Elucidating host cell response pathways and repurposing therapeutics for SARS-CoV-2 and other coronaviruses.

COVID-19, first reported in late 2019, is an ongoing pandemic that has been causing devastation across the globe. Although there are multiple vaccines that can prevent severe symptoms, effective COVID-19 therapeutics are still of importance. Using our proprietary in silico engine, we screened more than 22,000 unique compounds represented by over half a million gene expression profiles to uncover compounds that can be repurposed for SARS-CoV-2 and other coronaviruses in a timely and cost-efficient manner. We then tested 13 compounds in vitro and found three with potency against SARS-CoV-2 with reasonable cytotoxicity. Bortezomib and homoharringtonine are some of the most promising hits with IC50 of 1.39 µM and 0.16 µM, respectively for SARS-CoV-2. Tanespimycin and homoharringtonine were effective against the common cold coronaviruses. In-depth analysis highlighted proteasome, ribosome, and heat shock pathways as key targets in modulating host responses during viral infection. Further studies of these pathways and compounds have provided novel and impactful insights into SARS-CoV-2 biology and host responses that could be further leveraged for COVID-19 therapeutics development.

Amide-based inhibitors of p38alpha MAP kinase. Part 1: discovery of novel N-pyridyl amide lead molecules.

A novel series of N-pyridyl amides as potent p38alpha kinase inhibitors is described. Based on the structural similarities between the initial hit and a well-known imidazole pyrimidine series of p38alpha inhibitors, potencies within the newly discovered series were quickly improved by installation of an (S)-alpha-methylbenzyl moiety at the 2-position of the pyridine ring. The proposed binding modes of the new series to p38alpha were evaluated against SAR findings and provided rationale for further development of this series of molecules.

Amide-based inhibitors of p38alpha MAP kinase. Part 2: design, synthesis and SAR of potent N-pyrimidyl amides.

Optimization of a tri-substituted N-pyridyl amide led to the discovery of a new class of potent N-pyrimidyl amide based p38alpha MAP kinase inhibitors. Initial SAR studies led to the identification of 5-dihydrofuran as an optimal hydrophobic group. Additional side chain modifications resulted in the introduction of hydrogen bond interactions. Through extensive SAR studies, analogs bearing free amino groups and alternatives to the parent (S)-alpha-methyl benzyl moiety were identified. These compounds exhibited improved cellular activities and maintained balance between p38alpha and CYP3A4 inhibition.

Design and synthesis of piperazine-indole p38 alpha MAP kinase inhibitors with improved pharmacokinetic profiles.

Derivatives of the 4-fluorobenzyl dimethylpiperazine-indole class of p38alpha MAP kinase inhibitors are described. Biological evaluation of these compounds focused on maintaining activity while improving pharmacokinetic (PK) properties. Improved properties were observed for structures bearing substitutions on the benzylic methylene.

Piperidine-based heterocyclic oxalyl amides as potent p38 alpha MAP kinase inhibitors.

The design and synthesis of a new class of p38alpha MAP kinase inhibitors based on 4-fluorobenzylpiperidine heterocyclic oxalyl amides are described. Many of these compounds showed low-nanomolar activities in p38alpha enzymatic and cell-based cytokine TNFalpha production inhibition assays. The optimal linkers between the piperidine and the oxalyl amide were found to be [6,5] fused ring heterocycles. Substituted indoles and azaindoles were favored structural motifs in the cellular assay.

p38 MAPK inhibition reduces diabetes-induced impairment of wound healing.

In healthy tissue, a wound initiates an inflammatory response characterized by the presence of a hematoma, infiltration of inflammatory cells into the wound and, eventually, wound healing. In pathological conditions like diabetes mellitus, wound healing is impaired by the presence of chronic nonresolving inflammation. p38 mitogen-activated protein kinase (MAPK) inhibitors have demonstrated anti-inflammatory effects, primarily by inhibiting the expression of inflammatory cytokines and regulating cellular traffic into wounds. The db/db mouse model of type 2 diabetes was used to characterize the time course of expression of activated p38 during impaired wound healing. The p38α-selective inhibitor, SCIO-469, was applied topically and effects on p38 activation and on wound healing were evaluated. A topical dressing used clinically, Promogran™, was used as a comparator. In this study, we established that p38 is phosphorylated on Days 1 to 7 post-wounding in db/db mice. Further, we demonstrated that SCIO-469, at a dose of 10 µg/wound, had a positive effect on wound contraction, granulation tissue formation, and re-epithelialization, and also increased wound maturity during healing. These effects were similar to or greater than those observed with Promogran™. These results suggest a novel approach to prophylactic and therapeutic management of chronic wounds associated with diabetes or other conditions in which healing is impaired.

Pharmacological properties of SD-282 - an alpha-isoform selective inhibitor for p38 MAP kinase.

The effects of small-molecule p38 inhibitors in numerous models of different disease states have been published, including those of SD-282, an indole-5-carboxamide inhibitor. The aim of the present study was to evaluate the pharmacological activity of SD-282 on cytokine production in vitro as well as in 2 in vivo models of inflammation in order to illuminate the role of this particular inhibitor in diverse disease states. The results presented here provide further characterization of SD-282 and provide a context in which to interpret the activity of this p38 inhibitor in models of arthritis, pain, myocardial injury, sepsis and asthma; all of which have an inflammatory component. SD-282 represents a valuable tool to elucidate the role of p38 MAP kinase in multiple models of inflammation.

Importance of p38 mitogen-activated protein kinase pathway in allergic airway remodelling and bronchial hyperresponsiveness.

p38 mitogen-activated protein kinase (MAPK) plays an important role in the activation of inflammatory cells and in the proliferation of airway structural cells. We investigated the role of p38 MAPK by using a selective inhibitor of p38 alpha and beta isoforms, SD282, in a chronic model of 15 ovalbumin exposures in sensitised mice using two doses (30 and 90 mg/kg). Allergen exposure induced bronchial hyperresponsiveness to methacholine as measured by the concentration of methacholine needed to increase pulmonary resistance by 200% (PC200), eosinophilia in bronchoalveolar lavage fluid and increase in airway smooth muscle area and goblet cell hyperplasia. In addition, p38 MAPK activity as measured by phosphorylated p38 expression on Western blots was increased after allergen challenge, which was suppressed by SD282 at both doses. SD282 inhibited bronchial hyperresponsiveness, but had no effect on eosinophils in bronchoalveolar lavage fluid. It also reduced airway smooth muscle and goblet cell hyperplasia, but had no effect on serum immunoglobulin E. p38 MAPK is involved in the pathogenesis of bronchial hyperresponsiveness but not in eosinophilic inflammation or the allergic response; however, remodelling features such as airway smooth muscle or goblet cell hyperplasia are regulated through p38 MAPK. Furthermore, bronchial hyperresponsiveness induced by chronic allergen exposure may be related to the development of airway wall remodelling.

Preventive and therapeutic potential of p38 alpha-selective mitogen-activated protein kinase inhibitor in nonobese diabetic mice with type 1 diabetes.

Mitogen-activated protein kinases (MAPKs) and heat shock proteins (HSPs) are ubiquitous proteins that function within T cells in both normal and stress-related pathophysiological states, including type 1 diabetes. The nonobese diabetic (NOD) mouse spontaneously develops T cell-mediated autoimmune pancreatic beta cell destruction that is similar to type 1 diabetes in humans. Because p38 MAPKs have been shown to modulate T cell function, we studied the effects of a p38alpha MAPK-selective inhibitor, indole-5-carboxamide (SD-169), on the development and progression of type 1 diabetes in the NOD mouse. In preventive treatment studies, SD-169 significantly reduced p38 and HSP60 expression in T cells of the pancreatic beta islets. Following treatment, the incidence of diabetes as determined by blood glucose levels was significantly lower, and immuno-histochemistry of pancreatic beta islet tissue demonstrated significant reduction in CD5+ T cell infiltration in the SD-169 treatment group as compared with untreated NOD mice. In therapeutic studies using mildly and moderately hyperglycemic NOD mice, SD-169 treatment lowered blood glucose and improved glucose homeostasis. Furthermore, following cessation of SD-169 treatment, NOD mice showed significant arrest of diabetes. In conclusion, we report that this p38alpha-selective inhibitor prevents the development and progression of diabetes in NOD mice by inhibiting T cell infiltration and activation, thereby preserving beta cell mass via inhibition of the p38 MAPK signaling pathway. These results have bearing on current prophylactic and therapeutic protocols using p38alpha-selective inhibitors in the prediabetic period for children at high risk of type 1 diabetes, in the honeymoon period, and for adults with latent autoimmune diabetes.

A selective p38 alpha mitogen-activated protein kinase inhibitor reverses cartilage and bone destruction in mice with collagen-induced arthritis.

Destruction of cartilage and bone is a poorly managed hallmark of human rheumatoid arthritis (RA). p38 Mitogen-activated protein kinase (MAPK) has been shown to regulate key proinflammatory pathways in RA, including tumor necrosis factor alpha, interleukin (IL)-1beta, and cyclooxygenase-2, as well as the process of osteoclast differentiation. Therefore, we evaluated whether a p38alpha MAPK inhibitor, indole-5-carboxamide (SD-282), could modulate cartilage and bone destruction in a mouse model of RA induced with bovine type II collagen [collagen-induced arthritis (CIA)]. In mice with early disease, SD-282 treatment significantly improved clinical severity scores, reduced bone and cartilage loss, and reduced mRNA levels of proinflammatory genes in paw tissue, including IL-1beta, IL-6, and cyclooxygenase-2. Notably, SD-282 treatment of mice with advanced disease resulted in significant improvement in clinical severity scoring and paw swelling, a reversal in bone and cartilage destruction as assessed by histology, bone volume fraction and thickness, and three-dimensional image analysis. These changes were accompanied by reduced osteoclast number and lowered levels of serum cartilage oligomeric matrix protein, a marker of cartilage breakdown. Thus, in a model of experimental arthritis associated with significant osteolysis, p38alpha MAPK inhibition not only attenuates disease progression but also reverses cartilage and bone destruction in mice with advanced CIA disease.

p38 Inhibition attenuates the pro-inflammatory response to C-reactive protein by human peripheral blood mononuclear cells.

An active role for C-reactive protein (CRP) in inflammatory vascular diseases has been recently suggested. Monocytes play an important role in vascular pathology and are activated by p38 mitogen activated protein kinase (MAPK) dependent mechanisms in many inflammatory settings. Therefore, we investigated whether CRP directly promotes a pro-inflammatory phenotype in human peripheral blood mononuclear cells (HPBMC) via p38 MAPK signaling. CRP exposure leads to a rapid phosphorylation of p38 MAPK in HPBMC. CRP-induced p38 kinase activity in HPBMC was blocked by treatment with an inhibitor of p38 kinase, SD-282. CRP-induced the expression of tissue factor protein and the secretion of IL-6, IL-8, IL-1beta, TNFalpha and PGE(2). Co-exposure to CRP and SD-282 blocked the secretion of these pro-inflammatory and pro-thrombotic mediators. CRP treatment elevated IL-6, IL-8, IL-1beta, TNFalpha, COX-2 and TF mRNA expression. These effects of CRP also required p38 activity, since SD-282 blocked mRNA induction of each. Taken together these data suggest a mechanistic relationship between p38 MAPK signaling and CRP-induced pro-inflammatory and pro-thrombotic activities in HPBMC. Thus, p38 inhibition may represent a novel approach to attenuate inflammation and its consequences in cardiovascular disease.

Indole-based heterocyclic inhibitors of p38alpha MAP kinase: designing a conformationally restricted analogue.

p38alpha Mitogen Activated Protein Kinase (MAP kinase) is an intracellular soluble serine threonine kinase. p38alpha kinase is activated in response to cellular stresses, growth factors and cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). The central role of p38alpha activation in settings of both chronic and acute inflammation has led efforts to find inhibitors of this enzyme as possible therapies for diseases such as rheumatoid arthritis, where p38alpha activation is thought to play a causal role. Herein, we report structure-activity relationship studies on a series of indole-based heterocyclic inhibitors that led to the design and identification of a new class of p38alpha inhibitors.