SBIR-STTR Award

Cytoplasmic nucleic acids alert the immune system to invading pathogens and trigger a robust type I interferon (IFN) response via activation of the STING (stimulator of interferon genes) receptor. The sensor for cytoplasmic nucleic acids was recently discovered to be cyclic GMP-AMP synthase, an enzyme that produces a unique cyclic dinucleotide second messenger, cGAMP, that serves as an agonist for the STING receptor. Aberrant activation of the cGAS-STING pathway is rapidly emerging as an important underlying cause of debilitating and sometimes fatal autoimmune disorders including Aicardi–Goutieres Syndrome (AGS), a monogenic encephalopathy that is usually fatal before adulthood, and systemic lupus erythematosus (SLE). Development of cGAS inhibitors is clearly a therapeutic strategy that should to be explored. There are no drugs approved specifically for any of the IFN-driven autoimmune diseases, and current IFN-targeted therapies in clinical development are mostly biologicals; e.g., antibodies against IFN? or the type IFN receptor. Inhibiting cGAS, the molecular trigger for type I IFN induction, is likely to be more efficient than blocking downstream targets, and a small molecule cGAS drug would have obvious advantages in terms of cost, dosing and CNS exposure. Aberrant activation of cGAS by dsDNA is the trigger for a constitutive type I IFN response, resulting in autoimmune diseases such AGS and SLE with no curative therapies. We developed an HTS-compatible cGAS enzymatic assay and used it to screen a 100k diversity library resulting in the identification of four novel cGAS inhibitor chemotypes. Following the primary screen, we performed iterative rounds of SAR informed by computational modeling and medicinal chemistry expertise to prioritize two chemotypes with different mechanisms of action, and to increase potency into the nanomolar range. During the course of this work we uncovered interplay between cGAS sensivity to activation by nucleic acids and its modulation by small molecules that could inform the development of drugs with a favorable therapeutic window. The goals of this Phase I proposal are to use structure-based design to improve the potency and ADME/PK properties of the lead chemotypes and to demonstrate target engagement and efficacy in cells. We have made substantial progress toward these goals, including generation of a high resolution co-crystal X-ray structure for one of the lead chemotypes and demonstration of specific inhibition of cGAS-driven IFN gene expression in human monocytes. In Phase I, we will continue to improve the potency and other drug like properties of the two lead chemotypes using structure-based design enabled by a more powerful computational method. In addition, we will use stem cell-derived neural cell models and primary human peripheral immune cells to test inhibitor efficacy in a disease-relevant context. Successful completion of these aims will clearly establish feasibility for Phase II in vivo efficacy studies in a mouse model for AGS, a key milestone for clinical translation. Blocking cGAS with a small molecule could lead to a curative therapy for AGS and would likely spur development of other drugs targeting the cGAS/STING pathway with potential impact on millions of people suffering from debilitating autoimmune diseases. This is a multidisciplinary lead discovery effort that will combine BellBrook's extensive enzymology and screening expertise, medicinal chemistry and translational research expertise from David Maloney and Matt Boxer of Nexus Discovery Advisors (Frederick, MD - LOS and bios attached), structural and biophysical expertise from XTAL BioStructures (Natick, MA, LOS attached) and computational chemistry expertise from SilcsBio (Baltimore, MD – LOS attached). Dr. Keith Elkon, Co- director of the Center for Innate Immunity and Immune Disease and Head of Rheumatology at University of Washington School of Medicine is serving as a consultant (LOS and bio attached). Under a separate SBIR grant (R44GM123833) in collaboration with Dr. Elkon, BellBrook is developing methods for detecting cGAMP in cell and tissue samples to enable monitoring of cGAS inhibition in animal models, and eventually for stratification and monitoring of patients in clinical studies; e.g., AGS patients or SLE patients with high levels of cGAMP in PBMCs as candidates for cGAS inhibitors. The availability of a companion diagnostic would increase the potential medical impact and value of a cGAS inhibitor drug substantially.

Public Health Relevance Statement:
Narrative Aberrant activation of the innate immune machinery that senses DNA from microbial pathogens results in debilitating autoimmune diseases such as lupus and congenital encephalopathies. We are proposing to develop lead molecules targeting a recently discovered enzyme, cyclic GAMP synthase, that serves as the trigger for autoimmunity in response to self-DNA.

Project Terms:
ABCB1 gene; Adult; Affect; Agonist; analog; Animal Model; Antibodies; antimicrobial; antiviral immunity; Apoenzymes; aqueous; Autoantibodies; Autoimmune Diseases; Autoimmune Process; Autoimmunity; B-Lymphocytes; Baltimore; base; Binding; Biochemical; Biological; Biological Assay; Biological Availability; biophysical techniques; Biophysics; Brain; Cell Line; Cell model; Cells; Cessation of life; Clinic; clinical development; Clinical Research; clinical translation; Collaborations; companion diagnostics; Complex; computational chemistry; Computer Simulation; Computing Methodologies; cost; Crystallization; curative treatments; Cyclic GMP; design; Detection; Development; dimer; Dinucleoside Phosphates; Disease; disease phenotype; Diversity Library; DNA; DNA Damage; Dose; drug development; Drug Kinetics; Drug Targeting; ds-DNA; efficacy study; Encephalopathies; Enzymatic Biochemistry; Enzymes; Event; Gap Junctions; Gene Expression; Generations; Goals; Grant; Guanosine Triphosphate; Head; Human; Human Cell Line; Immune; Immune response; Immune system; Immune System Diseases; Immunity; improved; in vivo; in vivo evaluation; induced pluripotent stem cell; inhibitor/antagonist; Innate Immune Response; Interferon Type I; Interferon-alpha; Interferons; Invaded; Kinetics; Knock-out; Lead; Life; Ligands; Lupus; Medical; medical schools; Methods; Mitochondria; Modeling; Molecular; molecular dynamics; Monitor; monocyte; monomer; mouse model; multidisciplinary; Mus; nanomolar; Natural Immunity; neonatal encephalopathy; Neurons; neutrophil; novel; Nuclear; Nucleic Acids; pathogen; Pathogenesis; pathogenic microbe; Pathway interactions; Patient Monitoring; Patients; Periodicity; Peripheral; Peripheral Blood Mononuclear Cell; Permeability; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Phenotype; Plasma; Positioning Attribute; Production; Property; pseudotoxoplasmosis syndrome; receptor; relating to nervous system; Resolution; response; Rheumatology; Roentgen Rays; screening; Second Messenger Systems; sensor; Signal Induction; Signal Transduction; Sjogren's Syndrome; Small Business Innovation Research Grant; small molecule; Solubility; Stem cells; Stimulator of Interferon Genes; Stratification; Structure; Systemic Lupus Erythematosus; T-Lymphocyte; targeted treatment; Testing; Therapeutic; Therapeutic Intervention; Tissue Sample; Tissues; Transcription Factor AP-1; Transcriptional Activation; Translational Research; Translations; Treatment Efficacy; type I interferon receptor; Universities; Washington; Work

We are developing small molecule antagonists for cyclic GAMP synthase (cGAS) to identify a candidate drugmolecule for lupus. Systemic lupus erythematosus (SLE), or simply lupus, is the second most commonautoimmune disease next to rheumatoid arthritis; there are at least 300,000 patients in the U.S. alone and wellover a million globally. The unmet medical need is enormous: lupus patients suffer from a 67% increase inmortality rate with damage to major organs in 50% of cases; e.g., heart, lung, kidneys, and brain; lupus was the5th leading cause of death among young African American and Hispanic women in the U.S. from 2011-2015.There are no curative treatments for lupus, and only one drug (Benlysta) has been approved in the last 50 years. Lupus pathology is driven by type I interferons (IFNs), and the immune sensor, cyclic GAMP synthase(cGAS), is the trigger for type I IFN induction. DNA from dying cells binds to catalytically inactive cGAS to forman activated complex, triggering production of a unique cyclic nucleotide second messenger, cyclic GAMP(cGAMP). cGAMP binds to the STING protein to induce expression of type I IFNs, with autocrine and paracrineeffects that lead to activation of T- and B-cells and auto-antibody production, precipitating a vicious cycle of celldeath and autoimmunity. Using an innovative HTS assay developed under a separate SBIR, we discovered two promising cGASantagonist chemotypes (40783 and 50101) that function via distinct mechanisms and have made substantial progresson increasing their biochemical and cellular potency while maintaining ADME properties predictive of good oralbioavailability. Our structural data indicate that the 40783 chemotype has allosteric binding properties and maystabilize an inactive cGAS conformation, properties which we will leverage in Phase II to develop a highlyselective lead molecule with a long residence time. The 50101 chemotype appears to bind specifically to ahypersensitized cGAS-Mn-DNA complex, which could lead to an improved therapeutic window. In Phase II wepropose to: 1) further optimize the potency, selectivity and ADME properties of the two chemotypes and 2) test theirefficacy in an innovative model for UV-induced photosensitivity that replicates key aspects of SLE pathology andaligns closely with our clinical strategy. The animal efficacy studies will be performed in collaboration with Keith Elkon,Head of Rheumatology at University of Washington, Seattle, who developed the mouse photosensitivity model andhas pioneered research on the involvement of the cGAS/STING pathway in lupus. Most investigational lupus drugs target the downstream effects of type I IFNs, a strategy that is akin toplugging holes in a sinking ship. The development of drugs that target cGAS, the upstream molecular trigger fornucleic-acid driven type I IFN production could revolutionize the treatment of lupus along with a growing list of cGAS-driven autoimmune and inflammatory conditions.

Public Health Relevance Statement:
Narrative Aberrant activation of the innate immune sensor cyclic GAMP synthase leads to chronic overproduction of the inflammatory cytokines that cause extensive tissue damage in lupus. We are proposing to develop selective cyclic GAMP synthase antagonists with properties suitable for oral administration and test them for efficacy in an animal model of lupus-induced photosensitivity.

Project Terms:
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