Transcript sleeping sickness

Phosphodiesterase as drug
targets in kinetoplastid parasites
Maximilian Höselbarth
3.5.2016
Kinetoplastid parasites
• Uniflagellated protists, belonging to phylum
Euglenozoa
Fig. 1: Cartoon image of T. brucei (left) and light microscope image of live Trypanosome (right)
• Characteristic feature is the presence of
(mitochondrial) DNA rich granule called kinetoplast
• Species: Trypanosoma brucei & T. cruzi, Leishmania
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Kinetoplasts associated diseases (1)
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African Trypanosomiasis (Sleeping sickness)
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Species:
Vector:
Region:
Infections:
Symptoms:
Treatment:
T. brucei gambiense (98%) & T. b. rhodiense
Tsetse fly
Sub-Saharan Africa
~10,000 (2014) from 300,000 (1998)
Chancre, flu like symptoms, later neurological symptoms and death
2nd stage treatment highly complex with arsenic Melarsoprol (1950) &
combination medication
American Trypanosomiasis (Chagas disease)
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Species:
Vector:
Region:
Infections:
Symptoms:
– Treatment:
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T. cruzi
Triatomine bugs
South America & increasing worldwide cases
6-7 million (2015)
Acute phase: Fever, headaches, skin lesions, muscle pain among others
Chronic phase: Cardiac and digestive disorders, neurological symptoms, death
Benznidazole & Nifurtimox, efficiency decreases with delayed onset of therapy,
increasing parasitic refractoriness is observed
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Kinetoplasts associated diseases (2)
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Leishmaniasis
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Species:
Vector:
Region:
Infections:
Symptoms:
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Treatment:
Various (20+) Leishmania species
Sandflies
Worldwide
~1,3 million (2015)
3 forms: Visceral- (South Asia),
Cutaneous- (Middle East, South America)
& mucocutaneous Leishmaniasis (America)
Treatable and curable disease, mainly
caused by poor socioeconomic
conditions and malnutrition
Fig. 1: Geographical distribution of Leishmaniasis
Urgent need for novel, safe & well-tolerated and easily
applicable drugs due to increasing drug refractoriness,
limited & partly toxic drug armamentarium and no new
drug releases for decades
Besides drug development vector control remains one of the
most important methods to control disease distribution
Fig. 2: Cutaneous Leishmaniasis in patient
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Phosphodiesterase
• Enzymes, which cleave Phosphodiester bonds by
hydrolosis
Fig. 1: Cyclic Adenosinmonophosphate (cAMP)
• Many different types and families, most important
function is to catalyze reaction from cAMP to 5‘-AMP
– Antagonist of Adenylylcyclases
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PDE coupled cAMP signalling (1)
• cAMP is an important second messenger and
participates in many different signal
transduction cascades in the cell, including
regulation of glycogen, sugar and lipid
metabolism by activation of Proteinkinase A
• cAMP levels play also an important role in cell
proliferation/ cytokinesis as well as in cancer
development
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PDE coupled cAMP signalling (2)
Fig. 1: GPCR dependent signalling cascade. Illustration from Nature Reviews
- Agonist binding triggers
conformational change in
GPCR (7TM receptor)
- Substitution of GDP with
GTP in Gα subunit of
heterotrimeric G-protein
- Dissoziation of Gα from
Gβγ subunits
- Different „activated“ Gα
subunits trigger different
downstream responses
- Specific Gα subunit activates Adenylylcyclase, which catalyzes rection of ATP to cAMP
- High intracellular cAMP concentrations activate Proteinkinase A, which triggers
further downstream responses
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PDE families and inhibitors
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There are multiple families of essential
PDEs existing in all Trypanosoma species
Catalytic domains of parasitic PDEs are
highly conserved with human homologs,
making it possible to exploit vast
knowledge about human PDEs on
parasitic ones
Multiple human PDE inhibitors are on the
market:
– PDE4 inhibitor Roflumilast for COPD
– PDE5 inhibitor Sildenafil known as Viagra
and Tadafil & Vadenafil for erectile
dysfunction
– PDE3 inhibitor Cilostazol for intermittent
claudication (muscle pain in calve muscle)
Fig. 1: Differentiation of different famillies of
kinetoplastid PDEs
Fig. 2: (left) Sildenafil containing Vigra tablet of Pfiizer,
(right) package of Roflumilast from Nycoped pharma
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Trypanosomal PDEs
• Catalytic domains of PDEs are
highly conserved between human
and trypanosomes
• Despite high structural similarity the
clinically used drugs are highly
family sensitive
– Demonstrates that medicinal
chemistry was able to produce
inhibitors that are highly selective
for the respective PDE, with only
minimal effects on other closely
related PDEs
• This allows exploitation of existing
vast knowledge in developing
human PDE inhibitors against
parasitic PDEs
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Fig. 1: (A) Catalytic
domain of LmjPDEB1
(B) Comparison of 3D
structure of
LmjPDEB1
(cyan) & human
PDE4D (green) with
IBMX.
Figure from Seebeck
et al 2011.
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TbrPDEB1 Inhibitor identification
• TbrPDEB1 & 2 are the predominant controlling elements of intracellular
cAMP levels
• Disruption of TbrPDEB by RNAi causes dramatic increases of intracellular
cAMP levels and induces complete trypanosome cell lysis
• Scanning through a proprietary >400,000 compound library identified Cpd
A as a potent inhibitor of both PDEs
– Cpd A causes a rapid and sustained elevation of intracellular cAMP
levels and consequently inhibits cytokinesis
– No cross resistance with existing drugs was observed
Fig. 1: Racemic structures of Cpd A
with corresponding IC50 values
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Compound A effects (1)
Effects of Cpd A on intracellular cAMP levels in Trypanosomes
Fig. 1: (A) A-Cpd A, B-100 µM Etazolate, C-40 µM Dipyridamole, D – DMSO (B) Time dependent cAMP increase in
trypanosomes incubated with 1 µM Cpd A (filled squares) and DMSO only (squares). (C) Cpd A concentration
dependency on intracellular cAMP levels after 3 h incubation, *= p value=.01, ***= p value= .001. Figure from de
Koning et al 2012.
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Compound A effects (2)
Cpd A dramatically contributes to trypanosomal cell lysis
Fig. 1: (A) Trypanosomal cell lysis after exposure to various concentrations of Cpd A. Trypanosome were
incubated in propidium iodine medium and fluoresence was measured at 620 nm. (B) Growth inhibition and cell
lysis induced by Cpd A. Starting density of trypanosomal cultures at 4 x 105 cells/ml, negative control in inset.
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Summary
• Kinetoplasts are responsible for African sleeping sickness,
Chagas disease and Leishmaniasis and livestock infections
• Anti-parasitic drugs are old, ineffective and toxic
• PDEs regulate intracellular cAMP levels => important
regulators of human and parasitic metabolism and
cytokinesis
• Catalytic domains of PDE are highly conserved between
human and trypanosomes, making it possible to exploit
vast expertise
• Multiple human PDE inhibitors are on the market
• Cpd A was identified as a potent inhibitor of TbrPDEB and
causes trypanosomal cell lysis
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Thank you for your attention
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