Rational Design, Synthesis and Biological Evaluation of Benzimidazoles; Towards a Novel Therapy Selectively Targeting C. neoformans beta-tubulin

The Need:

Cryptococcus neoformans (C. neoformans) is a yeast like fungus which manifests as meningitis and less frequently pneumonia within the human body. In sub-Saharan Africa C. neoformans causes more deaths annually than tuberculosis. C. neoformans is the most common cause of fungal infection in patients with AIDS and there are an estimated 1 million cases of cryptococcal meningitis worldwide each year. The current drugs used to treat this disease are a 2 week course of amphotericin B and 5-flucytosine followed by a long term course of fluconazole. Amphotericin B and 5-flucytosine are associated with toxicity and fluconazole suppresses the growth of the fungus but does not completely remove it from the body. There is a therefore a great need for new drugs to treat C. neoformans infections. These drugs must have less toxicity and work better against the disease. The aim of this proposal is to complete the early stage drug discovery activities required to ultimately develop new drugs for cryptococcal meningoencephalitis that can be used in sub-Saharan Africa.

The Concept:

It has previously been reported within scientific papers that a class of compound called the benzimidazoles (widely used to treat worm infections in humans and as fungicides in agriculture) have good activity against C. neoformans in a laboratory and this has subsequently been confirmed by work here at the University of Liverpool (UoL). Benzimidazoles have been shown to interfere with cell division in C. neoformans through binding to beta-tubulin. Beta-tubulin is a protein that is essential for cell division and growth, when benzimidazoles bind to beta-tubulin the fungal cell can no longer grow. More recently, unpublished studies carried out at UoL have shown flubendazole to be active against C. neoformans in the mouse model. Humans also have beta-tubulin so it is essential that any new drug developed against the beta-tubulin target is selective for C. neoformans beta-tubulin. Medicinal chemistry (modification of the chemical structure of a drug) can be used to improve both selectivity for beta-tubulin and activity against C. neoformans.

The Solution:

Identification of an active class of compound, against a known target, with established whole cell C. neoformans and animal model activity provides an excellent starting point for medicinal chemistry modification. To date benzimidazole compounds have never undergone medicinal chemistry optimisation to establish beta-tubulin selectivity or to improve C. neoformans activity. The two most commonly use benzimidazole drugs (Flubendazole and albendazole) are extensively metabolised within the body and have a limited ability to move from the stomach into the blood stream. New compounds will be designed using information from computer models to predict how they are likely to behave within the body. In order to improve the safety profile of the newly designed compounds their selectivity for C. neoformans beta-tubulin over human beta-tubulin will be measured in the laboratory (alongside activity against C. neoformans) and computer models of the binding sites of the two tubulins used to look at how new compounds are likely to bind better with C. neoformans beta-tubulin. Once C. neoformans activity, selectivity for beta-tubulin and improved levels of circulating drug have been established the compounds will move into a mouse model of C. neoformans infection.

Impact:

The long term aim is to develop a single therapy that does not require subsequent long term treatment with fluconazole and that can be given both orally and by injection. Given the number of patients affected by C. neoformans this project has the potential to have a dramatic beneficial impact on individuals and societies.

Development:

Compounds demonstrating activity in the mouse model will form the basis of further funding applications to progress the new drugs through the drug development pipeline.

The aim of this proposal is to carry out the early stage drug discovery activities required to ultimately develop new agents for cryptococcal meningoencephalitis for use in sub-Saharan Africa. The project addresses the need for novel therapies to treat the systemic fungal infection C. neoformans that causes lethal meningoencephalitis. In sub-Saharan Africa C. neoformans causes more deaths annually than tuberculosis and is the most common cause of systemic mycosis in patients with AIDS. An estimated 1 million cases of cryptococcal meningitis occur worldwide each year.

It has been reported that selected benzimidazole compounds have good in vitro potency against C. neoformans and this has been confirmed through our own in house MIC screening. Benzimidazoles have been shown to disrupt mitosis in C. neoformans through binding to the beta-tubulin subunit of microtubules. Subsequently the C. neoformans beta-tubulin genes have been characterised. More recently, unpublished studies have shown flubendazole to be active in vivo against C. neoformans in the mouse model.

Identification of an active class of compound, against a known target, with established in vitro and in vivo activity provides a solid platform for a targeted medicinal chemistry programme. The benzimidazole template has never been optimised for C. neoformans activity or target selectivity before. Flubendazole and albendazole suffer from extensive drug metabolism and have limiting pharmacokinetic (DMPK) properties. As such new analogues design will be guided by DMPK predictive algorithms, whole cell activity, selectivity for C. neoformans beta-tubulin over human beta-tubulin (measured in vitro and modelled) and measured in vitro DMPK. Compounds satisfying set criteria values will move into an in vivo PK/PD mouse models of C. neoformans infection. Compounds active in the mouse model demonstrating suitable PK will form the basis of funding applications for candidate selection enabling studies and beyond.

Patients (Worldwide, particularly sub-Saharan Africa):

Cryptococcus neoformans is a fungus that is a common cause of lethal meningoencephalitis:
- In sub-Saharan Africa C. neoformans causes more deaths annually than TB.
- C. neoformans is the most common cause of systemic mycosis in patients with AIDS.
- An estimated 1 million cases of cryptococcal meningoencephalitis occur worldwide each year.

First line treatment for C. neoformans is a 2 week course of amphotericin B and 5-flucytosine followed by a long term course of fluconazole. Significant limitations of current agents include:
- Amphotericin B deoxycholate is potent, but associated with anaemia, infusional toxicity and nephrotoxicity. Patients must be closely monitored - not possible in much of sub-Saharan Africa.
- 5- Flucytosine is associated with hepatic and renal toxicity, cannot be used as monotherapy and there are global supply issues.
- Fluconazole is fungistatic and emergence of drug resistance may compromise efficacy.

The development of new orally bioavailable anti-cryptococcal agents with reduced toxicity and improved efficacy is imperative. Development of an agent that results in rapid fungicidal activity in the CNS. Providing a monotherapy that does not require subsequent long term treatment with fluconazole and that can be administered both orally and intravenously (depending on the condition of the patient) would have a huge impact worldwide with the potential to greatly improve the lives of over 1 million patients annually.

Commercial/Private Sector:

A novel therapy for the treatment of C. neoformans is limited in its commercial profitability due to the regions in which the disease is endemic. However, it would provide private sector funding bodies (e.g. Bill & Melinda Gates Foundation) with a vested interest in improving the lives of patients with HIV/AIDS with another tool with which to make a substantial difference to patient quality of life and morbidity rates. Private sector donors include foundations (charitable and corporate philanthropic organizations), corporations, faith-based organizations, international NGOs, and individuals. It is estimated that philanthropies provided US$636 million in 2012 to HIV activities internationally (1).

Contribution to the Nation's Health/Wealth:

Although HIV is the largest driver of cryptococcal disease and as such sub-Saharan Africa has been the global region most heavily affected, it is important to acknowledge other factors. Prior to the HIV era in the UK, the incidence of cryptococcosis per 1,000 persons increased from 1.4 in 1963-1968 to 7.4 in 1973-1978. This was predominantly attributable to disease in patients on immunosuppressive medications. Use of potent immunosuppressants (eg, corticosteroids, calcineurin inhibitors, cytotoxic agents, and monoclonal antibodies) for transplant conditioning or treatment of cancer and inflammatory conditions has continued to expand in higher income countries over the last 30 years. As such new therapies against C. neoformans have the potential to treat a small but important minority of cryptococcal illness in settings such as the UK where HIV-related cases are in decline (2).

Staff working on the project:

The skills and contact network of the members of our research group, in particular, the project RAs will be strongly enhanced by close experiment/theory co-working in a cutting-edge translational science area.

(1) https://kaiserfamilyfoundation.files.wordpress.com/2014/07/7347-10-financing-the-response-to-hiv-in-low-and-middle-income-countries.pdf
(2) D. J. Sloan and V. Parris, Clin Epidemiol. 2014; 6: 169-182.