ATP synthase: a new drug target for tuberculosis

The London Structural Biology Club (LSBC) is a network for students and researchers working in all aspects of structural molecular biology and based in London and the south-east of England. Once a term, members get together for an afternoon of research talks and discussion followed by refreshments (generally featuring pizza and beer). These meetings are often held at Birkbeck, and we have featured them on the PPS blog before (see this post from 2008 and this one from 2012).

The LSBC meeting for the summer term of 2016 was also held at Birkbeck, and hosted and chaired by Alfonso de Simone, a lecturer in NMR spectroscopy at Imperial College London. One of the four talks was given by Thomas Meier, also from Imperial College. Meier, who has worked at ETH Zurich, Switzerland, and the Max Planck Institute of Biophysics in Frankfurt, Germany, was appointed to a chair of structural biology at Imperial just over a year ago. His research concerns the structure and function of a tiny, complex ‘molecular machine’, ATP synthase (the link is to our page on that enzyme in section 10 of the PPS course). This enzyme is a ‘rotary ATP synthase’, catalysing the conversion of the electrochemical energy of ion transfer across the cell membrane into chemical energy stored in ATP. Meier and his group have solved the complete structure of this multi-subunit enzyme complex using a combination of X-ray crystallography and electron microscopy, and shed light on its role as a target for a novel class of drugs against tuberculosis.

ATP synthase is a highly dynamic enzyme complex, which makes it particularly difficult to study structurally. The complete enzyme comprises two motor sub-complexes; most of one, termed F_o, is embedded in the membrane and the other, F₁, is in the matrix. They are tightly coupled with each other and linked by subunits forming an outer stalk and an inner, central stalk. From a functional point of view, the ATP synthase consists of a rotor and stator part, both sharing subunits from the F₁ and the F_o sectors. The motors are driven either by the proton (or sometimes Na⁺) motive force to form ATP, or in opposite direction by the hydrolysis of ATP to pump ions across the membrane. Similar enzymes are found in the inner membranes of bacteria and mitochondria and in the thylakoid membranes of chloroplasts.

John Walker of the MRC Laboratory of Molecular Biology in Cambridge was awarded a share of the 1997 Nobel Prize in Chemistry for determining the structure of the bovine F1 motor using X-ray crystallography (e.g. PDB 1E79). This subcomplex consists of five different types of subunits; three pairs of similar alpha and beta subunits are arranged alternately around the rotor (gamma / delta / epsilon subunits), which harbours an asymmetric coiled coil domain in the gamma subunit. The nucleotides ADP and ATP can bind to the interfaces between the beta and alpha subunits; the central gamma subunit rotates in 120^o steps, causing conformational changes that in turn change the affinity of the three catalytic sites for the nucleotides. Rotation in one direction, driven by the energy of ion transfer across the membrane, leads to synthesis of ATP from ADP and phosphorus (P_i); rotation in the other direction will hydrolyse ATP back into ADP and P_i, thus releasing the energy required to pump ions.

The membrane-embedded F_o motor consists of the rotor part, a ring of identical c-subunits termed the c-ring and the stator part, a single-chain a-subunit that grants access and release pathways for ions. Each c-ring subunit is a helical hairpin with its N and C termini on the cytoplasmic side; the number of subunits is constant within a species but varies between species, as far as we know today, between 8 and 15 c-subunits. Meier’s group has solved the structures of a number of c-rings from different species of bacteria, helping to elucidate the rotor’s mechanism of action; essentially, protons (or in some cases Na⁺ ions) can reach one of the c-ring subunits by an ion pathway mediated by the stator a-subunit, where they lock to a free ion binding site on the c-ring, rotate with the c-ring for almost a complete 360^o turn to reach the second release pathway that leads to the other side of the membrane. The ion translocation causes rotation of the F_o ring and with it the complete central stalk that protrudes the F₁ headpiece.

Artist's impression of an ATP synthase molecule embedded in a membrane. Image © Laura Preiss, Max Planck Institute of Biophysics, Frankfurt, Germany

Until recently, ATP synthase has not been thought of as a drug target, principally because the structures and mechanisms of the bacterial and human enzymes are so similar. Now, however, it has emerged that it is the target for bedaquiline, the first novel drug to be approved for treating tuberculosis (TB) for over 40 years. And new drugs for TB are needed very badly: in 2015, over 9 million people contracted this disease and about 1.5 million died from it. Tens of thousands of TB cases each year are multidrug resistant (MDR) or even of the extremely drug resistant (XDR) variant for which no other clinically approved antibiotic is available anymore. Bedaquiline, however, is effective against both, MDR- and XDR-TB strains.

Functional analysis has shown that bedaquiline, which is a diarylquinoline, acts by binding to and halting the rotation of the Fo rotor. Meier and his co-workers have now solved the structure of the drug bound to the c-ring from a similar, non-pathogenic bacterial species, Mycobacterium phlei. This structure has nine c-subunits and shares over 80% sequence identity with the M. tuberculosis c-subunit variant (100% match at and around the drug binding surface). The crystal structure (PDB 4V1F; Preiss et al. (2015)) shows the drug occupying the proton-binding site on each of the nine subunits and thus preventing proton transfer. This stalls the rotation of the F_o motor, preventing rotation and thus the synthesis of ATP in F₁. Small differences between the structures of the proton-binding sites account for the exquisite specificity of bedaquiline for the F_o rings of mycobacteria and thus for its efficacy and safety as an anti-tubercular drug.

Meier ended his talk by explaining that TB-causing bacteria will eventually – perhaps sooner rather than later – develop resistance to bedaquiline, just as they have to every previous drug that has entered the clinic. There is therefore a pressing need to develop further drugs that act at the same target, and his group’s structural studies are proving useful in the search for bedaquiline analogues.

The London Structural Biology Club has a public Facebook group, which can be found here.

Commemorating World TB Day: Drugs from Plants

This post was written first for the Birkbeck College events blog and is re-posted here with permission.

World TB Day is held on 24 March every year, to mark the day in 1882 when Robert Koch, one of the fathers of microbiology, first announced that he had discovered the cause of tuberculosis (TB) – the bacterium now known as Mycobacterium tuberculosis (link to the TB proteome page in PPS section 5). Over 125 years since its discovery, and despite billions of dollars of investment in drug discovery, this bacterium is still a killer. The World Health Organisation estimates that about two billion people are infected with latent tuberculosis; in 2010, the last year for which full figures are available, over eight million people became ill with active tuberculosis, and 1.4 million people died from the disease. Two factors help make TB particularly deadly: it often occurs in people infected with the HIV virus, where it is one of the major causes of death, and drug resistant forms are becoming more common. In January 2012, Nature reported the identification in India of so-called “totally drug resistant” (TDR) tuberculosis, resistant to all anti-TB drugs in general use.

 Image of Mycobacterium tuberculosis bacteria

Photo credit: Janice Carr, Centers for Disease Control and Prevention, USA

In 2012 at Birkbeck, World TB Day coincided with the start of the College’s annual Science Week. Dr Sanjib Bhakta, head of the Mycobacteria Research Laboratory in the Department of Biological Sciences, organised a well-attended symposium on tuberculosis and its treatment. Besides two scientific presentations, the symposium featured a short video, Tuberculosis: The Real Story, highlighting the views of people affected by TB in the UK, and a panel discussion led by the grassroots volunteer organisation Results UK on some of the political challenges raised by tuberculosis.

Both science lectures focused on plants as a source of potential new drugs for tuberculosis. Professor Franz Bucar from the University of Graz in Austria highlighted the extreme chemical diversity of compounds that could be extracted from plants, particularly as compared to those found in the average synthetic compound library. Plants have always existed alongside their own microbial pathogens and have evolved natural antibiotics to protect themselves. Our ancestors, before the dawn of scientific medicine, used plant extracts to treat infectious disease, often quite successfully. The sub-discipline of ethnomedicine involves “mining” these traditional or historical remedies for pure chemicals that can be developed as, or modified into, drugs.

Bucar described a European herb, elecampane or Inula helenium, which is known to have been used to treat lung disease in the sixteenth century. He explained how a complex mixture of natural products derived from this plant had been tested against mycobacteria. Compounds found to have anti-mycobacterial activity were extracted and purified. Other plants have also yielded useful lead compounds; extracts of bark from a small tree with the Latin name of Berchemia discolor have even been shown to inhibit multi-drug resistant strains of Mycobacterium tuberculosis at useful concentrations.

Discovering antibacterial products in plant extracts, however, is only a first step towards drug discovery. Even when natural products like these compounds are found to be selective for bacterial over human cells, it is necessary to discover their mechanism of action; to modify them to optimize their activity; and, since plant sources are often scarce and extraction processes costly, to determine methods of synthesizing them in the laboratory.

The second scientific presentation was given by Dr. Bhakta himself and described current work in Birkbeck’s Mycobacteria Research Laboratory in searching for potential drugs for TB. These are needed not only to combat resistant forms of the bacteria but to improve current treatment regimens for “standard”, drug-sensitive TB. This requires a combination of four drugs to be taken for two months followed by two drugs for another four months, and many patients, particularly poorer and less well educated ones, fail to complete such a long and complex regimen. This in turn can lead to the development of further resistant strains.

Ideally, new drugs are required that target proteins not targeted by existing drugs, as resistance will be harder to develop. Mycobacteria have extremely complex cell walls, unlike those of other types of bacteria; they are essential for the bacteria to survive, and the enzymes used to synthesise them have no equivalents in mammalian genomes. These enzymes, therefore, have many of the characteristics of excellent drug targets.  Bhakta and his group have been exploring ways to inhibit the synthesis of the peptidoglycan that is one of the most important constituents of that cell wall. This molecule has been described as the bacterium’s “Achilles heel”, but no drugs targeting its synthesis have yet entered the clinic.

Mycobacteria synthesise peptidoglycan via a series of enzymes known as ligases, each of which adds a new link to the growing peptidoglycan chain. Bhaka’s group has focused on one of these ligases, termed MurE. This enzyme is essential for the bacterium to survive and it is conserved in all Mycobacterium tuberculosis strains. Working in collaboration with Professor Nick Keep, also in the Department of Biology, Bhakta solved the structure of MurE (PDB 2XTA) and showed it to have an active site that could in theory, at least, be occupied, and blocked, by a relatively small, “drug-like” molecule. He and his co-workers are now searching libraries of natural products for compounds that might inhibit this enzyme. They have identified promising MurE inhibitors from plants endemic to both Colombia and China, and are synthesizing analogues of these compounds for further testing.

It is unlikely that the next generation of anti-tuberculosis drugs will include any unchanged natural products. It is extremely likely, however, that natural products will yield the “scaffolds” on which these desperately needed drugs may be built, and perhaps one of these will be generated from within Bucar’s or Bhakta’s groups.