Objective
Accumulating data from human and rodent studies indicate a key role for stress pathways intrinsic to pancreatic β-cells in the development of type 1 diabetes (T1D). One of these pathways is the so called “ER stress”, characterized by dysfunction of a cellular organelle responsible for the adequate folding of “proteins for export”, including insulin. This leads to accumulation of misfolded proteins in the ER which, if not solved, will eventually trigger cell death. ER stress is present in islets from T1D patients, and work by the Principal Investigators of this project suggests that a crosstalk between β-cell ER stress and innate immunity mediators induces/amplifies insulitis and contributes to trigger T1D. Restoring the ER function to protect β-cells in T1D remains a major unmet need, and this is main objective of the present project.
An adequate calcium (Ca2+) concentration is crucial for ER function, and this is maintained by a specialized protein, the Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA2) pump which actively transports Ca2+ across the ER membrane, maintaining a steep Ca2+ gradient between the ER and cytosol. Our own data demonstrate marked loss of SERCA2 expression and activity in pancreatic islets from models of diabetes, resulting in ER Ca2+ depletion, ER stress, and reduced β-cell function and survival. Neurodon has developed a series of drug candidates that function as selective activators of SERCA2. Importantly, SERCA2 modulation with Neurodon activators prevent ER stress-induced apoptosis in β-cells, improve insulin secretion in islets from human donors with diabetes and delay the onset of diabetes in SERCA2 deficient NOD mice. Against this background, we hypothesize that small molecules capable of increasing SERCA2 activity will improve β-cell health in T1D. To test this hypothesis, and contribute to preserve β-cells in T1D, we will pursue three main aims:
Specific Aim 1: Perform hit-to-lead and lead optimization assays on our novel series of SERCA2 activators to improve biological activity and physical properties. We have identified a set of 30 drug-like allosteric SERCA2 activators that have confirmed efficacy in activating SERCA2 in a dose-responsive manner. We will now perform rational structure-based drug design to synthesize optimized analogues with improved potency and pharmaceutical properties. These studies will generate active lead compounds to be utilized for efficacy studies in Aims 2 and 3.
Specific Aim 2: Evaluate the efficacy of SERCA2 allosteric activators using in vitro models of T1D and ER stress. Top performing SERCA2 activators from Aim 1 will be tested using in vitro models of T1D and ER stress-induced diabetes. Selection of compounds and dosages will initially be determined using human insulin-producing EndoC-βH1 cells. Subsequent experiments will be performed in human islets and iPSC-induced β-cells from non-diabetic donors treated with pro-inflammatory cytokines as a model of T1D, in iPSC-induced β-cells from donors with Darier-White disease (SERCA2 haploinsufficiency), and in iPSC-induced β-cells from donors with mutations in the gene YIPF5, which result in ER stress-induced neonatal diabetes. Endpoints of interest will include ER stress, HLA class I expression, proinflammatory gene expression, insulin secretion, and β-cell death.
Specific Aim 3: Determine the in vivo effect of SERCA2 activation in preclinical mouse models of T1D. The in vivo efficacy of the most promising SERCA2 activators will be tested in wild-type (WT) and SERCA2 haploinsufficient NOD mice (NOD-S2+/-). Endpoints of interest will include insulin secretion, β-cell mass, ER stress activation, islet immune cell infiltration, and diabetes incidence/prevention.
Background Rationale
There is a key role for intrinsic β-cell pathways during the development of type 1 diabetes (T1D). Endoplasmic reticulum (ER) stress become activated early during the evolution of T1D and contribute to β-cell death. The ER plays a vital role in β-cell function, including insulin production, processing, and secretion. Alterations in β-cell ER Ca2+ impact both ER and mitochondrial function, leading to activation of ER stress signaling pathways, increased β-cell immunogenicity and eventual β-cell death. Importantly, markers of ER stress are present in human islets of organ donors with T1D and in β-cells of NOD mice, and PIs of the present project (CE-M and DLE) obtained data suggesting a marked loss of SERCA2 (a pump crucial for the preservation of ER Ca2+) expression and activity in models of diabetes, resulting in ER Ca2+ depletion, ER stress, and reduced β-cell function and survival. These findings suggest that interventions focused on the preservation and restoration of ER Ca2+ may improve β-cell health and survival in T1D. Neurodon has developed a series of drug candidates that function as selective activators of SERCA2 and that prevent ER stress-induced apoptosis in β-cells, improve insulin secretion in islets from human donors with diabetes and delay the onset of diabetes in SERCA2 deficient NOD mice.
Against this background, Neurodon will collaborate closely with Indiana University (IU) and Universite Libre de Bruxelles (ULB) Schools of Medicine to test the hypothesis that small molecules capable of increasing SERCA2 activity will improve β-cell health in T1D.
Description of Project
Pancreatic β-cells increase several-fold the synthesis of insulin during stimulation by glucose and other nutrients. Insulin synthesis/folding takes place in the endoplasmic reticulum (ER), a key organelle for the production of secreted proteins. When faced with an increased demand, β-cells trigger adaptive mechanisms to upgrade the functional capacity of the ER. These mechanisms are known as the unfolded protein response (UPR). Prolonged or excessive UPR can cause β cell death. Cytokines produced by immune cells invading the islets in type 1 diabetes (T1D) induce β-cell ER stress and apoptosis. The UPR is activated in islets from persons with T1D, and work by the Principal Investigators of this project suggests that a crosstalk between the UPR and innate immunity induces/amplifies insulitis and contributes to trigger T1D. Restoring the ER function to protect β-cells in T1D remains a major unmet need.
Normal ER function requires high concentrations of calcium (Ca2+) within the lumen of the organelle. The Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA2) pump actively transports Ca2+ across the ER membrane, maintaining a steep Ca2+ gradient between the ER and cytosol. Inhibition of SERCA2 expression and activity seems to be an “Achilles’ heel” of a β-cell’s ER function, and our own data demonstrate marked loss of SERCA2 expression and activity in models of diabetes, resulting in ER Ca2+ depletion, ER stress, and reduced β-cell function and survival. Neurodon has developed a series of drug candidates that function as selective activators of SERCA. Importantly, SERCA2 modulation by the Neurodon activators prevent ER stress-induced apoptosis in β-cells, improve insulin secretion in islets from human donors with diabetes and delay the onset of diabetes in SERCA2 haploinsufficient NOD mice. Against this background, we hypothesize that small molecules capable of increasing SERCA2 activity will improve β-cell health in T1D. To test this hypothesis, Neurodon will work in close collaboration with Indiana University (IU) and Universite Libre de Bruxelles (ULB) Schools of Medicine to achieve the following aims:
Specific Aim 1: Perform hit-to-lead and lead optimization assays on our novel series of SERCA2 activators to improve biological activity and physical properties. We have identified a set of 30 drug-like allosteric SERCA2 activators that have confirmed efficacy in activating SERCA2 in a dose-responsive manner. We will now perform rational structure-based drug design to synthesize optimized analogues with improved potency and pharmaceutical properties. These studies will generate active lead compounds to be utilized for efficacy studies in Aims 2 and 3.
Specific Aim 2: Evaluate the efficacy of SERCA2 allosteric activators using in vitro models of T1D and ER stress. Top performing SERCA2 activators from Aim 1 will be tested using in vitro models of T1D and ER stress-induced diabetes. Selection of compounds and dosages will initially be determined using human insulin-producing EndoC-βH1 cells. Subsequent experiments will be performed in human islets and iPSC-induced β-cells from non-diabetic donors treated with pro-inflammatory cytokines as a model of T1D, in iPSC-induced β-cells from donors with Darier-White disease (SERCA2 haploinsufficiency), and in iPSC-induced β-cells from donors with mutations in the gene YIPF5, which result in ER stress-induced neonatal diabetes. Endpoints of interest will include ER stress, HLA class I expression, proinflammatory gene expression, insulin secretion, and β-cell death.
Specific Aim 3: Determine the in vivo effect of SERCA2 activation in preclinical mouse models of T1D. The in vivo efficacy of the most promising SERCA2 activators will be tested in wild-type (WT) and SERCA2 haploinsufficient NOD mice (NOD-S2+/-). Endpoints of interest will include insulin secretion, β-cell mass, ER stress activation, islet immune cell infiltration, and diabetes incidence/prevention.
Anticipated Outcome
We expect that the proposed studies will provide compelling pre-clinical justification to pursue additional studies to select one SERCA2 activator for future human trials aiming to protect pancreatic β-cells in the early stages of T1D. Thus, the ultimate outcome of the presently proposed project will be the validation and prioritization of the best SERCA2 stimulator for a future clinical trial.
Of note, Eizirik and Evans-Molina are members of the prioritization committees of respectively INNODIA and TrialNet, and can present the proposals for the use of agents aiming to protect β-cells in clinical trials, once convincing pre-clinical data – hopefully to be generated in the present project – become available.
Relevance to T1D
Pioneering research by the applicants and others indicate that pathways intrinsic to the β-cells play a key role for their deleterious dialog between β-cells and the immune system that contributes to progressive β-cell loss and eventually clinical T1D. Most of the research efforts for the discovery of novel treatments for T1D has focused on the immune system, and innovative therapies that protect β-cells and block the deleterious β-cell-immune system dialogue remain to be discovered. The presently proposed new therapy, focusing on SERCA2 stimulation, is based on solid preliminary data and has the potential to remove one of the key “words” of the β-cell-immune system dialogue, namely β-cell ER stress. This is expected to decrease auto-antigen presentation and β-cell death. Of particular relevance, there is a growing trend in the field to identify combined therapeutic approaches for the prevention of T1D, including both agents that modulate or “re-educate” the immune system and that protect β-cells. In this context, the lack of drugs that restore dysfunctional β-cells remains a major unmet need. If confirmed, our hypothesis that SERCA2 stimulators can protect and/or restore β-cells in the early stages of T1D will provide a new and valuable pharmaceutical adjuvant to prevent - or at least delay - the outbreak of disease.