- Research Overview
- Chemical Biology and Medicinal Chemistry
- Chemical Biology and Medicinal Chemistry Overview
- Discovering Enzyme Substrates and Functions
- Discovering Protein Ligands to Probe and Alter Function
- Discovering Enzyme Activators
- Analyzing Mechanisms of Drug Resistance via Chemical Biology
- Analyzing Enzyme Conformational Dynamics, Substrate Binding, and Catalysis
- Effective Drug Targeting of Pathogens via Medicinal Chemistry
- Computational Chemistry and Biology
- Computational Chemistry and Biology Overview
- Modeling protein regulation via allostery and post-translational modifications
- Visualizing and integrating bioinformatics and biomolecular data
- Modeling membrane permeation to optimize pharmacokinetics
- Determining enzyme function by predicting substrate specificity
- Physical Biology
- Protein and Cellular Engineering
- Protein and Cellular Engineering Overview
- Monitoring enzyme activity and disease biomarkers
- Generating human proteome antibodies via phage display and directed evolution
- Globally analyzing and dissecting apoptosis
- Proximity tagging of protein-protein interactions
- Investigating cellular interactions in tissues
- Creating fluorescent probes targeting the genome and key bio-pathways
- De novo design of catalytic and membrane proteins
- Probing and modulating membrane proteins
Effective Drug Targeting of Pathogens via Medicinal Chemistry
Examples of our research and methods include
Fighting the flu: Developing inhibitors against drug-resistant viral proteins
Flu epidemics cause millions of cases of severe illness and between 250,000 and 500,000 deaths annually worldwide. In addition, pandemics and more virulent forms, such as “bird flu,” are cause for concern since vaccine development tends to lag behind the emergence of new subtypes. Two antiviral drugs used for decades to treat the flu (amantadine, rimantadine) are no longer effective due to drug-resistant mutations in the viral protein they target—the M2 proton channel.
Department researchers lead a multi-institution, interdisciplinary effort to develop new drugs to inhibit the mutated protein.
Despite its current drug resistance, M2 remains a prime target because it plays a crucial role in viral replication: its transmembrane domain (the drug target) forms a pore-like structure that facilitates the passage of protons through the viral envelope, yielding acidification that frees viral RNA to infect host cells and replicate.
But S31N, the predominant, drug-resistant M2 mutation, is especially challenging because the mutation reduces the size of an already small drug-binding site in the ion channel pore and changes the polarity of adjacent channel-lining residues.
To address this, department scientists have:
- Used nuclear magnetic resonance (NMR) spectroscopy to determine how amantadine binds with the non-mutant (wild type) M2 to understand its inhibitory mechanism and binding orientation.
- Developed new robust assays for M2 inhibition, suitable for high-throughput screening of large chemical libraries.
- Combined the above with other medicinal chemistry approaches to discover inhibitors of S31N mutants and iteratively improve the top binder’s affinity. This molecule serves as both a probe (locking the mutant protein into a bound conformation that allows NMR analysis) and as a lead for new drug development.
Tackling TB: Characterizing major pathogen enzymes as drug targets
Mycobacterium tuberculosis (MTB), the bacterial species that causes tuberculosis, latently infects a new person every second, leading to nine million new cases of active disease and about 1.5 million deaths each year worldwide. Recent decades have seen the development of multiple and extremely drug-resistant strains.
MTB invades and survives inside immune system macrophage cells in the lungs. Both tasks require scavenging cholesterol from host cells, then breaking it down using the carbon atoms for energy and to synthesize lipids used to maintain its robust cell walls.
Department researchers have identified several of MTB’s 20 cytochrome P450 (CYP) enzymes that initiate the breakdown of cholesterol. In fact, deleting the enzymes leads to the toxic accumulation of a substrate (cholest-4-en-3-one) that blocks the bacteria’s growth.
Department scientists are identifying the sites and mechanisms of action of this intermediate substrate as potential drug targets. They are also developing inhibitors of the two main CYPs involved in the degradative oxidation of cholesterol side-chains for use in cell wall lipids.
To achieve this, they are:
- Reconstituting in vitro an MTB CYP found to be critical for infection (in mice) to study its role incorporating cholesterol side-chain carbon atoms into the bacteria’s cellular lipids.
- Synthesizing and evaluating a series of substrate analogs—cholesterol side-chains—as mechanistic probes of important MTB CYP enzymes.
- Performing high-throughput screening of small molecules and subsequent analysis to determine key chemical structures affecting the ligand selectivity of MTB CYP130, a potential drug target.
Ultimately, scientists here seek to determine the structures, substrates, and biological roles of all 20 MTB CYPs, to provide new drug targets.
Halting herpes: Allosteric inhibition at a viral protein interface
Treatments for the eight human herpes viruses face both rising rates of resistance and dose-limiting toxicity. This includes the herpes virus that causes Karposi’s sarcoma (KSHV), the most common cancer in patients with AIDS. KSHV’s protease enzyme is vital to viral replication, however its active sites have proven extremely difficult to effectively target with inhibitors since they are extremely shallow.
But department researchers analyzing KSHV proteases found they only became active after a pair of inactive monomer precursors come together. The resulting complex, known as a dimer, plays a vital role in the virus’ replication by stabilizing the conformation of the enzyme's active sites.
The twin monomers are joined via an interface of alpha helical structures. Screening a library of alpha helix mimetics, scientists here identified a small molecule (dimer disrupter 2, or DD2) that, chemically refined, bound to a newly discovered allosteric site at the interface that blocks a key conformational change, so the two monomers cannot combine and activate.
All eight human herpes viruses, including those that cause mononucleosis, shingles, genital herpes, and cytomegalovirus (a serious health threat to infants and immuno-compromised patients), employ dimeric proteases that appear to be vulnerable to similar allosteric inhibition.
Further development of DD2, modifying the molecule to make it more potent (effective at lower doses) and better able to cross cell membranes, is proceeding in combination with high-throughput screening of tens of thousands of compounds and fragments to discover, analyze, and optimize other inhibitor leads.
Inhibition at protein-protein interfaces, while difficult, is a promising route for tackling drug resistance. The amino acids that drugs would bind to at a dimer interface are less likely to mutate—perhaps because any mutation could result in loss of dimerization and therefore loss of activity.
Developing parasite metabolite-targeted drug delivery
Malaria is a life-threatening parasitic disease that afflicts about 200 million people each year and causes roughly one death every minute worldwide, disproportionately affecting African children.
Seeking more effective treatments, including delivering new classes of highly potent drugs selectively to the parasite, department researchers are developing a new approach for antimalarial therapy.
The new approach takes advantage of two factors:
- In malaria, parasites invade red blood cells and digest the hemoglobin protein, leaving behind concentrations of toxic, free heme in their digestive compartments. (Heme is a reactive iron-containing molecule rarely found in its free, unbound state in healthy cells.)
- The current anti-malarial drug artemisinin, and also newer synthetic analogs in late-stage clinical trials as of 2013, are thought to exert their anti-malarial effects via initial reaction with this free heme.
Department scientists have designed molecules in which an initial heme-promoted reaction produces an anti-malarial effect while simultaneously severing a chemical linkage that releases and activates a second drug inside the parasite.
Studies have now successfully demonstrated this approach in mice. Targeted drug delivery cured the parasitic infection and also reduced the overall exposure to the partner drug, notably reducing side effects and improving safety. Ongoing work includes simplifying these molecules so they can be produced less expensively and exploring applications of this targeted drug delivery approach in other parasitic diseases and in cancer.