Combining molecular modelling with experiments: Sulfonylureas and glinides as new PPARγ agonists

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1 Combining molecular modelling with experiments: Sulfonylureas and glinides as new PPARγ agonists Marco Scarsi Biozentrum - Swiss Institute of Bioinformatics

2 Drug discovery and development years of work 5-6 years 5-6 years 1-2 years Preclinical research Candidate drugs Clinical tests Drug Registration and marketing

3 Pre-clinical drug research: Real and virtual Target identification Molecular DB screening Optimization toxicology metabolism Candidate drugs Target Optimization Molecular DB identification toxicology screening metabolism Target Optimization Molecular DB identification toxicology screening 1-2 years 1 year 3-4 metabolism years Candidate drugs Candidate drugs

4 Virtual screening? DB Does the molecule bind to the protein?

5 Virtual screening Principles of molecular mechanics: Atoms as basic units Chemical bonds cannot be broken: They can be stretched, bent, torqued. Good to simulate non-covalent binding

6 AutoDock The potential generated by the protein is calculated on a grid Each ligand is flexibly sampled on the grid (conformational search)

7 AutoDock: algorithm Lamarckian Genetic Algorithm The searcher modifies the phenotype, which is allowed to update the genotype Lamarck: an adaptation of an individual to its environment can be inherited by its offsprings

8 Our work Target selection: PPARγ Virtual screening of known drugs Experimental validation Drug redirection Identify new PPARγ ligands among known drugs TheraSTrat was interested in side-effects of known drugs

9 Nuclear receptors Ligands: Nuclear receptor Response Element

10 PPARγ Pharmacological target for type-ii diabetes (several drugs on the market) It controls lipid metabolism and glucose homeostasis Lots of experimental data available

11 PPARγ agonists Fatty acid (endogenous) Thiazolidinedione (drug) Tyrosine-based agonist (candidate drug)

12 X-ray: PPARγ bound to farglitazar HIS 323 TYR 473 SER 289 HIS 449 Farglitazar: potent synthetic agonist (nm) Mutations is SER289, HIS323, TYR473, HIS449 strongly reduce activity

13 Agonists aligned (X-ray)

14 Is there anything else binding to PPARγ? Virtual screening

15 Compound libraries TheraSTrat AG database: ~8000 compounds most marketed drugs proprietary Chembank database: ~6000 compounds bioactive compounds freely available

16 Virtual screening on a grid Docking a ligand to the receptor [BC] 2 PC Desktop Grid (UD MP, Win32) Basel Grid Manager CSCS (PBS) Ticino [BC] 2 HPC cluster (SGE, x86-32) Basel Vital-IT HPC cluster (LSF, Itanium 64) Lausanne

17 Docking results Sulfonylureas and glinides bind to PPARγ Gliquidone Repaglinide Farglitazar (x-ray)

18 Glipizide Nateglinide Glimepiride Mitiglinide

19 Why are sulfonylureas and glinides so interesting as putative PPARγ agonists?

20 Type II Diabetes: drug therapies PPARγ agonists Bind to PPARγ? Sulfonylureas, Glinides Bind to sulfonylurea receptor Reduce insulin resistance Improve insulin secretion Type II diabetes treatment

21 Experimental validation 3 experiments: biochemistry cell biology Binding to receptor displacement of labeled ligand Activation of receptor transactivation assays Activation of metabolic pathways expression of PPARγ-regulated genes

22 PPARγ Binding Assays PPARγ + fluorescent labeled high-affinity ligand Competitor assay

23 Results of Binding Assays Sulfonylureas Glinides Gliquidone IC 50 = 8μM Nateglinide IC 50 = 316μM Ar(%) Compound (µm) Ar(%) Compound (µm)...and...and Glimepiride IC 50 = 125μM Repaglinide IC 50 > 1.5mM Glipizide IC 50? Mitiglinide IC 50?

24 Activation of receptor Transactivation assay Ligand PPAR Expression Vector Measure ligand effect on synthetic target gene PPAR PPARE RXR Reporter vector LUC light emitted by luciferin/luciferase reaction measured by photometer

25 Results of transactivation assays 16 Sulfonylureas Glinides 14 4 Fold activation Fold activation Gliquidone Glipizide Glimepiride Repaglinide Nateglinide Mitiglinide Concentration (µm) m Concentration (µm) Sulfonylureas and glinides activate PPARγ in the mm range

26 PPARγ-dependent gene activation Mouse pre-adipocyte cells Measure expression of selected genes induced by PPARγ signaling: Adiponectin ap2 GLUT4

27 PPARγ-dependent gene activation % of induction rosiglitazone glipizide 100 microm nateglinide 50 microm gliquidone 10 microm pioglitazone 10 microm adiponectin ap2 GLUT4 Sulfonylureas and Glinides Known PPARγ activator

28 Experiments: Summary biochemistry Sulfonylureas and glinides: bind to PPARγ cell biology activate PPARγ and enhance transcription

29 Clinically relevant? PPARγ activation observed at concentrations of μm Do these drugs ever reach these plasma concentrations? Yes (gliquidone, glipizide, nateglinide)

30 Type II Diabetes: drug therapies Glitazones, TZDs Sulfonylureas, Glinides Activate PPARγ Bind to sulfonylurea receptor Reduce insulin resistance Improve insulin secretion type II diabetes treatment

31 Conclusion: Chemical carboxylic acids thiazolidinediones sulfonylureas Same acidity: carboxylic acids pka ~4.8 thiazolidinediones pka ~6.5 sulfonylureas pka ~5.3 Same network of H-bonds known PPARγ agonists new

32 Conclusions: Pharmacological Sulfonylurea and glinide drugs can: Enhance insulin secretion (SU receptor) Reduce insulin resistance (PPARγ) Possible to design new drugs targeting PPARγ and the SU receptor A "favorable side effect"?

33 A broad sinergy Michael Podvinec Adrian Roth Renate Looser Urs A. Meyer Torsten Schwede Christoph Ruecker Hubert Hug TheraSTrat Hugo Albrecht Sander Kersten

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35 Patent? Hard to patent: Patentable: Disease 1 Disease 1 Disease 2

36 Pre-clinical research: real and virtual Target identification Highthroughput screening Leads Lead optimization Toxicology, metabolism Highthroughput Leads Lead Toxicology, Target identification optimization screening metabolism Highthroughput year Target 1-2 years 1 Leads Lead Toxicology, identification 2-3 optimization years 1-2 years screening metabolism Candidate drugs Candidate drugs Candidate drugs