Protein-protein interaction disruptors as therapeutic targets (an example of a drug discovery project) Yvonne Lai, Ph.D. Department of Psychological and Brain Sciences Indiana University, Bloomington Navigator, Indiana CTSI (Bloomington campus) NMDAR Signaling Cascade Image from Cayman Chemical Protein-protein interaction disruptors of NMDAR-dependent signaling Tat-NR2B9c (NA-1) Peptide inhibitor Pain behavior NMDAR -----------PSD95----------nNOS-------------NOS1AP--------MAPK------PTSD (CAPON) Depression Stroke Small molecule inhibitors: IC87201/ZL006 Drug Development Target Discovery Lead Discovery Assay development High-throughput screen Lead Optimization Candidate selection Preclinical Evaluation Full Development Clinical Trials Current collaborative Team Pain efficacy: Andrea Hohmann (IUB)-Wan-hung Lee Neurotoxicity: Andy Hudmon (IUSM)-Aarti Chawla PTSD efficacy: Anantha Shekhar (IUSM)-Stephanie Fitz Chemistry: Ganesh Thakur (Northeastern University)-Pushkar Kukarni nNOS-NOS1AP disruption Michael Courtney (University of Southern Finland)-Lilli Li Small molecule nNOS-PSD95 protein-protein interaction inhibitors IC87201: • Inhibited NMDA-induced nNOS-dependent NO formation • Efficacious in chronic pain model (Florio, BJP 2009; Lai, Hohmann unpublished) • Decrease symptoms of PTSD in a rat preclinical model (Shekhar, unpublished) • Efficacious in one depression model (Doucet 2013) ZL006 (analog of IC87201): • Blocked focal cerebral ischemic damage in mice and rats (Zhou, Nat Med 2010) • Efficacious in one depression model (Doucet 2013) Model Neuronal cells Cerebral Ischemia Neuropathic pain Fear conditioning Plasma Side effect Profile NOS enzyme Spatial memory Motor movement Effect Measured Cytotoxicity in vitro cGMP (NO marker) Infarct volume IC87201 (~EC50) <0.5 mM 3 mM N.D. ZL006 (~EC50) 0.08 mM N.D. 1.5 mg/kg (iv) Tactile allodynia Freezing time Drug level (rat) 2 mg/kg (ip) 4 mg/kg (ip) 6mM-2mg/kg (i.p.) N.D. 10 mg/kg (ip) 9mM-1.5mg/kg (i.v.) Catalytic activity Morris water maze Rotarod No effect (10 mM) N.D. No effect (4 mg/kg) No effect (10 mM) No effect (10 mg/kg) No effect (10 mg/kg) Additional profile of IC87201/ZL006 • Efficacious systemic or i.t., found in CSF and brain tissues • No significant inhibition of 35 target (Receptorgram, MDS Pharma) • No effect on basal pain sensation • No motor ataxia • No effect on novel object recognition • No effect on Morris water maze • No effect on other PDZ P-P interactions (e.g. Erb4-PSD95) HTS (NIH clinical library) NGP-202-01 (2966) NOS-NOS1AP 100 50 NOS-PSD95 NOS-NOS1AP 150 100 %Control %Control NGP-105-01(3281) NOS-PSD95 150 50 0 0 0 20 40 60 80 0 20 Well # NGP-202-02 (3055) Target Discovery Lead Discovery NGP-105-02 (3361) NOS-PSD95 NOS-NOS1AP 100 150 60 80 50 NOS-PSD95 NOS-NOS1AP 100 %Control %Control 150 40 Well # 50 0 0 0 20 40 60 80 0 20 Well # NGP-202-03(3144) 150 40 60 80 Well # NGP-105-03 (3441) NOS-PSD95 NOS-NOS1AP 200 NOS-PSD95 NOS-NOS1AP 150 %Control %Control 100 50 Assay development 50 0 0 0 20 40 60 80 0 20 40 60 80 Well # NGP-202-04 (3233) NGP-105-04 (3521) NOS-PSD95 NOS-NOS1AP 150 100 %Control %Control 150 High-throughput screen 100 50 0 NOS-PSD95 NOS-NOS1AP 100 50 0 0 20 40 Well # 60 80 0 20 40 60 Well # Alpha Screen using NIH clinical library (~800 cpds); nNOS-PSD95 vs nNOS-NOS1AP (Fan, Courtney, Hohmann, Lai, unpublished) 80 Virtual screen? Target Discovery Lead Discovery Virtual Screen Model of PSD95 PDZ2 and the nNOS PDZ complex (PDZ2 is superimposed on the α-syntrophin structure. From Tochio 2000) Drug Development Preclinical Evaluation Side effect profile What are the critical No Go side effects for treatment of pain and PTSD? Additional: • Memory models? • Toxicity profile? • Oral bioavailability? Drug Development CTSI Core Facilities Target Discovery Lead Discovery Lead Optimization Preclinical Evaluation HTS: Chemical Genomics IUB-Light Microscopy Imaging Center (high content screen) Need for library: For unusual targets (e.g. P-P interactions) For cell based or more complicated assays Computational modeling Virtual screening Chemical design and synthesis Full Development Funding CTSI Core facility grant (collaboration with Purdue’s BAL) IURCG collaborative grant (Hohmann, IUB and Hudmon, IUSM) NIH likes our innovative approaches: • Funded: o R21/NIDA (pain efficacy)-Hohmann PI; Lai, Co-PI • Expected to be funded: o R21/NIMH (PTSD)-Shekhar, PI; Lai, Co-PI o SBIR phase I (two years)-Lai, PI What other funding resources? VC, Angel funds? Collaboration with industry? Collaboration between start ups with academic universities: What is the model? How to optimize expertise and minimize duplications?