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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 03.05.2016 Maximilian Hoeselbarth 2 Kinetoplasts associated diseases (1) • African Trypanosomiasis (Sleeping sickness) – – – – – – • 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) – – – – – Species: Vector: Region: Infections: Symptoms: – Treatment: 03.05.2016 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 Maximilian Hoeselbarth 3 Kinetoplasts associated diseases (2) • • • Leishmaniasis – – – – – Species: Vector: Region: Infections: Symptoms: – 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 03.05.2016 Maximilian Hoeselbarth 4 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 03.05.2016 Maximilian Hoeselbarth 5 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 03.05.2016 Maximilian Hoeselbarth 6 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 03.05.2016 Maximilian Hoeselbarth 7 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 Fig. 2: (left) Sildenafil containing Vigra tablet of Pfiizer, (right) package of Roflumilast from Nycoped pharma 03.05.2016 Maximilian Hoeselbarth 8 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 03.05.2016 Maximilian Hoeselbarth 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. 9 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 03.05.2016 Maximilian Hoeselbarth 10 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. 03.05.2016 Maximilian Hoeselbarth 11 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. 03.05.2016 Maximilian Hoeselbarth 12 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 03.05.2016 Maximilian Hoeselbarth 13 Thank you for your attention 03.05.2016 Maximilian Hoeselbarth 14