Tuberculosis (TB) is a commonly fatal bacterial infection caused by fungal bacteria, usually Mycobacterium tuberculosis. Humans contract TB mainly thorough airborne transmission when TB infected persons cough, sneeze, or otherwise expectorate. The infection is usually in the lungs, but is sometimes found in other organs, e.g., eyes. Once in the lung, the bacteria can enter the blood stream, eventually even the lymphatic system, the kidneys and the brain. TB infection can be treated effectively with antibiotics, but with great difficulty. Mycobacterium tuberculosis is one of the most recalcitrant bacteria towards many drugs.
Drug treatment of any infection is about exploiting a feature in the life-cycle of the infecting agent that is hopefully differentiated from the host. In the case of TB one attractive feature is the bacteria’s need for the chemical, mycothiol (MSH). All organisms need some manner of protection from toxins and the damaging effects of reactive oxygen species that are toxic by-products of aerobic life. While many species use glutathione for this purpose, pathogenic mycobacteria use MSH. MSH scavenges compounds that would be fatal to the bacteria, e.g., formaldehyde, various electrophiles including reactive oxygen, and antibiotics. Without MSH producing enzymes Mycobacterium tuberculosis could not survive, so inhibitors of these enzymes are very attractive drug targets. MSH is not used in normal human physiology, thus there are no human enzymes that synthesize it. So presumably a substance that inhibits MSH production in the bacteria would have little to no adverse interactions with normal human enzymes.
Historically drug discovery has relied on trial-and-error testing of compounds on cell cultures, organ models or whole test animals; and matching the apparent effects to treatments. Rational drug design, on the other hand, starts with some developed knowledge about the physics and chemistry of some cell or molecule involved in the pathology. In many cases this means use of three-dimensional structural information obtained from such techniques as X-ray crystallography and NMR spectroscopy, two very common physics-based methods.
An enzyme is just a large molecular scaffold that brings chemical groups together to affect some chemical transformation. Knowing the three-dimensional structure of an enzyme, be it from x-ray crystallography or NMR spectroscopy, is very important in understanding its chemistry at the atomic and electronic level, as you can see what functional chemical groups are brought together to conduct the chemical transformations that explain the products seen in bulk assays. Knowledge of the geometries (energy minimal configurations), flexibilities (force constants), and dynamics of the protein backbone, its side chains, the reactants, products, cofactors, and other small molecules that may be around, e.g., water, all provide constraints on the enzyme’s exact chemical mechanism and provide insight into the rational design of inhibitors (potential drugs).
In their current work, Broadley, et. al., were able to show a new crystal form of a key MSH synthesizing enzyme from Mycobacterium tuberculosis. Their new crystal structure shows the enzyme with the normal reaction product, acetate, coordinated to the metal-ion co-factor in the active site. Furthermore, in this new structure it is seen how key amino acid side chains make critical conformational changes to physically accommodate the enzyme substrate (chemical reactant), and also to participate in the enzyme’s molecular chemistry.
In particular they show how Tyr142 (with its phenol chemical group) can rotate around its C-alpha—C-beta bond to stabilize the acetate product in the pocket. This suggests that there is rotation between the conformation they found and the conformation seen in other structures of the same enzyme. Furthermore this rotation plays a role in the ultimate product release from the enzyme. That is, this side chain initially holds the product in place as it is formed, then swings “open” to release it from the enzyme. Moreover, when holding the substrate in place in the “closed” conformation, it actually helps lower the activation energy of chemical reaction. In enzyme chemistry, structural movements on the order of 1/10th of a nanometer (1 angstrom) or even less can play a significant role in chemical mechanisms. The new structure found in this work shows how nano-scale protein movements can lead to a new mechanistic construct that is different from ones previously suggested for this enzyme.
Finally, biophysical findings such as these can help guide trial and error drug discovery efforts. In this work Broadley, et. al. suggest that glycerol itself can be a competitive inhibitor to the enzyme. (Their crystal structure has it in the enzyme’s active site.) And they also have models of substrates in the active site. One can easily think of synthesizing series of analogues to glycerol and the natural substrate and doing quick assays to see if they prevent MSH synthesis. The follow-on work intended by this research team includes elucidating more protein crystal structures with substrates bound in the active site. This can provide tighter and tighter constraints for trial and error drug development efforts.