Dynamin Drug Discovery Program for Epilepsy

Transcription

1 Dynamin Drug Discovery Program for Epilepsy Introduction Bio-Link presents a unique drug discovery program focused on modulation of the GTPase activity of dynamin for the treatment of epilepsy and other CNS indications. An extensive collaboration involving the Children s Medical Research Institute (CMRI), the University of Newcastle and the University of Melbourne has shown that inhibition of dynamin can prevent synaptic vesicle endocytosis (SVE), leading to anti-convulsant activity with in vivo epilepsy models. Safety of modulating dynamin is supported by clinical experience utilising CNS drugs that potently modulate dynamin through off-target activity. Executive Summary A rational and multi-disciplinary academic drug discovery program focused on developing selective and potent dynamin inhibitory drugs for treatment of refractory epilepsy. This unique drug discovery platform includes: A novel mechanism of action for treatment of refractory epilepsy; A novel target, the neuronal isoform of dynamin I; A broad set of Structure Activity Relationship (SAR) data supported by homology modeling of the target GTP binding site in dynamin I; Lead chemical scaffolds have exhibited potent in vivo efficacy in 6Hz and amygdala kindling seizure or epilepsy models; A high throughput in vitro and in-cell screening platform for identification of isoform-selective dynamin inhibitors; Extensive translational and clinical capability and expertise in profiling and testing investigational anti-convulsant compounds; Correlative clinical data supporting the safety of modulation of dynamin; Legal certainty for partnering and licensing of IP; Potential opportunities to leverage Australian government funding sources; A virtual biotech drug discovery program, involving extensive collaborations between fundamental biologists focused on cell signalling and dynamin biology, neurobiologists, medicinal chemists, in vivo biologists and clinicians; Bio-Link is seeking an industry partner for preclinical and clinical development of dynamin inhibitor drug candidates for epilepsy. 1

2 Epilepsy Market and The value of the epilepsy market in the major developed markets was approximately US$3.4 billion in 212 and is expected to register a Compound Annual Growth Rate (CAGR) of 3.9% to reach $4.5 billion by Epilepsy is a collection of diverse disorders that together affect about 1% of the general population. The disease is characterised by neuronal hyper-excitability, and is associated with head trauma, tumors or with mutations in around 8 genes involved in nerve communication or energy production. A recent study of epileptic encephalopathies by the EuroEPINOMICS-RES consortium together with the Epilepsy Phenome/Genome Project and Epi4K Consortium found 429 associated de novo mutations, including de novo mutations in the dynamin isoform 1 gene, DNM1, in five individuals 2. Present epilepsy therapies are moderately successful in some patients, although most drugs have side-effect tolerability problems. Unfortunately, approximately % of patients receive no therapeutic benefits from current treatments, representing a large unmet medical need 3. Further, no currently available drugs are disease-modifying, i.e. there is treatment of the underlying disease. Most existing and investigational anti-epileptic drugs (AEDs) are designed to target Channelopathies : K + channels, Na + channels, Ca 2+ channels, acetylcholine receptor channels and GABAA receptor channels to reduce excitatory synaptic transmission in epileptic patients. The mechanism of action for most currently used drugs can largely be attributed to inhibition of these channels. Overall, by reducing excitatory transmission or increasing inhibitory transmission, all current AEDs either directly or indirectly target the synaptic vesicle cycle 3 (see below). Dynamin is critical to the synaptic vesicle cycle but no clinical AED targets dynamin. The development of drugs with novel and known mechanisms of action has the potential to address the refractory patient population 3, by delivering safe and effective new treatment options. The Dynamin Drug Discovery Program for Epilepsy is focused on this goal. A Novel for Refractory Epilepsy: Modulating Synaptic Vesicle Endocytosis (SVE) Dynamin inhibition represents a unique strategy for treatment of epilepsy that is based on a novel mechanism of action - modulation of synaptic vesicle endocytosis (SVE). This is a novel route to reducing synaptic transmission in an activity-dependent manner, i.e. after a lag phase, rather than constantly. SVE retrieves empty synaptic vesicles for later refilling after neurotransmitter release that occurs during synaptic firing. The GTPase dynamin I is the most attractive pharmacological target among a large collection of proteins involved in endocytosis. It is a key driver of SVE with two functions; it initiates multiple protein-protein interactions for SVE to recruit the required machinery and it provides mechanochemical force for internalisation of new synaptic vesicles. Blocking dynamin can eliminate all SVE while other molecular targets only reduce the process. 2

3 omparison of and mg/kg s in ECT Corneal Comparison Stimulation of and 4 mg/kg Valproate s EC 5 Stimulation Amplitude (ma) 5 Mg 2+ /GTP Assembly Assembly SH3 Domains 4 2 A B ns EC 5 Stimulation Amplitude (ma) K + SV2A Na + P value.879 Kainate AMPA Ca 2+ Glutamate NMDA Receptor P value <.1 Ca 2+ Dynamin Valproate D Valproate E Comparison of and mg/kg s Comparison of and mg/kg s in ECT Corneal Comparison Stimulation of and mg/kg MR178.B Adjusted EC 5 Stimulation Amplitude (ma) mg/kg P value.37 ** EC 5 Stimulation Amplitude (ma) mg/kg EC 5 Stimulation Amplitude (ma) P value < P value.269 * GTP binding Lipid binding Protein Interaction, Phosphorylation C Oligomerisation Comparison of and mg/kg s BSE G Domain EC 5 Stimulation Amplitude (ma) 5 GTP GDP + Pi 4 Lead Scaffold 2 BSE (Time (s)) Stalk PtdIns(4,5)P 2 PH P value.879 ns Stalk BSE PRD Control (Glutamate Release) Inhibition F Switch II Switch I P-loop G4 loop Top view Switch I Ser61 Val64 Gly62 Switch II Gly6 Pro138 Mg ++ Pro58 Ser41 P-loop Arg59 Ser45 Lys44 Ala42 Gln239 Ser46 Lys26 Comparison of and mg/kg MR178.B Adjusted Ser238 EC 5 Stimulation Amplitude (ma) Asn Leu29 G4 loop Val235 Asp28 P value <.1 Normal Seizure Transmission Figure 1. Dynamin function is critical to synaptic vesicle endocytosis, a mechanism required for synaptic transmission and internalisation of neurotransmitter-filled vesicles. Using in silico modelling and proprietary SAR data, the Program has developed potent compounds to inhibit dynamin function in SVE. These compounds have demonstrated anticonvulsant activity in a range of in vivo animal models. A.) Cartoon showing the role of Dynamin in synaptic vesicle endocytosis during synaptic transmission; B.) Domain diagram of dynamin; C.) An experiment in rat synaptosomes demonstrating that glutamate release is decreased over time after inhibition of dynamin I function. Importantly, release of glutamate occurs at normal levels for the first 6 seconds due to pre-existing synaptic vesicle stores. When vesicle stores are exhausted because of dynamin (SVE) inhibition, an activity dependent run-down of glutamate release is observed. This supports the view that dynamin inhibition will prevent or modulate seizure activity associated with uncontrolled ongoing synaptic transmission that occurs during a seizure, but not normal synaptic transmission that is associated with short discrete neuronal firing (See Anggono et al., (26)); D.) with the GTPcompetitive compound MR178B2 produces dose-dependent protection against seizures in the 6Hz mouse corneal stimulation model (n = 15),** p=.37, p=.1; E.) of amygdala kindled rats with MR178B2 produces dose-dependent decrease in seizure class and duration (n = 6-7); F.) Ribbon model of dynamin I with docking modeling (including bound Mg-GTP) of the GTP binding pocket into which the Drug Discovery Program is targeting small molecule inhibitors. 3

4 Epileptic seizures are characterised by uncontrolled cycles of exocytosis and endocytosis at nerve synaptic terminals. During a seizure, massive uncontrolled discharges (exocytosis) of transmitter molecules are compensated by endocytosis (SVE), which retrieves empty synaptic vesicles for re-filling and reuse (Figure 1A). This cycle perpetuates the seizure and is required for its spread across the brain (seizure propagation). It is hypothesised that reduction of endocytotic activity will reduce the availability of synaptic vesicles for these exo endocytosis feedback loops, exhausting the available vesicle pool and stopping or preventing seizure propagation. Using a rationally designed dynamin I peptide inhibitor, our cell based studies have demonstrated that inhibition of SVE leads to activity-dependent run-down in synaptic transmission (Figure 1C., Anggono et al., 26 4 ). This provides in vitro proof-of-principle that suppression of a seizure is possible through modulation of SVE. The therapeutic utility of dynamin inhibition in epilepsy is supported by multiple in vivo studies utilising small molecule dynamin inhibitors in a spectrum of epilepsy animal models. This is exemplified by results utilising the rationally designed small molecule MR178B2 (Figure 1D,E), which shows that dynamin inhibitors are able to decrease seizure severity and duration in animal models of refractory epilepsy. Through rational drug design and screening of a portfolio of chemically distinct compounds, the Program has developed a leading expertise in the SAR and modeling of dynamin inhibition (Fig 1F.), and a library of potent compounds, including candidates that exhibit potent and selective dynamin I activity. A number of compounds have been evaluated in vivo, and induce anti-convulsant efficacy in the 6Hz and amygdala kindling animal models. The lead scaffold is supported by profiling data, SAR across a library of analogs and encouraging pharmacokinetic properties. Support for the Clinical Safety of Dynamin Inhibition Although dynamin represents a novel target, the Program has evidence that a number of clinically used (non-epilepsy) CNS drugs have inhibitory off-target activity on this enzyme at similar in vitro potencies to the Program s proprietary drug candidates. Moreover, animal studies have demonstrated that these drugs also exhibit anti-convulsant activity in vivo. This data supports the potential safety (and efficacy) of dynamin as a therapeutic target and provides insights into other possible applications for dynamin inhibitors in disease indications for which these clinically utilised drugs are prescribed. Application of Dynamin Modulators for Broader Therapeutic Indications In addition to epilepsy, dynamin modulators that work via different mechanisms of action may be applicable to a range of diseases with significant unmet medical needs. CMRI and The University of Newcastle have developed potent compounds and in vivo proof of principle efficacy data relevant to a number of other disease indications, including kidney disease, cancer and infectious diseases. 4

5 Drug Development Team The Dynamin Drug Discovery Program represents a unique and extensive collaboration of leading medicinal chemists, fundamental biologists, translational biologists and clinicians from a range of institutions, working together under a Collaborative Research and Commercialisation Agreement to develop dynamin modulator therapeutic candidates. The lead principle investigators in this Program are Professor Phil Robinson (CMRI), Professor Adam McCluskey (University of Newcastle) and Professor Terence O Brien (University of Melbourne). The lead commercial institution of the collaborative parties is CMRI. Bio-Link is a biotechnology business development company assisting CMRI to secure commercial partners for the Dynamin Drug Discovery Program. Government Subsidies An industry collaboration with CMRI may be eligible to access Australian government funding to subsidise further drug discovery work, de-risking any upfront investment from a biopharma partner. A Commercial Opportunity for Development of Therapeutic Drugs for Epilepsy Bio-Link is seeking an industry partner for preclinical and clinical development of dynamin inhibitor drug candidates for epilepsy. Christian Toouli PhD, Executive Director PO Box 666, Glebe, 237, NSW, Australia, Ph c.toouli@bio-link.com References: 1 GBI Research (March, 214): Epilepsy Therapeutics in Major Developed Markets to New AEDs with Novel Mechanisms of Action Signal a Shift in Patterns; 2 EuroEPINOMICS-RES Consortium, Epilepsy Phenome/Genome Project and Epi4K Consortium (214) De Novo Mutations in Synaptic Transmission Genes Including DNM1 Cause Epileptic Encephalopathies. American Journal of Human Genetics 95, 36-37; 3 Bialer, M. & White, H.S. (2) Key factors in the discovery and development of new antiepileptic drugs. Nat Rev Drug Discov 9, 68-82; 4 Anggono et al., (26), Nature Neuroscience, 9(6):