we pursue with this approach are the design of peptides that can modulate protein-protein interactions, the design of transmembrane proteins with desired ion-transport properties, and the design of metal or small molecule binding sites.

For amino acid motifs, we use a tool recently developed in our lab, Suns, which has a built-in PyMol interface enabling the interactive building of motifs with the search results. For backbone motifs, we use MadCat, which efficiently compares the distance-map of a query motif to a database of pre-computed distance-maps for thousands of proteins from the PDB. Additionally, we have recently developed SuperCodons, a tool that allows the construction of a randomized DNA library that closely matches a chosen amino acid distribution. We have also developed the knowledge-based E(z)-3D Transmembrane Protein Orientation Potential, which predicts the most favorable orientation within a membrane for transmembrane proteins.