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Robert Freeze,1 Philip Hughes,1,2 Timothy Haystead,1,2 Scott Scarneo1 1Eydisbio, Inc, Durham, NC, USA; 2Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC, USACorrespondence: Scott Scarneo, Email scott.scarneo@eydisbio.comPurpose: Joint pain is one of the most commonly reported pain types in the United States. In the case of patients suffering from inflammatory diseases such as osteoarthritis (OA) and gout, persistent inflammation due to long-term overexpression of several key cytokines has been linked to neuronal hypersensitivity and damage within the joints. Ultimately, a subset of patients develop chronic pain. Pharmacologic treatment of joint pain involves the use of analgesics such as acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs), opioids, antidepressants, as well as intra-articular injections of corticosteroids and hyaluronic acid. However, NSAIDs are short-acting and fail to alleviate severe pain, opioids are generally ineffective at managing chronic pain, and all therapeutic options involve increased risks of serious side effects.Methods: We explored the therapeutic and analgesic effects of transforming growth factor-β-activated kinase 1 (TAK1) inhibition in both the monoiodoacetate (MIA) and monosodium urate (MSU) models of joint pain as an innovative strategy for alleviating chronic inflammatory pain. Mechanical allodynia (Von Frey), weight-bearing and histological changes were measured in separate groups of rats receiving either the selective TAK1 inhibitor, HS-276, gabapentin or vehicle.Results: Our data support that TAK1 inhibition effectively prevented the development of mechanical allodynia and differential weight-bearing in the MIA model. In the MSU model of gouty arthritis, treatment with HS-276 significantly reduced mechanical allodynia and knee edema in female rats, but not male rats. Histological evaluation of effected joints in both models showed that HS-276 treatment significantly reduced disease-induced degradation of the joint.Conclusion: Our results support that TAK1 is a critical signaling node in inflammatory joint diseases such as OA and gouty arthritis. Selective pharmacological inhibition significantly attenuated several aspects of the disease, including joint degeneration and mechanical pain. Thus, TAK1 is a novel therapeutic target for the treatment of painful inflammatory joint diseases.Perspective: This article reports on the therapeutic potential of TAK1 in the treatment of chronic inflammatory joint diseases such as OA and gout. Using the selective TAK1 inhibitor, HS-276, we show the therapeutic and analgesic effects of TAK1 inhibition in two preclinical murine models of inflammatory joint pain.Keywords: TAK1, joint pain, inflammation, kinase, analgesics

Poor survival and lack of treatment response in glioblastoma (GBM) is attributed to the persistence of glioma stem cells (GSCs). To identify novel therapeutic approaches, we performed CRISPR/Cas9 knockout screens and discovered TGFβ activated kinase (TAK1) as a selective survival factor in a significant fraction of GSCs. Loss of TAK1 kinase activity results in RIPK1-dependent apoptosis via Caspase-8/FADD complex activation, dependent on autocrine TNFα ligand production and constitutive TNFR signaling. We identify a transcriptional signature associated with immune activation and the mesenchymal GBM subtype to be a characteristic of cancer cells sensitive to TAK1 perturbation and employ this signature to accurately predict sensitivity to the TAK1 kinase inhibitor HS-276. In addition, exposure to pro-inflammatory cytokines IFNγ and TNFα can sensitize resistant GSCs to TAK1 inhibition. Our findings reveal dependency on TAK1 kinase activity as a novel vulnerability in immune-activated cancers, including mesenchymal GBMs that can be exploited therapeutically., (© 2024. The Author(s).)

Evidence in SARS-CoV-2 patients have identified that viral infection is accompanied by the expression of inflammatory mediators by both immune and stromal cells within the pulmonary system. However, the immunogenicity of individual SARS-CoV-2 proteins has yet to be evaluated. The SARS-CoV-2 virus consists of 29 proteins, categorized either as nonstructural proteins (NSP's), structural proteins (SP's) or accessory proteins. Here we sought to evaluate the immunogenicity of NSP 1, 7, 8, 9, 10, 12, 14, 16 and the SP's spike protein (full length, S1, S2 and receptor binding domain subunits), nucleocapsid and membrane SARS-CoV-2 proteins against THP-1 and human peripheral blood mononuclear cells (PBMCs). Our results indicate that various SARS-CoV-2 proteins elicit a proinflammatory immune response indicated by increases in cytokines TNF, IL-6 and IL-1β. Our results support that SARS-CoV-2 membrane protein induced a robust increase of TNF, IL-6, IL-1β and IL-10 expression in both THP-1 and human PBMC's. Further evaluation of intranasal membrane protein challenge in male and female BALB/c mice show increases in BALF (bronchalveolar lavage fluid) proinflammatory cytokine expression, elevated cellularity, predominantly neutrophilic, and concomitant peribronchiolar and perivascular lymphomononuclear and neutrophilic inflammation. Our results suggest that individual membrane associated SARS-CoV-2 proteins induce a robust immune response that may contribute to viral induced cytokine release syndrome (CRS) in the lungs of moderate to severe COVID-19 patients. We posit that SARS-CoV-2 membrane challenges in immune-competent mice can serve as an adequate surrogate for the development of novel treatments for SARS-CoV-2 induced pulmonary inflammation, thereby avoiding expensive live virus studies under BSL-3 conditions., (© 2023. The Author(s).)

The origin of chronic pain is linked to inflammation, characterized by increased levels of proinflammatory cytokines in local tissues and systemic circulation. Transforming growth factor beta-activated kinase 1 (TAK1) is a key regulator of proinflammatory cytokine signaling that has been well characterized in the context of cancer and autoimmune disorders, yet its role in chronic pain is less clear. Here, we evaluated the ability of our TAK1 small-molecule inhibitor, takinib, to attenuate pain and inflammation in preclinical models of inflammatory, neuropathic, and primary pain. Inflammatory, neuropathic, and primary pain was modeled using intraplantar complete Freund's adjuvant (CFA), chronic constriction injury (CCI), and systemic delivery of the catechol-O-methyltransferase (COMT) inhibitor OR486, respectively. Behavioral responses evoked by mechanical and thermal stimuli were evaluated in separate groups of mice receiving takinib or vehicle prior to pain induction (baseline) and over 12 days following CFA injection, 4 weeks following CCI surgery, and 6 hours following OR486 delivery. Hindpaw edema was also measured prior to and 3 days following CFA injection. Upon termination of behavioral experiments, dorsal root ganglia (DRG) were collected to measure cytokines. We also evaluated the ability of takinib to modulate nociceptor activity via in vitro calcium imaging of neurons isolated from the DRG of Gcamp3 mice. In all 3 models, TAK1 inhibition significantly reduced hypersensitivity to mechanical and thermal stimuli and expression of proinflammatory cytokines in DRG. Furthermore, TAK1 inhibition significantly reduced the activity of tumor necrosis factor (TNF)-primed/capsaicin-evoked DRG nociceptive neurons. Overall, our results support the therapeutic potential of TAK1 as a novel drug target for the treatment of chronic pain syndromes with different etiologies. PERSPECTIVE: This article reports the therapeutic potential of TAK1 inhibitors for the treatment of chronic pain. This new treatment has the potential to provide a greater therapeutic offering to physicians and patients suffering from chronic pain as well as reduce the dependency on opioid-based pain treatments., (Copyright © 2023 United States Association for the Study of Pain, Inc. Published by Elsevier Inc. All rights reserved.)

Rheumatoid arthritis (RA) is a complex autoimmune disease characterized by hyperactive immune cells within the joints, which leads to inflammation, bone degeneration, and chronic pain. For several decades, frontline immunomodulators such as the anti-tumor necrosis factor (TNF) biologics adalimumab (Humira), etanercept (Enbrel), and infliximab (Remicade) have successfully managed disease progression for many patients. However, over time, patients become refractory to these treatments requiring chronic disease to be managed with conventional and more problematic disease modifying antirheumatic drugs such as methotrexate and hydroxychloroquine, and corticosteroids. Due to the large proportion of patients who continue to fail on frontline biologic therapies, there remains an unmet need to derive novel alternative targets with improved efficacy and safety profiles to treat RA. Recent advances in the field have defined novel targets that play important roles in RA pathology, including the Janus activated kinase (JAK) and transforming growth factor beta activated kinase-1 (TAK1). Although three inhibitors of the JAK signaling pathway have been approved for the treatment of moderately to severely active RA in patients who failed on one or more anti-TNFs, at present, no FDA approved TAK1 treatments exist. Our recent discovery of a highly potent and selective, orally bioavailable TAK1 inhibitor has provided insight into the therapeutic potential of this protein kinase as a novel target for RA. Here, we show the distinct cytokine signaling of tofacitnib (Xeljanz; JAK1/3 inhibitor) compared to HS-276 (TAK1 inhibitor) in lipopolysaccharide (LPS) challenged THP-1 cells. Furthermore, in the collagen induced arthritis pre-clinical mouse model of RA, both tofacintib and HS-276 attenuated disease activity score and inflammatory cytokines in the serum. Overall, our results delineate the distinct cytokine signaling of JAK1/3 and TAK1 targeted therapies in vitro and in vivo and suggest that selective TAK1 inhibitors may provide superior therapeutic relief in RA with fewer adverse events., (© 2023 Eydis Bio, Inc and The Authors. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Selective targeting of TNF in inflammatory diseases such as rheumatoid arthritis (RA) has provided great therapeutic benefit to many patients with chronic RA. Although these therapies show initially high response rates, their therapeutic benefit is limited over the lifetime of the patient due to the development of antidrug antibodies that preclude proper therapeutic benefits. As a result, patients often return to more problematic therapies such as methotrexate or hydroxychloroquine, which carry long-term side effects. Thus, there is an unmet medical need to develop alternative treatments enabling patients to regain the benefits of selectively targeting TNF functions in vivo. The protein kinase TAK1 is a critical signaling node in TNF-mediated intracellular signaling, regulating downstream NF-κβ activation, leading to the transcription of inflammatory cytokines. TAK1 inhibitors have been developed but have been limited in their clinical advancement due to the lack of selectivity within the human kinome and, most importantly, lack of oral bioavailability. Using a directed medicinal chemistry approach, driven by the cocrystal structure of the TAK1 inhibitor takinib, we developed HS-276, a potent ( K i = 2.5 nM) and highly selective orally bioavailable TAK1 inhibitor. Following oral administration in normal mice, HS-276 is well tolerated (MTD >100 mg/Kg), displaying >95% bioavailability with μM plasma levels. The in vitro and in vivo efficacy of HS-276 showed significant inhibition of TNF-mediated cytokine profiles, correlating with significant attenuation of arthritic-like symptoms in the CIA mouse model of inflammatory RA. Our studies reinforce the hypothesis that TAK1 can be safely targeted pharmacologically to provide an effective alternative to frontline biologic-based RA therapeutics.

There are no validated, objective diagnostic or prognostic biomarkers for sports-related concussion (SRC), which hinders evidence-based treatment for concussed athletes. While quantitative electrophysiology (EEG) and diffusion tensor imaging (DTI) are promising technologies for providing objective biomarkers for concussion, the degree to which they are related has not been systematically investigated in concussed athletes. This study examined whether diffusion metrics differentiated concussed athletes with prolonged recovery ( n  = 18) from non-conccused athletes ( n  = 13) and whether observed diffusion alterations related to EEG. Collegiate athletes ( N  = 31) completed EEG, neurocognitive, and magnetic resonance imaging. White matter diffusivity differed between the groups in multiple white matter tracts, including the corpus callosum, cingulum bundle, thalamic radiations, and inferior fronto-occipital, inferior longitudinal, and uncinate fasciculi, but not after correction for multiple comparisons. The enhanced Brain Function Index (eBFI), a measure that combines EEG and neurocognitive data, significantly correlated with altered diffusion in the concussed athletes. These preliminary findings suggest that the absolute deviation of diffusion metrics in concussed versus non-concussed athletes may have clinically utility. Results also suggested that the eBFI may be sensitive to early changes from sports-related concussion.