THE COMBINED USE OF COMPARATIVE SYSTEMS BIOLOGY AND STRUCTURE-BASED APPROACHES

1. THE COMBINED USE OF COMPARATIVE SYSTEMS BIOLOGY AND STRUCTURE-BASED APPROACHES FOR THE DEVELOPMENT OF SELECTIVE ENZYMES INHIBITORS AS LEAD DRUGS AGAINST TRYPANOSOMATID PARASITES

2. The medical problem Various species of the protozoan family Trypanosomatidae are responsible for serious, often fatal diseases of mankind in (sub)tropical countries. • Trypanosoma brucei African trypanosomiasis or “sleeping sickness” • Trypanosoma cruzi American trypanosiomiasis or “Chagas’ disease” in South and Central America • 20 different Leishmania species Various manifestations of leishmaniasis in almost all (sub)tropical areas of the world The current possibilities for proper treatment of the diseases are limited : available drugs are expensive, not very efficient and/or toxic at their therapeutic doses

3. Prevalence of Human African Trypanosomiasis (HAT) or Sleeping Sickness HAT is endemic in 36 countries in sub-Saharan Africa

4. Transmission • Via the bite of bloodsucking male and female tsetse flies that transfer the parasites from human to human. • Cattle and other wild mammals act as reservoir hosts of the parasites. • Tsetse flies can acquire parasites by feeding on these animals, or on an infected person. • Inside the human host, trypanosomes multiply and invade most tissues.

5. The life cycle of Trypanosoma brucei

6. Symptoms of sleeping sickness • Infection leads to malaise, lassitude and irregular fevers. • Early symptoms include fever and enlarged lymph glands and spleen. • Early signs are followed by a range of symptoms including headache, anaemia, joint pains and swollen tissues; • Advanced symptoms include neurological and endocrine disorders. 

7. Current treatment of sleeping sickness • Pentamidine is not effective against late-stage disease and some parasite strains are resistant.  • Suramin has to be administered intravenously and can  have adverse side-effects. • Melarsoprol, an arsenical drug, is used against late-stage disease, Between 4% and 12% of patients die. • A newer drug, eflornithine, has shown to be efective against only one form of HAT and requires 400g / treatment.

8. Similar bad situations exists in treatment of Chagas Disease and the various forms of leishmaniasisNew, effective, not toxic, affordable drugs against the diseases caused by trypanosomatid parasites are badly needed New drugs are needed

9. Aim: Combating trypanosomes without harming their hosts Despite the morphological difference, trypanosomes are biochemically very similar to their hosts

10. Glycolysis as drug target Glycolysis of trypanosomes is an attractive target for drugs, because: • The parasites, when living in the human bloodstream, rely entirely on glycolysis for their ATP supply • but…… • Glycolysis is also essential for many mammalian host cells. • Therefore, high drug selectivity is required to spare the host.

11. How to achieve high drug selectivity? The discovery of selective anti-trypanosome drugs may involve the exploitation of parasite versus host differences in the: • structure of enzymes • - structure-based drug design • organization of metabolic networks • - network-based drug design

12. Trypanosome glycolysis as drug target Despite the importance of glycolysis for the human host too, it is an attractive drug target in trypanosomes because: • The glycolytic pathway of trypanosomes is organized in a unique manner; the majority of its enzymes are sequestered in peroxisomes hence called glycosomes • It is anticipated that this peculiar organization and the long evolution of trypanosomes independent of other eukaryotes have resulted in a pathway with unique network properties and enzymes with unique structural features

13. 1 - 2 ADP ATP Glycolysis in bloodstream-form T. brucei CYTOSOL glucose GLYCOSOME glucose ATP ADP glc-6-P fru-6-P ATP MITOCHONDRION ADP fru-1,6-BP H2O GA-3-P DHAP DHAP Pi Pi NADH SHAM T NAD+ glycerol-3-P glycerol-3-P O2 G-1,3-BP ADP ADP ATP ATP glycerol G-3-P glycerol G-3-P G-2-P PEP pyruvate

14. Glycosomes of bloodstream-form T. brucei visualised by serial sectioning • Three major types of organelles are shown: the nucleus, the single mitochondrion and the microbodies • Glycolytic enzymes are sequestered in some 65 microbodies, called glycosomes that are distributed throughout the cytoplasm Glycosome Nucleus Mitochondrion From Tetley and Vickerman, 1991

15. The major proteins associated with glycosomes are glycolytic enzymes Glycosomes of T. brucei in situ SDS-PAGE of glycosomal preparations

16. Structure-based design of trypanocidal drugs • exploits the properties of enzymes to identify targets which would provide the highest drug specificity and selectivity • is based on a comparative structural analysis of parasite and host enzymes

17. Structure-based design of trypanocidal drugs design/ select redesign to improve protein 3D structure purification human selective inhibitor enzyme enzymological studies synthesize gene cloning 3D structure +sequencing trypanosome determine IC50 enzyme overexpression crystallography determine structure enzyme-inhibitor evaluate in trypanosomes evaluate in animals preclinical toxicology clinical trials drugs

18. Additional approaches used in drug discovery work - Catalytic mechanism-based design - Database mining - Parallel synthesis (combinatorial chemistry) - Selection from medicinal plant extracts - Chemical library screening - Combinations of various of the above mentioned methods

19. Network-based design of trypanocidal drugs • exploits the properties of metabolic and gene expression networks to identify targets which would provide the highest drug selectivity • is based on Metabolic Control Analysis

20. Metabolic Control Analysis e1 e2 ei Metabolic Control Analysis (MCA) is a tool for quantitative analysis of a metabolic pathway S I1 I2 P • Thequalitativecategory ofrate-limitingis replaced in MCA by aquantitativescale for the influence of an enzyme on the flux through a pathway : theflux controlcoefficient • flux control coefficient = % change of flux % change of enzyme activity • Control can be shared by enzymes of a pathway _ often enzymes have a flux control coefficient in between 0 and 1 _ the sum of the control coefficients over all enzymes in a metabolic pathway is 1

21. Metabolic Control Analysis (2) 150 a c 100 b % flux a : rate limiting CJ = 1 b : not rate limiting CJ = 0 c : 0 <CJ < 1 50 0 150 0 100 50 % enzyme activity • flux control coefficient CJ = % Change of flux • % Change of enzyme activity

22. Metabolic Control Analysismethodology Flux control coefficients of enzymes in a pathway can be determined by measuring the flux upon titration of the activity of each enzyme individually, while keeping the other conditions constant by : • genetic means • using specific inhibitors • using a computer model of the pathway

23. Structure-based design of trypanocidal drugs Is structure-based design of selective inhibitors of trypanosomatid glycolytic enzymes feasible?Do these enzymes differ sufficiently from their human counterparts?

24. Most trypanosomatid glycolytic enzymes possess unique properties • All glycolytic enzymes of T. brucei have been purified; their kinetic properties have been determined. • The genes of all enzymes involved in glycolysis of bloodstream-form T. brucei have been cloned and sequenced. • Most T. brucei glycolytic enzymes differ considerably in structural and kinetic properties from their homologues in other organisms

25. Amino-acid identity (%) to the corresponding human enzyme Glycolytic enzymes of Trypanosoma brucei Enzyme Glc transporter 19 37 HXK 57 PGI 21 PFK 48-49 ALD 52 TIM 55 GAPDH 47 PGK no homology PGAM 59-62 ENO 49-51 PYK 29 gGPDH (NADH) 50 GK 23 mGPDH (FAD) TAO no homology

26. Regulation of glycolysis in T. brucei The available enzyme kinetics data suggest that glycolysis in bloodstream-form T. brucei is essentially unregulated. No or little activity regulation has even been found for hexokinase and phosphofructokinase. Activity regulation is only found for the last step of the pathway, pyruvate kinase.

27. Properties of Hexokinase • Trypanosomatid HXK is a glycosomal enzyme that is:not inhibited by glucose 6-phosphatenot inhibited by ATPnot stimulated by ADP or AMP • Conclusion: HXK is essentially unregulated

28. Properties of Phosphofructokinase • Trypanosomatid PFK is a glycosomal enzyme that is:not regulated by the classical regulators of other PFKsnot regulated by fructose 2,6-bisphosphateactivated by AMP, not by ADP and GDP • Conclusion: PFK is essentially unregulated

29. Properties of Pyruvate Kinase • Trypanosomatid PYK is:- a cytosolic enzyme- an allosteric enzyme, regulated by the classical heterotropic regulators of PYK- uniquely activated by fructose 2,6-bisphosphate, the allosteric activator of PFK in other organisms

30. Trypanosomatid glycolytic enzymesavailable crystal structures Trypanosoma brucei Trypanosoma cruzi Leishmania mexicana PGI PFK ALD ALD TIM TIM TIM GAPDH GAPDH GAPDH PGK PGAM ENO PYK GPDH Protein crystallography groups involved: Hol,Wierenga, Oliva/Garrett/Thiemann, Walkinshaw/Gilmore, Rigden, Sygush

31. Comparison ofTrypanosoma and human glycolytic enzymes revealed many possibilities for structure-based drug design • The core structure and active site of most glycolytic enzymes are very well conserved, but important differences were found for PFK and PGAM. • Seemingly minor differences, yet potentially exploitable for development of selective inhibitors, were found in the active site of some enzymes such as ALD and ENO. • Major differences could be found elsewhere in the structure: - at the surface (all enzymes) - at subunit interfaces (most enzymes) - in allosteric effector-binding sites (e.g. PYK) - in cofactor-binding sites (e.g. GAPDH)

32. Structure-based design of trypanocidal drugs Structure-based design of selective inhibitors of trypanosomatid glycolytic enzymes seems feasible.Most of the trypanosomatid enzymes differ considerably in structural and functional properties from their human counterparts. What about the network design? Could additional selectivity expected from differences between the organization of the glycolytic pathway design in human and trypanosomes?

33. Network-based design of trypanocidal drugs • exploits the properties of metabolic and gene expression networks to identify targets which would provide the highest drug selectivity • is based on Metabolic Control Analysis

34. The available information about the organization of the pathway and kinetics of the glycolytic enzymes, allowed addressing the following questions relevant for network-based drug design: 1. How is the glycolytic flux controlled? Has the remarkable lack of activity regulation of trypanosomatid HXK and PFK, and the compartmentation of the pathway, an effect on the flux control? 2. Which enzymes need little inhibition to reduce the glycolytic flux and kill the trypanosomes? A first approach to address these questions was made by constructing a mathematical model of the system and using it in computer simulations, based on the principles of ‘Metabolic Control Analysis’.

35. Mathematical model of T. brucei glycolysis • A mathematical model of glycolysis in bloodstream-form T. brucei was developed on the basis of all available kinetic data 1 • The model predicted correctly the experimentally observed: • glycolytic flux under (aerobic and anaerobic) physiological conditions, and at different glucose concentrations • cellular metabolite concentrations under aerobic and anaerobic conditions • inhibition of anaerobic glycolysis by glycerol • The model was used to determine the rate-limiting steps of the glycolytic flux 2 • 1 Bakker et al., J.Biol.Chem., 1997; 2 Bakker et al., J.Biol.Chem., 1999.

36. 1 0.8 0.6 0.4 0.2 0 0 2 4 6 8 10 Which steps control the glycolytic flux ? CJ CJ glucose transport + +: ALD : 0.16 GAPDH : 0.14 PGK : 0.09 GPDH : 0.10 physiological range Extracellular [glucose] (mM) No control was exerted by HXK, PFK, PYK or ATP utilization

37. Identification of drug targetsin T.brucei glycolysis Reaction % inhibition required for 10% flux reduction 50% flux reduction Glucose transport 11 51 HXK 77 93 PFK 87 93 ALD 44 76 GAPDH 53 84 PGK 61 85 PYK 94 97 GPDH 56 83

38. Conclusions from glycolytic flux-control analysis by computer simulation • Glycolysis in bloodstream-form T. brucei can be understood in terms of the kinetics of the glycolytic enzymes. • Under physiological conditions, most control resides in glucose uptake. Some control is exerted by ALD, GAPDH, PGK and GPDH. • Trypanosome glycolysis can be inhibited efficiently at several enzymatic steps (e.g. glucose transport, aldolase, GAPDH, PGK and GPDH). • Surprisingly, HXK, PFK and PYK exert no control. Nearly full inhibition of these kinases is required to achieve a substantial decrease of the glycolytic flux.

39. glucose m i t o c h o n d r i o n ? ? 1 0.68 glucose Distribution of flux control in glycolysis of bloodstream-form Trypanosoma brucei Data obtained by MCA studies using the model based on enzyme-kinetic properties. ATP supply, not ATP demand controls the process. cytosol ? glycosome glucose ATP 0.03 2 2 ADP ADP Glc-6-P 17 0.001 3 ATP AMP Fru-6-P ATP 0.005 4 ADP Fru-1,6-BP 0.05 5 0.01 6 O H DHAP DHAP GA-3-P 2 + NAD 0.06 7 ? 13 14 NADH Gly-3-P 1,3-BPGA Gly-3-P 0.5 O 2 ADP 0.07 8 15 ATP glycerol 3-PGA 2 ADP ? 0 18 3-PGA 9 ATP AMP 2-PGA glycerol 10 PEP 0.01 0.001 ADP 11 16 ATP pyruvate 12 pyruvate glycerol

40. To which host cells should we compare the trypanosomes? • live in the same environment as trypanosomes (blood) • are completely dependent on glycolysis for ATP synthesis, as trypanosomes • a kinetic model is available* (*Schuster and Holzhütter, Eur.J. Biochem, 229, 1995, 403-418) Erythrocytes

41. glucose m i t o c h o n d r i o n ? ? 1 0.00 0.68 glucose cytosol ? Selectivity by differential control glycosome glucose ATP 0.03 0.02 2 2 ADP ADP Glc-6-P 17 0.001 0.00 3 ATP AMP Fru-6-P ATP 0.02 0.005 4 ADP Fru-1,6-BP -0.01 0.05 5 Erythrocyte model Demand control Trypanosome model Supply control 0.01 6 O H DHAP DHAP GA-3-P 2 + NAD 0.07 0.00 0.00 7 ? 13 14 NADH Gly-3-P 1,3-BPGA Gly-3-P 0.5 O 2 0.07 ADP 0.00 8 15 ATP glycerol 3-PGA 2 ADP ? 0.00 0 18 3-PGA 9 ATP AMP 2-PGA glycerol 10 PEP 0.94 0.01 0.03 ADP 11 0.001 16 ATP pyruvate 12 pyruvate glycerol

42. ( ) J C i parasite ( J ) C i host Selectivity (% flux inhibition parasite) / (% flux inhibition host) network structure Inhibition = control  elasticity  uptake  affinity ( ) v * e i host d [ I ] K dJ / J ( parasite ) I / K . . . parasite parasite i i Selectivit y = = ( ) v parasite dJ / J ( host ) d [ I ] * K e i host i 1 4 2 4 3 I / K 1 4 2 4 3 1 4 2 4 3 host 1 4 4 2 4 4 3 i uptake structure control elasticity 1 4 4 4 4 2 4 4 4 4 3 network

43. v *  (parasite) i I / Ki v *  (host) i I / Ki Network selectivity of competitive inhibitors (MCA) Against competitive with network selectivity Glc transport glucose 5.7·10-17.2 ·103 4.1 ·103 Hexokinase glucose 311.08 34 ATP 0.0151.08 0.016 Aldolase Fru16BP 8.5 ·10-2-7.0 ·100-6.0 ·10-1 GAPDH GAP 1.1 ·100-7.6 ·101 -8.6 ·101 NAD 1.8 ·10-1-7.6 ·101 -1.4 ·101 PGK 13BPGA 4.4 ·10-15.8 ·101 2.5 ·101 ATP 1.2 ·10-15.8 ·101 6.8 ·100 J C ) i ( parasite J C ) ( i host Selectivity towards trypanosome Selectivity towards erythrocyte

44. ( ) J C i parasite ( J ) C i host Selectivity (% flux inhibition parasite) / (% flux inhibition host) network structure Inhibition = control  elasticity  uptake  affinity ( ) v * e i host d [ I ] K dJ / J ( parasite ) I / K . . . parasite parasite i i Selectivit y = = ( ) v parasite dJ / J ( host ) d [ I ] * K e i host i 1 4 2 4 3 I / K 1 4 2 4 3 1 4 2 4 3 host 1 4 4 2 4 4 3 i uptake structure control elasticity 1 4 4 4 4 2 4 4 4 4 3 network

45. [I]/ K i High concentrations of glucose transport inhibitors (competitive) 100 Trypanosome model 80 60 Erythrocyte model ATP synthesis flux (% of uninhibited flux) 40 20 0 0 200 300 500 400 100 600

46. High concentrations of hexokinase inhibitors (competitive with ATP) 100 Trypanosome model 80 Erythrocyte model 60 ATP synthesis flux (% of uninhibited flux) 40 20 0 0 20 40 60 80 100 [I]/ K i

47. Criterium for selectivity at high inhibitor concentrations Determine Ki, Ery / Ki, Tryp (structural selectivity) at which trypanosome metabolism can be inhibited by 95% at 5% inhibition of erythrocyte metabolism  Sets the goal for structure-based drug design

48. Selectivity at high concentrations (competitive) Against competitive with Ki, Ery / Ki, Tryp * Glc transport glucose 1.7 Hexokinase glucose - ATP 9095 Aldolase Fru16BP < 58 GAPDH GAP < 46 NAD 0.23 PGK 13BPGA 270 ADP 1.2 * Ki ratio at which trypanosome metabolism can be inhibited by 95 % at 5 % inhibition of erythrocyte metabolism The challenge for the structure-based drug design depends strongly on the target enzyme chosen and the nature of the inhibitor

49. Predictions from comparative in silico network-based drug design studies Comparison of the properties of the glycolytic network between trypanosome and host cells with regard to: 1. control exerted by each enzyme 2. in situ sensitivities (‘elasticities’) of each enzyme towards inhibitors 3. uptake of inhibitors 4. inhibition constants and nature of inhibition can identify uniquely vulnerable steps with the greatest potential for selectively inhibiting the flux through the trypanosome’s pathway. The preliminary analysis indicates the glucose transporter, GAPDH and PGK as promising steps for competitive inhibitors (the latter enzymes with cofactor analogues as inhibitors).

50. All the predictions are based on computer modeling • How accurate is the model? • The model is based on kinetic data obtained from analyses of purified enzymes and specific enzyme activities as measured in lysates of trypanosomes. Do these data reflect the in vivo situation? • Experimental verification of the predictions of the model requires glycolytic flux measurements by cells in which activities of individual enzymes and transporters are altered by inhibitors or genetic means.