Objective
Our objective for this JDRF grant is to produce new drugs that prevent stressed insulin-producing pancreatic beta cells from committing suicide under the duress of being attacked by the immune system. Beta cells are hard-working and yet fragile by their very nature. Their loss leads to the requirement for lifelong insulin injections in patients with type 1 diabetes. However, insulin injections (or pumps) are no match for the exquisite control that one's own beta cells provides to control blood glucose levels, both during fasting and after meals. This grant is predicated on what is now a rich body of studies showing that beta cells have the tendency to self immolate during stressed states. This particular type of stress starts in a cellular compartment called the endoplasmic reticulum, where insulin first starts being produced, and this effect culminates in another cellular compartment called the mitochondria, which trigger a signal for the beta cells to commit suicide by a process called apoptosis. We have identified a crucial protein in the endoplasmic reticulum called IRE1 that begins this apoptosis process, and we are making chemicals that inhibit IRE1's tendency to push these beta cells towards apoptosis. Thus our objective is to arrive at new potential drugs called kinase inhibitors—to test in the clinic for patients with new-onset type 1 diabetes—through modifying the internal response to endoplasmic reticulum stress by sparing these stressed from self-destruction though a pathway involving IRE1 kinase in the "unfolded protein response" towards apoptosis.
Background Rationale
Type 1 Diabetes (T1D) is a disease that upon auto-immune attack of pancreatic islets results in loss of blood glucose control when a critical fraction of insulin-producing, pancreatic islet -cells become dysfunctional and then die. We are asking here: why—and how—do these immune-targeted beta-cells die? We are also asking: what can we do to intervene during critical pathogenic periods of T1D to prevent, and perhaps even to reverse this disease, by sparing these beta-cells from death leading to T1D? We are learning that immune cell infiltration may not be sufficient to cause islet beta-cell death, but may also cause the highly stressed beta-cells to commit suicide during disease progression. Many clinical trials have attempted to halt T1D progression through targeting the immune system.
Recently, upon its approval by the FDA, the anti-CD3 monoclonal antibody (Teplizumab), has emerged as an immune cell-modulating prophylactic therapy to delay onset to T1D in high-risk who have developed antibodies to beta-cells. But disease-modifying treatments are still needed to spare stressed beta-cells from suicide in the “honeymoon” period when beta-cells may be potentially saved and even regenerated in new-onset T1D patients. By their very nature, beta-cells work at the limits of their protein secretory capacity and are therefore prone to exhaustion. Beta-Cells are essentially protein secreting "factories" that continuously produce and secrete large quantities of insulin product to maintain normal blood glucose levels. Faced with this enormous burden, protein-folding and structural maturation can easily become saturated in the -cell endoplasmic reticulum (ER)—the first cellular organelle (cellular compartment) of the secretory pathway—where insulin matures, causing a type of stress we call “ER stress”.
ER stress activates the “unfolded protein response (UPR)”, which is an ancient intracellular signaling pathway that attempts to remedy the ER stress. Under manageable levels of ER stress, the UPR temporarily halts production of secretory proteins such as insulin, and injects molecular chaperones and other protein modification enzymes into the ER to reduce ER stress. Such an “Adaptive” UPR restores cellular health. But under levels of ER stress that are too high to be contained, the UPR becomes hyperactive, leading to inflammation and death of the highly ER stressed -cells. We call this set of destructive outputs, the “Terminal” UPR (‘T’-UPR). We learned that in mouse models of T1D the initial autoimmune attack precipitates islet inflammation, drives up ER stress, causing A- to T-UPR conversions in beta-cells leading to beta-cell apoptosis and diabetes.
The most ancient arm of the UPR is controlled by IRE1alpha, an ER transmembrane protein containing bifunctional kinase/endoribonuclease (RNase) catalytic activities. We found that IRE1 controls binarily opposite cell fate outcomes, depending on the force of upstream ER stress—i.e., IRE1alpha operates as a life-death switch. Unfolded ER proteins cause IRE1alpha to dimerize and trans-autophosphorylate, which in turn activates its RNase to initiate frame-shift splicing of XBP1 mRNA. Low-level XBP1 mRNA splicing produces XBP1s (‘s’= spliced) transcription factor, whose mRNA targets promote adaptive protein secretion. But under irremediable ER stress, sustained IRE1 autophosphorylation—driven by IRE1alpha homo-oligomerization—hyperactivates IRE1’s RNase to trigger massive endonucleolytic decay of ER-localized mRNAs depleting beta-cell cargo (e.g., insulin) and protein folding components (e.g., chaperones, oxidoreductases, and PC1/3 and PC2 prohormone convertases). Additionally, we found that IRE1alpha RNase hyperactivation causes the cleavage of select micro-RNAs such as mir17(at the pre-miR level) that normally derepress genes whose protein products cause sterile inflammation and senescence, such as TXNIP. Thus, IRE1alpha’s T-UPR outputs cause loss of differentiated beta-cell identity, inflammation, and promote apoptosis.
Thus, we are targeting IRE1alpha’s T-UPR outputs while maintaining the adaptive A-UPR outputs, with new kinase inhibitory molecules we are developing, with a pathway to the clinic for T1D.
Description of Project
Type 1 diabetes is caused by the breakdown of the delicate balance of the immune system that normally prevents it from targeting cells in one's own body versus targeting towards outside threats such as viruses and bacteria. In Type 1 diabetes, it is specifically the pancreatic beta cells (which make insulin, a hormone necessary for glucose control and proper metabolism) that become exposed and targeted by the overactive immune system. Through many decades of work, scientists have learned how this breakdown of immune tolerance occurs as the beta cells in patients are invaded by immune cells from their own body. This starts a disease process in which the immune cells cause inflammation and initiate the death of beta cells. This 'homicide" of beta cells by immune cells and how to prevent it has long the been the focus of immunology research. After many years, this research has resulted in the drug development of an antibody infusion called Teplizumab that when given to the relatives of patients with type 1 diabetes, and who are genetically at risk for also eventually getting the disease, prolongs the time that it will take for these at-risk individuals to develop type 1 diabetes. However, it is also emerging the beta cells themselves, when targets by these immune cells, commit "suicide" under the stress of the invasion by the immune. This stress-induced suicide occurs by the beta cells "talking to themselves" in an internal cellular conversation called "signaling". The signaling normally occurs through a molecular system called the Unfolded Protein Response that allows insulin to be properly produced. However, we learned that when the Unfolded Protein Response is hyperactive, it will cause the beta cells to commit suicide. Thus, we have identified a protein in the Unfolded Protein Response—called IRE1—whose function starts pushing beta cells towards suicide, and we are making drugs called "kinase inhibitors" that specifically will turn off this internal IRE1 signaling that leads to beta cell suicide and type 1 diabetes. Our work may even have the potential to work in combination with immune therapies and lead to better outcomes in patients with type 1 diabetes, and perhaps those at risk.
Drug development is a long process of target identification, finding and vetting molecules that extract the desired outputs from the target (in this case IRE1), and moving in discrete stages towards clinical trials to the FDA. Our new candidate molecules need further work in human pancreatic beta cell samples to be sure that we are extracting these desired outputs that keep the beta cells alive and producing insulin. We already have some initial data of what we call "target engagement" with our new kinase inhibitors, but we need much more data to show their effectiveness in human pancreatic samples with newer and more refined kinase inhibitors that we are developing through this grant application to JDRF. Here in two stages—called "aims"—we chart out what we intend to do bring these kinase inhibitors to a clinical trial in humans for type 1 diabetes as new drugs that have the potential to prevent and possibly even reverse the disease.
Anticipated Outcome
Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by hyperglycemia due to progressive immune-mediated destruction of pancreatic -cells. The high secretory burden of these insulin-producing professional secretory cells predisposes them to high levels of endoplasmic reticulum (ER) stress. Recent work from our laboratories has shown that the activation of ER stress response pathways such as the unfolded protein response—UPR—may be an initial causal event in the development of T1D. In this collaborative SRA grant to JDRF we will pharmacologically target the UPR with next generation chemical matter—kinase inhibitors called PAIRs—as new beta-cell sparing therapeutic approaches for T1D. Our anticipated outcome is that by disrupting the T-UPR, beta cells will be spared under regimens of high and unmanageable ER stress that lead to diabetes under autoimmune attack of the beta cells, which self-immolate under duress.
We anticipate that new PAIRs we are devising using biochemical and cellular systems will be efficacious in at least three models of type 1 diabetes, leading to progressive advances towards the clinic. The first is using human pancreatic slices from the network of pancreatic organ donors (nPOD). As we show in our research proposal, we already have found that advanced PAIRs can rescue these pancreata and preserve beta-cell function by preserving the secretion of insulin upon a glucose challenge (GSIS) during ER stress. The second is using isolated human pancreatic islets, obtained from our own islet harvesting facility at UCSF, or through commercial sources. These are purified sources of beta cells that can be cultured, subjected to ER stress agents, and hyperglycemia, then interrogated with our PAIR compounds to confirm target engagement of IRE1, and downstream gene expression changes. The physiological effects of engaging isolated human islets with PAIRs will be monitored in these ex vivo (test tube) systems. Finally, we wish to under how our dug candidates perform inside live animals. To this end, we will employ immunodeficient mice, called NSG, that can tolerate the transplantation of human islets under their kidney capsule. We have learned to perform survival surgery on these mice using human islets that substitute for the mice's own islets, which we can ablate. Thus, the human islets in a live animal setting provide a measure of glucose control (which we measure using random and fasting blood glucose levels). The anticipated outcomes on each of these three metrics is that potential, selective, orally-bioavailable PAIRs will progress down a testing cascade leading from biochemistry (kinase/RNase), cellular systems that overexpress IRE1, to the human-relevant models.
Where our project is ultimately innovative is that it translates discoveries we have made from atomic resolution X-ray crystal models, to biochemical assays, to reductionist cellular models, and finally into human islet studies and live animals.
The ultimate direction of this work is to partner with external partners to head towards an investigational new drug (IND) application to the FDA for patients suffering from type 1 diabetes .
Relevance to T1D
The relevance of our project t Type 1 Diabetes is direct and timely. The premature death of -cells, which work at the limits of their protein secretory capacity and are therefore prone to secretory exhaustion, is ultimately the cause of type 1 diabetes, even though immune infiltration is what incites the disease in the first place. -Cells are essentially protein secretory factories that live under a strict metabolic mandate to continuously produce and secrete large quantities of pristine insulin product (even in healthy states, about one million insulin molecules are produced by each -cell per minute) to maintain normal blood glucose levels. So all this becomes a “perfect storm” when the immune system invades the pancreatic islets. Already faced with this enormous insulin secretory burden, even in healthy states, protein-folding and structural maturation can easily become saturated in the -cell endoplasmic reticulum (ER)—the first organelle of the secretory pathway—wherein insulin precursor proinsulin matures, causing “ER stress”. As beta cells are lost through “homicide” by invading immune cells (both innate and adaptive cells), the remaining beta cells have to pick up the slack and work even harder, pushing them into vicious cycles leading inexorably to cell dysfunction and death.
All the aforementioned processes are “active” processes, which is possibly good news from a therapeutic standpoint because we can potentially inactivate these active processes. The grant application here to JDRF moves forward two concepts to the clinic for treatment of type 1 diabetes. The first is the identification of a cellular target—IRE1—in the unfolded protein response with mechanistic rationale from our lab going back over seventeen years with our several publications (whose findings have been replicated and built upon by many labs all the world). The second is the new chemical matter we describe in this grant application, whose advancement will lead to potential drug candidates that have disease-modifying potential towards an IND to treat patients with T1D by testing in future clinical trials.
Finally, we end with the notion that these beta-cell sparing compounds we are developing may possibly synergize with the new immunomodulatory therapy (anti-CD3 antibody) that was just approved by the FDA for at-risk patients in T1D. In conclusion, our mechanism of action is orthogonal to that of Teplizumab’s, which targets immune processes of -cell destruction. Future experiments combining UPR/ER stress modulators with anti-CD3 antibody therapies, though not proposed here, may suggest the potential of synergistic actions in T1D patients (and those at risk for the disease).