Elsevier

Drug Discovery Today

Volume 22, Issue 7, July 2017, Pages 1008-1016
Drug Discovery Today

Review
Keynote
Targeting isocitrate lyase for the treatment of latent tuberculosis

https://doi.org/10.1016/j.drudis.2017.04.012Get rights and content

Highlights

  • Isocitrate lyase (ICL) is essential to growth and survival of Mycobacterium tuberculosis (Mtb).

  • ICL is involved in Mtb glyoxylate and methylisocitrate cycles, and may play a part in Mtb antibiotic resistance development.

  • The roles of the two Mtb ICL isoforms are not fully understood.

  • Reported ICL inhibitors not suitable as drug candidates; challenges include polarity, size of binding pocket and selectivity.

  • Further work is required to fully validate ICL as a therapeutic target.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis that can remain dormant for many years before becoming active. One way to control and eliminate TB is the identification and treatment of latent TB, preventing infected individuals from developing active TB and thus eliminating the subsequent spread of the disease. Isocitrate lyase (ICL) is involved in the mycobacterial glyoxylate and methylisocitrate cycles. ICL is important for the growth and survival of M. tuberculosis during latent infection. ICL is not present in humans and is therefore a potential therapeutic target for the development of anti-TB agents. Here, we explore the evidence linking ICL to persistent survival of M. tuberculosis. The structure, mechanism and inhibition of the enzyme is also discussed.

Introduction

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis 1, 2. M. tuberculosis can live inside the human body for years without causing disease – resulting in a syndrome that is known as latent TB. TB has a latency period that is longer than any other infectious disease 3, 4, 5. It is estimated that at least a quarter of the world’s population is infected by the bacteria [6], of which around 5–15% will develop active TB in their lifetime, a probability that increases dramatically if the infected individual becomes immunocompromised.

The World Health Organisation End TB Strategy aims to reduce the mortality rate by 90% and the incidence rate by 80% by 2030 [7]. Treatment of latent TB infection, especially for people from high-risk groups such as those who are infected by HIV, is a viable strategy to control the disease because M. tuberculosis can only spread from people who have developed active pulmonary TB 8, 9, 10. Current medication regimens used to treat latent TB infection require high patient compliance, which typically involves regular (sometimes daily) intake of antimicrobial drugs for up to 9 months 11, 12. In addition, these drugs can induce severe hepatotoxicity and other unpleasant side effects [13]. The development of more-effective and less toxic drugs to treat latent TB infection is therefore required if the goals set out by the WHO are to be met.

Section snippets

ICL in M. tuberculosis

Isocitrate lyase (ICL) is an Mg2+-dependent enzyme that catalyses the reversible lysis of a Csingle bondC bond of d-isocitrate to form glyoxylate and succinate (Fig. 1a) [14]. ICL is present in bacteria (including mycobacteria), fungi and plants, but not in humans or animals [15]. In M. tuberculosis there are two known isoforms of ICL: ICL1 and ICL2, which are encoded by the genes icl1 and aceA (also known as icl2), respectively 16, 17. Exceptions can be found in some mycobacterial species including M.

ICL and the glyoxylate cycle

One of the most well-known roles of ICL is its involvement as the first enzyme in the glyoxylate cycle, which was first identified from cell-free extracts of Pseudomonas aeruginosa in 1953 [24]. The glyoxylate cycle is an alternative pathway to the tricarboxylic acid (TCA) cycle (Fig. 2) 25, 26. The early steps of the glyoxylate cycle resemble those in the TCA cycle, in which acetyl-coenzyme A (CoA) is converted into d-isocitrate via citrate and cis-aconitate. The major point of difference

ICL and the methylcitrate cycle

During infection, the pools of carbon that M. tuberculosis can utilise include fatty acids from the host as well as the bacteria’s internal lipid reserves [39]. Although natural animal fatty acids are composed of an even number of carbons, bacteria including mycobacteria possess the ability to synthesise odd-chain fatty acids [40]. However, β-oxidation of odd-chain fatty acids is potentially harmful to the bacteria, because the process generates propionyl CoA and propionate 41, 42, 43, both of

ICL and its potential role in antibiotic tolerance of M. tuberculosis

Recently, ICL was linked to the development of antibiotic resistance in M. tuberculosis. By using metabolomics and gene expression analyses it was demonstrated that ICL was activated when M. tuberculosis was subjected to sublethal doses of three different anti-TB drugs: rifampicin, streptomycin or isoniazid [50]. M. tuberculosis with an icl1 deletion showed a 100-fold increase in sensitivity to these antibiotics [50]. Furthermore, studies that used a M. tuberculosis mutant complemented with a

Structure and catalytic mechanism of ICL

The exact mechanism by which ICL converts isocitrate into glyoxylate and succinate is not fully understood, but a retro Claisen-type condensation pathway has been inferred (Fig. 1b) [52]. The first step involves deprotonation of the isocitrate hydroxyl group followed by fragmentation of the isocitrate to form glyoxylate and succinate 53, 54. Mutagenesis and bioinformatics studies showed that the highly conserved KKCGH sequence motif at the enzyme active site (residues 189–193 in ICL1, also

Inhibitors of ICL

Given the central role ICL has in the glyoxylate and methylisocitrate cycles, ICL is a current inhibition target for antimicrobial applications including (but not limited to) latent TB. However, despite considerable efforts by academia and industry, no compounds have progressed through to the clinical trial stage. There are three major challenges in targeting ICLs: (i) the polar nature of the ICL binding pocket; (ii) the small size of the natural substrates; and (iii) the need to target ICL1

Concluding remarks

ICL is an attractive inhibition target for the treatment of latent TB because it is vital for bacterial survival and, in addition, humans do not possess this enzyme. Existing ICL inhibitors are mimics of the native substrate (or products) that keeps the enzyme in the closed confirmation, as observed in the inhibitor-bound structures of ICL1. The inherent toxicity of these inhibitors is probably caused by their binding to other human enzymes, for example those that take isocitrate or succinate

Acknowledgement

We thank the University of Auckland for a Doctoral Scholarship (R.P.B) and the Maurice and Phyllis Paykel Trust for funding.

Ram Bhusal is carrying out PhD research under the supervision of Dr Ivanhoe Leung and A/Prof. Jonathan Sperry at the University of Auckland. His PhD is focused on the structural, mechanistic and inhibition studies of Mycobacterium tuberculosis isocitrate lyase. Before joining the University of Auckland, he obtained his undergraduate degree in Pharmacy from Pokhara University, Nepal, and his MSc in Medicinal Chemistry from Wonkwang University, South Korea. He also has experience working as a

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    Ram Bhusal is carrying out PhD research under the supervision of Dr Ivanhoe Leung and A/Prof. Jonathan Sperry at the University of Auckland. His PhD is focused on the structural, mechanistic and inhibition studies of Mycobacterium tuberculosis isocitrate lyase. Before joining the University of Auckland, he obtained his undergraduate degree in Pharmacy from Pokhara University, Nepal, and his MSc in Medicinal Chemistry from Wonkwang University, South Korea. He also has experience working as a lecturer and pharmacist in his home country, Nepal. His long-term interest is in teaching and research.

    Ghader Bashiri received his PhD in structural biology at the University of Auckland in 2009. He is currently a Sir Charles Hercus Fellow, awarded through the Health Research Council of New Zealand. His research investigates proteins of biomedical significance, with a focus on bacterial biochemistry and physiology. As part of the Maurice Wilkins Centre, a national Centre of Research Excellence, he utilises a structure-based drug discovery approach with the ultimate goal of developing a new generation of anti-TB agents.

    Jonathan Sperry obtained his BSc (Hons) in biological and medicinal chemistry in 2002 from the University of Exeter, UK. He conducted his PhD under the supervision of Professor Chris Moody at the same institution, before moving to New Zealand where he spent 3.5 years as a postdoctoral researcher with Distinguished Professor Margaret Brimble at the University of Auckland. He took up a lectureship at the same institution in 2009, where he is currently an Associate Professor and a Royal Society of New Zealand Rutherford Discovery Fellow.

    Ivanhoe Leung attained his MChem in Chemistry in 2007 from the University of Oxford, UK, as a member of St Peter’s College. He completed a DPhil in the laboratories of Profs Christopher J. Schofield FRS and Timothy D.W. Claridge at the same institution. After his DPhil he spent a further 2 years in the same groups as a postdoctoral research assistant. He joined the School of Chemical Sciences at the University of Auckland in September 2014, where he is currently a senior lecturer. His research focuses on the application of biophysical techniques to a variety of problems in chemistry and biology.

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