Analyzing Mechanisms of Drug Resistance via Chemical Biology

There are four research areas in the Department of Pharmaceutical Chemistry. Analyzing mechanisms of drug resistance via chemical biology is a research challenge within chemical biology and medicinal chemistry.

The challenge

Drug resistance occurs when medications exert evolutionary selection pressure on disease agents. Pathogens and cancer cells with metabolic defense mechanisms or mutations in drug-targeted proteins will survive treatments and replicate, until they become the dominant type. The rational design of novel and more effective therapies requires a detailed understanding of these resistance mechanisms.

Examples of our research and methods include

Battling tuberculosis: dormancy via sensor kinases in Mycobacterium tuberculosis

GAF sensory domain tuberculosis

The GAF sensory domain of a transmembrane kinase sensor in tuberculosis bacteria. The yellow/red ball-and-stick figure depicts the domain’s heme cofactor, which binds gases (O2, NO, CO), allowing the bacteria to detect immune system defenses and, if necessary, go into a dormant, drug-resistant state.

Mycobacterium tuberculosis (MTB), the bacterium that causes tuberculosis, latently infects up to a third of the world’s population, leading to nine million new cases of active disease and 1.5 million deaths each year.

A prime reason for the difficulty in treating and eradicating MTB is that it has a two-component regulatory system for detecting immune system response—specifically reacting to the emission of nitric oxide and carbon monoxide by macrophages and the oxygen deficiency (hypoxia) induced when immune cells seal off the invaders in granulomas. Under such conditions, MTB switches to a dormant metabolic state, which makes it far less susceptible to drugs.

Department scientists are characterizing the MTB signaling system in order to find inhibitors against the bacteria’s latent state. They reconstitute in vitro the two different transmembrane kinase sensors to study how they bind the different gases before signaling a second protein inside the bacterium, which activates genes that put it into hibernation.

Researchers generate mutant sensors to parse the roles of specific residues and also study differences in the activation and ligand preferences of the two sensors.

Such research helps determine the mechanism by which the identity of the gas is interpreted as an off- (O2) or on- (NO, CO) dormancy signal. This knowledge will guide the search for sensor inhibitors that short-circuit MTB’s hibernation system so that it remains in active form and can be more readily treated with antibiotics.

Combatting staph: Multi-drug resistant pathogens via rRNA methylation

Many antibiotics work by binding to a key location on the bacteria’s ribosome, preventing this cellular machinery from forming peptide bonds between amino acids to make proteins, thus killing the organism.

But pathogenic bacteria, including Staphylococcus aureus, have evolved or acquired a gene, cfr, which encodes an enzyme that can render them resistant to antibiotics, including last-line synthetic treatments such as linezolid.

The Cfr enzyme confers drug resistance by installing a methyl group at the ribosome location where the antibiotics normally bind and prevents them from doing so.

preventing antibiotics from binding

A methyl group (CH3) added by the Cfr enzyme to an amino acid (adenosine 2503) of ribosomal RNA (rRNA) at the peptidyl transfer center (PTC) prevents antibiotics such as linezolid from binding there to block bacterial protein production.

Department scientists have conducted the first biochemical studies of Cfr.

They found that this enzyme uses a unique mechanism to build the methyl group on ribosomal RNA (rRNA). This mechanism combines the use of two molecules of S-adenosyl methionine (SAM). One is used as a source of a methyl group that post-translationally modifies Cfr itself and is then partly transferred to the rRNA. A second SAM molecule is cleaved to generate a radical that activates the resulting methyl group for addition to rRNA.

Understanding of this unique mechanism may be used for development of inhibitors of Cfr that synergize with ribosome-targeting antibiotics in the treatment of bacterial infections.

Understanding bacterial mercury resistance via acquired operons

cysteinerich active site of organomercurial lyase binding mercury

Close-up of cysteine(Cys)-rich active site of organomercurial lyase (MerB) binding mercury (Hg).

Some bacteria have become resistant to mercurial antiseptics by acquiring groups of genes (operons) that express enzymes that sequester and neutralize the toxic metal ions.

These include mercuric ion reductase (MerA), which adds electrons (reduces) to counter the metal’s cationic properties so that it harmlessly diffuses out of cells, rather than binding to and interfering with crucial proteins. Some operons also generate organomercurial lyase (MerB), which adds protons to break the bonds between mercury cations and organic molecules.

To understand how this mercury detoxification pathway works, department researchers structurally analyzed key domains of MerA using nuclear magnetic resonance (NMR) spectroscopy. Such analysis included the mobile cysteine-bearing, tail-like structures that, along with other transporter molecules, help the enzyme gather mercury ions into its active site. (The sulfur in cysteine’s thol side chains helps bind mercury.)

In addition, mutants are expressed with key residue deletions and stop-flow measurement is used to capture their effect on catalytic rates. Findings included MerB’s use of a proton from aspartate to break cationic bonds.

Mercury is not the only metal ion, toxic (chromium) or vital (copper), that is protein-regulated in cells. Because some of the structural domains and interactive dynamics used by bacteria to eliminate mercury are similar to those other metallochaperones, fully mapping out one pathway may aid our understanding of many others.