Dr Bernard Rawlings, University of Leicester

In vivo production of
analogues of amphotericin B
Dr Bernard J. Rawlings
Department of Chemistry
University of Leicester
In close collaboration with:
Dr Patrick Caffrey
School of Biomolecular and Biomedical Science
and Centre for Synthesis and Chemical Biology
University College, Dublin
BJR Oct 14
(Mycosamine)
A polyketide (polyene) produced by
the soil bacterium Streptomyces nodosus
Antifungal used in clinic against
systemic mycoses
for 60 years (NO resistance).
Now, also used against leishmaniasis
Mode of action: Binding to ergosterol
(with sterol can oligomerise to form transmembrane pores
causing uncontrolled release of ions and small molecules)
VERY EFFECTIVE ANTIBIOTIC
(does NOT need making more effective!)
Problem is toxicity (side effects):
Must be delivered intraveneously (small doses, in hospital, over 1 month)
Will bind to cholesterol in human membranes/blood cells causing anaemia
Low water/serum solubility
causes aggregation and precipitation in kidney (nephrotoxicity)
Liposomal formulation (Ambisome), less toxic
but very expensive (£1,000 per day, Fungizone £10/day)
Used extensively in NHS, but on ‘pharmacoeconomic’ basis
In recent years,
frontline treatment for visceral leishmaniasis
500,000 new cases of VL each year (2004)
50,000 deaths per year
Used in Bihar (India) and Nepal replacing antimonials
These are some of the poorest parts of the world.
WHO subsidises use of ‘Ambisome’
(US$3,000 per treatment reduced to US$200)
but still far too expensive
Courtesy of P. Bates
Univ Lancaster
Visceral
leishmaniasis
Vector is the sand fly:
- a silent killer!
Mucocutaneous
leishmaniasis
Chemical semisynthesis has provided improvements
but far too expensive for clinical use
AIM OF OUR (PC + BJR) PROGRAMME:
THROUGH HARNESSING THE BIOSYNTHETIC
POWER OF S. NODOSUS
PRODUCE COMMERCIALLY VIABLE
ANALOGUES OF AMPHOTERICIN B
THAT RETAIN THERAPEUTIC POWER
but with reduced toxicity
Less toxicity, less side effects, higher but fewer doses
less time hospitalised.
Dr Patrick Caffrey (Dublin) has sequenced the
amphotericin B gene cluster (113 kb)
Currently performing gene
disruption/deletion/substitution experiments
Sends mutants to Leicester
We grow, isolate, purify and characterise
amphL
Less soluble
amphDIII
No sugar
inactive
PKS product
less soluble
‘KR16 mutant’
More soluble in water
equipotent with apph B
Then add a glycosyltransferase…
but currently low yields
Use hybrid glycosyltransferase to replace
mycosamine with persosamine in amphotericin B
regioisomer
(4 mgs isolated)
19-(O)-Perosaminyl-amphoteronolide B
Most analogues produced in low quantities
VERY TEDIOUS LABOUR INTENSIVE TO OBTAIN mgs
‘amphNM’ mutant
*
Relatively good titre
Relatively soluble, and easy to purify
(from membrane lipids, saccharides and other polyenes)
Also investigating use of functionalised polymers
Several growths, 100 mg ‘purified’ (Simon Walmsley)
Chemical semisynthesis (Burke, USA), 11 steps 1.35%, gave 9 mg.
(disk diffusion with yeast or Candida, equipotent to Amph B)
Simon Walmsley
Dr Patrick Caffrey and Group
University College, Dublin
Acknowledgements:
Dr Charles Borissow (Oct 06 to Feb07)
Dr Barry Murphy (Apr 07 to Oct 2009)
Bunmi Ibrahim (PhD)
Simon Walmsley (PhD current)
Resul Haser (PhD current)
Mick Lee (Technical)
Dr Gerry Griffiths (NMR)
Dr Graham Eaton (MS)
MChem, MSc and summer students:
Jessica Hearn (MChem)
Natalie Lambourn (MChem)
Emma J. Gibson (MChem)
Katherine (Katie) Anderson (Summer)
Katherine (Kat) Pugh (Summer)
Daniella Pariza (MSc)
Laskarini Tsoulkani (MSc)
Xi Chen (MSc)
Peter Kanengoni (MChem)
Kah-Mun Ng (MChem)
Vincent Leung (MChem)
Todd Cowan (MSc)
Zaeem Omar (MChem, current)
Financial assistance:
BBSRC

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