1. Phosphodiesterase as drug targets in kinetoplastid parasites
Maximilian Höselbarth

2. Kinetoplastid parasites
Uniflagellated protists, belonging to phylum Euglenozoa
Characteristic feature is the presence of (mitochondrial) DNA rich granule called kinetoplast
Species: Trypanosoma brucei & T. cruzi, Leishmania
Pictures from:
Fig. 1: Cartoon image of T. brucei (left) and light microscope image of live Trypanosome (right)
Maximilian Hoeselbarth

3. Kinetoplasts associated diseases (1)
African Trypanosomiasis (Sleeping sickness)
Species: T. brucei gambiense (98%) & T. b. rhodiense
Vector: Tsetse fly
Region: Sub-Saharan Africa
Infections: ~10,000 (2014) from 300,000 (1998)
Symptoms: Chancre, flu like symptoms, later neurological symptoms and death
Treatment: 2nd stage treatment highly complex with arsenic Melarsoprol (1950) & combination medication
American Trypanosomiasis (Chagas disease)
Species: T. cruzi
Vector: Triatomine bugs
Region: South America & increasing worldwide cases
Infections: 6-7 million (2015)
Symptoms: Acute phase: Fever, headaches, skin lesions, muscle pain among others
Chronic phase: Cardiac and digestive disorders, neurological symptoms, death
Treatment: Benznidazole & Nifurtimox, efficiency decreases with delayed onset of therapy, increasing parasitic refractoriness is observed
Maximilian Hoeselbarth

4. Kinetoplasts associated diseases (2)
Leishmaniasis
Species: Various (20+) Leishmania species
Vector: Sandflies
Region: Worldwide
Infections: ~1,3 million (2015)
Symptoms: 3 forms: Visceral- (South Asia),
Cutaneous- (Middle East, South America)
& mucocutaneous Leishmaniasis (America)
Treatment: Treatable and curable disease, mainly
caused by poor socioeconomic
conditions and malnutrition
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. 1: Geographical distribution of Leishmaniasis
Visceral Leishmaniasis:
- fever, weight loss, enlargement of the spleen and liver, and anaemia
- Fatal if untreated
Cutaneous L.:
- Most common form
- Skin lesions resulting in life long scars and disabilities
Mucocutaneous L.:
- partial or total destruction of mucous membranes of the nose, mouth and throat (Latin America only)
Picture from:
Fig. 2: Cutaneous Leishmaniasis in patient
Maximilian Hoeselbarth

5. Maximilian Hoeselbarth
Phosphodiesterase
Enzymes, which cleave Phosphodiester bonds by hydrolosis
Many different types and families, most important function is to catalyze reaction from cAMP to 5‘-AMP
Antagonist of Adenylylcyclases
Picture from:
Fig. 1: Cyclic Adenosinmonophosphate (cAMP)
Maximilian Hoeselbarth

6. 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
Maximilian Hoeselbarth

7. PDE coupled cAMP signalling (2)
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
Picture from Nature reviews cancer
Fig. 1: GPCR dependent signalling cascade. Illustration from Nature Reviews
Specific Gα subunit activates Adenylylcyclase, which catalyzes rection of ATP to cAMP
High intracellular cAMP concentrations activate Proteinkinase A, which triggers further downstream responses
Maximilian Hoeselbarth

8. PDE families and inhibitors
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
COPD = Chronic obstructive pulmonary disease
Fig. 2: (left) Sildenafil containing Vigra tablet of Pfiizer,
(right) package of Roflumilast from Nycoped pharma
Maximilian Hoeselbarth

9. Maximilian Hoeselbarth
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
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.
IBMX is a non-specific inhibotr of human PDEs
Maximilian Hoeselbarth

10. 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
Maximilian Hoeselbarth

11. Maximilian Hoeselbarth
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= Figure from de Koning et al 2012.
Maximilian Hoeselbarth

12. Maximilian Hoeselbarth
Compound A effects (2)
Cpd A dramatically contributes to trypanosomal cell lysis
Propidium Iodine intercalates with DNA like EtBr, then changes its absorption spectrum in emission to 620 from 590 nm. Propidium Iodine is only able to penetrate proliferated membranes of dead cells, alive one wont be affected.
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.
Maximilian Hoeselbarth

13. Maximilian Hoeselbarth
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
Maximilian Hoeselbarth

14. Thank you for your attention
Maximilian Hoeselbarth