DHFR- a target for TB drugs?

Structure of DHFR
01 April 2017

This month’s featured structure from our 2017 calendar is the enzyme dihydrofolate reductase (DHFR) from the bacterium that causes tuberculosis (TB), Mycobacterium tuberculosis. DHFR is an essential enzyme for life as it reduces dihydrofolate to tetrahydrofolate, a cofactor for several enzymes involved in synthesising amino acids and DNA bases. Humans also have this enzyme, which is targeted by the chemotherapy drug methotrexate, however the human and bacterial versions, while similar, do contain subtle differences.

This difference makes DHFR a key drug target for treatment of tuberculosis, along with a number of other diseases. There is large amount of research focussing on finding suitable drugs which will stop the TB DHFR doing its job while leaving the human DHFR alone. In fact, there are currently 15 structures of DHFR from M.tuberculosis in the PDB archive, many of which have small-molecule inhibitors bound. With the emergence of drug resistant bacterial strains, the task of combatting this disease is only growing more significant, particularly in the developing world.

An historic disease

Tuberculosis is one of the oldest known infectious diseases that is still common today. It has likely been around in some form since the advent of humanity, with cases similar to the symptoms of TB even observed in images on Assyrian clay tablets from as early as the 7th century BC. Some of the first documented studies of the disease were carried out by Hippocrates around the 5th century BC. Hippocrates postulated that TB was hereditary, but Aristotle, who also wrote about the disease, was one of the few minds at the time who correctly suggested it to be contagious. A combination of increasing population density and poor living conditions aided the spread of TB, with the number of cases peaking in the 19th century. The lack of understanding about suitable treatment did not help, particularly as it was thought to be non-contagious and so infected patients were not isolated from healthy individuals. In 1882 Robert Koch discovered the underlying bacterium, which he called the tubercle bacillus. This was a significant step in the understanding of TB, for which he won the Nobel Prize for Medicine in 1905. From around the time of this discovery, the rates of tuberculosis begun to decline as the improvement in sanitation and isolation of patients helped prevent further spread of the disease.

It was still a significant time before a successful treatment for TB was found. The Bacillus Calmette-Guerin (BCG) vaccine was created using an attenuated strain of a related organism Mycobacterium bovis, from cows. Though the first medical use of this vaccine was in 1921, it was much later before it was widely used due to dangers associated with the early vaccine. The first effective drug to treat TB was the antibiotic Streptomycin, discovered in 1953 by the group of Selman A. Waksman. Streptomycin, which binds to the ribosome and inhibits protein synthesis, is still used for treatment of TB today, though the emergence of drug resistant strains mean that it is often used in conjunction with a number of other antibiotics. With the continuing rise in drug resistant strains, there is a definite requirement for more drugs to be developed and structures of potential drug targets such as DHFR are vital in the search for new antibiotics.

A bacterial flak jacket

M.tuberculosis is a human pathogen, seemingly unable to live outside the host for long. Transmission of the disease is not by physical contact, instead requiring air droplets from the infected person (from coughing and sneezing etc.) to be inhaled into the lungs. Once within the lungs, the immune system of the host responds by producing macrophages that ‘eat’ the cells. However, the bacteria cannot be killed or digested by the defence cells of the host as the bacterium possesses a waxy cell coating, composed mainly of mycolic acid, which prevents fusion of these immune cells. Even more important for M.tuberculosis survival is that, although its waxy coat renders it impervious to the antibacterial action of the immune cells, it does not block the transfer vesicles that contain nutrients from which it benefits. For the host immune system it is a bit like the enemy jumping on your tanks and throwing a party.

Drug targets

There are nearly 2 million deaths per year worldwide attributed to TB. Though the advent of antibiotics had greatly reduced the number of cases by the 1960s, the appearance of drug resistant strains, along with the increasing population density within developing countries, means that Tb is on the rise once more. This has necessitated the combined use of multiple antibiotics in order to treat the infection, however multi-drug resistant strains have now also begun to appear. The waxy coat on the cell surface also plays a role in drug resistance. Containing both hydrophobic and hydrophilic components, this cell coat can effectively prevent many chemicals from permeating into the cell. In addition, any molecules that do get through may be degraded or pumped back out of the cell by antibiotic resistance proteins. New drugs are currently being developed in order to combat these mechanisms of resistance, with a lot of research directed into finding molecules that inhibit synthesis of the waxy coat. Though much work is being done to find new treatments, it is likely that the bacterium will evolve new mechanisms of resistance.

The artwork

The image from the calendar is of the structure of dihydrofolate reductase from M. tuberculosis, based on PDB entry 1dg7. The protein is displayed in a modern, pop art style, in contrast with the ancient history of this disease. The use of Ben-Day dots in the background is a parallel to the physiology of the lung interior where the disease propagates.