The Confo body technology, a new platform to enable fragment screening on GPCRs

Transcription

1 The Confo body technology, a new platform to enable fragment screening on GPCRs Christel Menet, PhD CSO Miptec, 216

2 Confo Therapeutics Incorporated in June 215 Located in Brussels, Belgium Confo Therapeutics builds a portfolio of first-in-class programs on pathway selective drugs for unmet medical need 2

3 GPCR, the largest known gene superfamily Despite the success of drugs targeting GPCRs only a limited amount of GPCRs have been validated as therapeutic targets GPCRs = proven class of targets 6 of top 2 drugs >6bn revenues generated Wide array of pathological processes implicated 8% of this large class of targets remains commercially unexploited High failure rate in GPCR drug discovery: many deemed undrugable using current methodologies 3

4 Introduction GPCR the largest known gene superfamily GPCRs in vertebrates are commonly divided into five families on the basis of their sequence and structural similarity rhodopsin (family A), secretin (family B), glutamate (family C), adhesion and Frizzled/Taste2 GPCRs are the largest family of membrane proteins and mediate most cellular responses to hormones and neurotransmitters, as well as being responsible for vision, olfaction and taste Family A Family B Family C 4

5 History of GPCR screening GPCR a difficult target: many still undruggable Cell based assays with high rate of false positives Single Pathway ie. coupled calcium HTS-Brute force HTS to screen large number of compounds (millions) If no hits, then screen more compounds Hits = large molecules difficult to optimize Fragment based drug discovery Mutation points allow better stability and sensitive approaches like SPR, NMR New technologies for structural work: more and more structures available in PDB SBDD, structure based drug design approach. HTS on signaling pathways 5

6 Biological respons (%) Conformational complexity of GPCR transmembrane signaling GPCRs are membrane receptors translating an extracellular stimulus into an intracellular phenotype via a change of conformation Inverse agonist Basal states Agonist Full agonist Partial agonist Log drug concentration Basal activity Inverse agonist G protein GDP GTP G protein G βϒ + G α G βϒ + G α 6

7 Complexity of GPCR transmembrane signaling GPCRs are membrane receptors translating an extracellular stimulus into an intracellular phenotype via a change of conformation Kobilka et al, Nature, 29 7

8 Complexity of GPCR transmembrane signaling Ligands or compounds can activate selectively a signaling pathway Natural ligands or synthetic compounds can be validated for: triggering a desired signaling pathway inducing a desired therapeutic effect Disease 1 Disease 2 G α S L1 L2 L2 GIP G α i/o βarr G α 12/13 Side effect Side effect Jak G α q/11 PDZ PKA GRK βarr PKC Clathrin βarr Dyn 8

9 Functional selectivity of (biased) ligands Ligands or compounds can activate selectively a signaling pathway and avoid unwanted effects L1 L2 L3 Pathway 2 Pathway 2 Pathway 1 Pathway 1 Pathway 3 9

10 New drug discovery approach We lock targets in the desired signaling conformation in Pharma s current challenge Basal conformation masks druggable ligand binding sites and makes them inaccessible for screening with conventional technologies Orthosteric ligands Full agonists Partial agonists Biased agonists Inverse agonists Antagonists Occluded structural features of binding sites for pathway selective agonists revealed Allosteric modulators PAMs NAMs Allosteric agonists Nanobody Basal states Desired signaling conformation Conventional drug discovery Proprietary CONFO technology 1

11 Confobody-enabled conformational drug discovery Reference tools to stabilize the desired signalling conformation Non-prominent druggable conformation C H 2 C H 3 Heavy chain-only antibody Conventional antibody 15 kda antigen binding fragment of a camelid heavy chain-only Ab High affinity Confobodies can be raised against virtually any target Preferentially binds discontinuous conformational epitopes of native (membrane) proteins 11

12 Confobodies bind conformational epitopes Lysozyme NH COO - Nanobody Confobody 12

13 Starting point of our competitors CONFO technology Exploiting the conformational complexity of GPCRs for drug discovery Tailored methods to produce Confobodies Drugable binding sites for pathway selective agonists Confolinks constrain desired conformers Basal states = Ensemble of ligand-free conformations Confobody Desired signalling conformation Confoscore predicts the functional profile of each compound Confoscreen enables screening for differentiating NCEs or NBEs Confostructure enables active state crystal structure 13

14 Confobodies enable crystallization of GPCR in active state 14

15 Technologies known for GPCR crystallization These past years have seen a number of new technology to crystallize GPCRs Crystal structure of the β 2 - adrenoceptor G s protein complex (PDB ID: 3SN6) illustrates some of the protein engineering strategies available for structural studies of GPCRs D. Milic, Frontier in pharmacology,

16 Confobodies stabilize unique conformations of GPCRs Confobodies enable a number of crystal structures in active state Rasmussen et al. (211) Nature 469, 175 Kruse et al. (213) Nature 4, Huang et al. (215) Nature 524, Burg et al. (215) Science 347,

17 Turning on a GPCR Carazolol Inverse agonist BI Super agonist G protein or Nb TM5 TM6 17

18 Confobodies stabilizing the desired GPCR signaling conformation Nanobodies bias the pharmacology of the target receptor 18

19 Turning on a GPCR 19

20 Confobodies enable screening on GPCR in active state 2

21 Case study G protein mimicking Confobodies lock GPCR signaling conformations hβ2ar agonist Extracellular Cytoplasmic Cb8 Asthma & bradycardia Rasmussen et al. (211) Nature 469, 175 Experiment performed by Jan Steyaert s lab (VIB-VUB) 21

22 Case studies Gs-mimicking confobodies lock the signaling conformation of hβ2ar Extracellular Cytoplasmic hβ2ar Cb8 hβ2ar G α sg β G γ Rasmussen et al. (211) Nature 469, 175 Rasmussen et al. (211) Nature 477, 549 Experiment performed by Jan Steyaert s lab (VIB-VUB) 22

23 Confofusions are constitutively constrained drugable GPCR conformers The locked GPCR is very sensitive to agonists % [ 3 H]DHA β2ar β2ar 28 Irrelevant Nb Nb8 Druggable active conformation log[epinephrine] Prominent conformation 23

24 Screening approach on locked GPCR Conformational fragment screening on constrained GPCRs? Can we avoid the traditional HTS with high number of compounds and high rate of false positives? Nanobody 24

25 % [ 3 H]DHA % [ 3 H]DHA % [ 3 H]DHA Confoscreen Proprietary comparative assay can be performed at a single compound concentration Epineprine Log agonist concentration (M) Alprenolol Log antagonist concentration (M) ICI 118, Log inverse agonist concentration (M) β2ar β2ar Desired signalling conformation Basal conformations Irrelevant Cb Gs-mimicking Cb8 Experiment performed by Jan Steyaert s lab (VIB-VUB)

26 Confobody-enabled fragment screening compounds of the Maybridge R3 fragment library at 2µM Agonist like Inverse Agonist like Experiment performed by Jan Steyaert s lab (VIB-VUB) 26

27 Confobody-enabled fragment screening Screening on basal conformer identifies 1 fragment Agonist like Inverse Agonist like Experiment performed by Jan Steyaert s lab (VIB-VUB) 27

28 Fragment based Confoscreen on β2ar Proprietary comparative assays that are highly sensitive for agonists and predict the activity profile of each compound [ 3 H] antagonist binding (DHA) % 8% Irrelevant Cb 6% Cb8 4% 2% agonists antagonists inverse agonists compounds of the Maybridge R3 fragment library were comparatively screened at 2µM in a competitive radioligand displacement assay on the Gs signalling conformation (green bars) versus the basal conformations (red bars) of β2ar Experiment performed by Jan Steyaert s lab (VIB-VUB) 28

29 Confobody-enabled structure-based drug design Availability of structure in active state allows design of new compounds β2ar active state Possibility of extension Experiment performed by Jan Steyaert s lab (VIB-VUB) 29

30 Confobody-enabled structure-based drug design Simple elaboration of the fragments Ex: simple benzyl amine clogp Acylation 5 R NH 2 R H N O R Reductive amination R H N R Fragments MW Elaborated Fragments Experiment performed by Jan Steyaert s lab (VIB-VUB) 3

31 Confobody-enabled structure-based drug design Simple elaboration of fragments increases the affinity keeping high ligand efficiency LE: ligand efficiency Known pharmacophore Fragments with new chemistry Elaborated fragments,5,4,3 Increase potency Experiment performed by Jan Steyaert s lab (VIB-VUB) Affinity on active state IC 31

32 Example of cell activity after elaboration Fragment 1 elaborated to potent and pathway selective compounds 75 BC399 ADRB2 HitHunter camp Assay Agonist mode Max = Slope = Min = EC = R2 =.9976 Affinity active state (IC, M) Confoscore Fragment E BC E BC399 ADRB2 PathHunter β-arrestin GPCR Assay Agonist mode Max = 15 Slope =.9524 Min = EC = R2 =.9446 µm µm Multiple pathway screening at DiscoverX: 2nd messenger (camp & Calcium) Arrestin Recruitment Receptor Internalization PathHunter Total GPCR Internalization Assay Agonist mode 32

33 Examples of cell activity after elaboration Fragment 1 elaborated to potent and pathway selective compounds Affinity active state (IC, M) Confoscore Elaborated compound HitHunter camp Assay PathHunter β-arrestin Assay PathHunter Total GPCR Internalization Assay Fragment E-5 17 No data No data No data BC399 ADRB2 BC399 ADRB2 BC E KG E Max BC45 = Slope = Min ADRB2 = EC = R2 = Max = Slope = 1.74 Min = EC =.7751 R2 = Max = 15 Slope =.9524 Min = EC = R2 =

34 Examples of cell activity after elaboration Fragment 2 elaborated to potent and pathway selective compounds Affinity active state (IC, M) Confoscore Fragment 2 2. E-5 38 BC E BC45 ADRB2 HitHunter camp Assay Agonist mode 75 BC45 ADRB2 PathHunter β-arrestin GPCR Assay Agonist mode Max = Slope = 1.74 Min = EC =.7751 µm R2 =

35 Examples of cell activity after elaboration Fragment 2 elaborated to potent and pathway selective compounds Affinity active state (IC, M) Confoscore Elaborated compound HitHunter camp Assay PathHunter β-arrestin Assay PathHunter Total GPCR Internalization Assay Fragment 2 2. E-5 38 No data No data No data BC E BC41 3,1 E BC45 ADRB Max = Slope = 1.74 Min = EC =.7751 R2 BC41 =.9952 ADRB Max = Slope = Min = EC = R2 = BC45 ADRB BC41 ADRB Max = 9.24 Slope = 1.9 Min = EC =.8351 R2 =

36 Examples of cell activity after elaboration Fragment 3 elaborated to potent and pathway selective compounds Affinity active state (IC, M) Confoscore HitHunter camp Assay PathHunter β-arrestin Assay PathHunter Total GPCR Internalization Assay Fragment 7.4 E-5 49 No data No data No data BC E No data GC E BC442 ADRB GC Max ADRB2 = 9 Slope = Min = EC =.1468 R2 = BC442 ADRB GC Max ADRB2 = Slope = Min =.2761 EC = 1.7 R2 = GC6 1.9 E GC E KG E No data GC ADRB2 Max = 8 Slope = Min = EC =.93 R2 = Max = Slope = GC7 Min = EC =.2993 ADRB2 R2 = KG ADRB2 Max = 9 Slope = Min = EC = R2 = Max = 9 Slope = Min = EC =.8662 R2 = GC6 Max = Slope = 1.69 ADRB2 Min =.253 EC = R2 = GC7 Max = Slope = 2.32 ADRB2 Min =.8914 EC = R2 = KG177 - ADRB Max = Slope = Min =.98 EC = R2 = Max = 15 Slope = Min =.778 EC = R2 =

37 Nanobody-enabled HTS on β2ar k Axxam (2µM) Agonist like Inverse Agonist like 37

38 Thanks to.. 38

39 Reference ligands: confobody and cell based assays Data produced in DiscoverX assay Confobody binding 39