First-in-class MKK4 inhibitors enhance liver regeneration and prevent liver failure.

Diminished hepatocyte regeneration is a key feature of acute and chronic liver diseases and after extended liver resections, resulting in the inability to maintain or restore a sufficient functional liver mass. Therapies to restore hepatocyte regeneration are lacking, making liver transplantation the only curative option for end-stage liver disease. Here, we report on the structure-based development and characterization (nuclear magnetic resonance [NMR] spectroscopy) of first-in-class small molecule inhibitors of the dual-specificity kinase MKK4 (MKK4i). MKK4i increased liver regeneration upon hepatectomy in murine and porcine models, allowed for survival of pigs in a lethal 85% hepatectomy model, and showed antisteatotic and antifibrotic effects in liver disease mouse models. A first-in-human phase I trial (European Union Drug Regulating Authorities Clinical Trials [EudraCT] 2021-000193-28) with the clinical candidate HRX215 was conducted and revealed excellent safety and pharmacokinetics. Clinical trials to probe HRX215 for prevention/treatment of liver failure after extensive oncological liver resections or after transplantation of small grafts are warranted.

Small-molecule inhibition of MAP2K4 is synergistic with RAS inhibitors in KRAS-mutant cancers.

The Kirsten rat sarcoma viral oncogene homologue KRAS is among the most commonly mutated oncogenes in human cancers, thus representing an attractive target for precision oncology. The approval for clinical use of the first selective inhibitors of G12C mutant KRAS therefore holds great promise for cancer treatment. However, despite initial encouraging clinical results, the overall survival benefit that patients experience following treatment with these inhibitors has been disappointing to date, pointing toward the need to develop more powerful combination therapies. Here, we show that responsiveness to KRASG12C and pan-RAS inhibitors in KRAS-mutant lung and colon cancer cells is limited by feedback activation of the parallel MAP2K4-JNK-JUN pathway. Activation of this pathway leads to elevated expression of receptor tyrosine kinases that reactivate KRAS and its downstream effectors in the presence of drug. We find that the combination of sotorasib, a drug targeting KRASG12C, and the MAP2K4 inhibitor HRX-0233 prevents this feedback activation and is highly synergistic in a panel of KRASG12C-mutant lung and colon cancer cells. Moreover, combining HRX-0233 and sotorasib is well-tolerated and resulted in durable tumor shrinkage in mouse xenografts of human lung cancer cells, suggesting a therapeutic strategy for KRAS-driven cancers.

Ready-to-use nanopore platform for label-free small molecule quantification: Ethanolamine as first example.

In recent decades, nanopores have become a promising diagnostic tool. Protein and solid-state nanopores are increasingly used for both RNA/DNA sequencing and small molecule detection. The latter is of great importance, as their detection is difficult or expensive using available methods such as HPLC or LC-MS. DNA aptamers are an excellent detection element for sensitive and specific detection of small molecules. Herein, a method for quantifying small molecules using a ready-to-use sequencing platform is described. Taking ethanolamine as an example, a strand displacement assay is developed in which the target-binding aptamer is displaced from the surface of magnetic particles by ethanolamine. Non-displaced aptamer and thus the ethanolamine concentration are detected by the nanopore system and can be quantified in the micromolar range using our in-house developed analysis software. This method is thus the first to describe a label-free approach for the detection of small molecules in a protein nanopore system.

Targeting Lewy body dementia with neflamapimod-rasagiline hybrids.

Lewy body dementia (LBD) represents the second most common neurodegenerative dementia but is a quite underexplored therapeutic area. Nepflamapimod (1) is a brain-penetrant selective inhibitor of the alpha isoform of the mitogen-activated serine/threonine protein kinase (MAPK) p38α, recently repurposed for LBD due to its remarkable antineuroinflammatory properties. Neuroprotective propargylamines are another class of molecules with a therapeutical potential against LBD. Herein, we sought to combine the antineuroinflammatory core of 1 and the neuroprotective propargylamine moiety into a single molecule. Particularly, we inserted a propargylamine moiety in position 4 of the 2,6-dichlorophenyl ring of 1, generating neflamapimod-propargylamine hybrids 3 and 4. These hybrids were evaluated using several cell models, aiming to recapitulate the complexity of LBD pathology through different molecular mechanisms. The N-methyl-N-propargyl derivative 4 showed a nanomolar p38α-MAPK inhibitory activity (IC50 = 98.7 nM), which is only 2.6-fold lower compared to that of the parent compound 1, while displaying no hepato- and neurotoxicity up to 25 µM concentration. It also retained a similar immunomodulatory profile against the N9 microglial cell line. Gratifyingly, at 5 µM concentration, 4 demonstrated a neuroprotective effect against dexamethasone-induced reactive oxygen species production in neuronal cells that was higher than that of 1.

Targeting SARS-CoV-2 Main Protease (MPro) with Kinase Inhibitors: A Promising Approach for Discovering Antiviral and Anti-inflammatory Molecules against SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infected over 688 million people worldwide, causing public health concern and approximately 6.8 million deaths due to COVID-19. COVID-19, especially severe cases, is characterized by exacerbated lung inflammation with an increase of pro-inflammatory cytokines. In addition to antiviral drugs, there is a need for anti-inflammatory therapies to treat all phases of COVID-19. One of the most attractive drug targets for COVID-19 is the SARS-CoV-2 main protease (MPro), an enzyme responsible for cleaving polyproteins formed after the translation of viral RNA, which is essential for viral replication. MPro inhibitors, therefore, have the potential to stop viral replication and act as antiviral drugs. Considering that several kinase inhibitors are known for their action in inflammatory pathways, this could also be investigated toward a potential anti-inflammatory treatment for COVID-19. Therefore, the use of kinase inhibitors against SARS-CoV-2 MPro may be a promising strategy to find molecules with dual activity─antiviral and anti-inflammatory. Considering this, the potential of six kinase inhibitors against SARS-CoV-2 MPro were evaluated in silico and in vitro, including Baricitinib, Tofacitinib, Ruxolitinib, BIRB-796, Skepinone-L, and Sorafenib. To assess the inhibitory potential of the kinase inhibitors, a continuous fluorescent-based enzyme activity assay was optimized with SARS-CoV-2 MPro and MCA-AVLQSGFR-K(Dnp)-K-NH2 (substrate). BIRB-796 and Baricitinib were identified as inhibitors of SARS-CoV-2 MPro, presenting IC50 values of 7.99 and 25.31 µM, respectively. As they are also known for their anti-inflammatory action, both are prototype compounds with the potential to present antiviral and anti-inflammatory activity against SARS-CoV-2 infection.

MKK4 Inhibitors-Recent Development Status and Therapeutic Potential.

MKK4 (mitogen-activated protein kinase kinase 4; also referred to as MEK4) is a dual-specificity protein kinase that phosphorylates and regulates both JNK (c-Jun N-terminal kinase) and p38 MAPK (p38 mitogen-activated protein kinase) signaling pathways and therefore has a great impact on cell proliferation, differentiation and apoptosis. Overexpression of MKK4 has been associated with aggressive cancer types, including metastatic prostate and ovarian cancer and triple-negative breast cancer. In addition, MKK4 has been identified as a key regulator in liver regeneration. Therefore, MKK4 is a promising target both for cancer therapeutics and for the treatment of liver-associated diseases, offering an alternative to liver transplantation. The recent reports on new inhibitors, as well as the formation of a startup company investigating an inhibitor in clinical trials, show the importance and interest of MKK4 in drug discovery. In this review, we highlight the significance of MKK4 in cancer development and other diseases, as well as its unique role in liver regeneration. Furthermore, we present the most recent progress in MKK4 drug discovery and future challenges in the development of MKK4-targeting drugs.

Isolation and Identification of Phytocompounds from Maytenus dhofarensis and Their Biological Potentials.

Maytenus dhofarensis Sebsebe (Celestraceae) is a naturally growing shrub in Oman. It is not a reputed medicinal plant in Oman, but it is regionally endemic and causes shivering attacks on goats that graze on it. The chemical investigation of the hexane and chloroform extracts of the fruits and stems of M. dhofarensis afforded dihydro-ß-agarofuran-type sesquiterpene pyridine alkaloid (1), lupanyl myristoate (2) and lignanolactone (3). Compounds (1-3) are new isolates from M. dhofarensis. The structures of these compounds were assigned through comprehensive IR, NMR, and ESI-MS analyses, and the relative configurations of compounds 1 and 3 were deduced from density function theory (DFT) calculations and NMR experiments. Compound 1 was assayed against the kinase enzyme and showed no inhibition activity for p38 alpha and delta at a 10 µM test concentration. Compound 3 inhibited the 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH) by 69.5%, compared to 70.9% and 78.0% for gallic acid and butylated hydroxyanisole, respectively, which were used as positive controls.

Isostearic acid is an active component of imiquimod formulations used to induce psoriaform disease models.

Topical imiquimod based creams are indicated as immune stimulants for papillomas and various skin neoplasms. Imiquimod is considered a TLR7 ligand. These creams are also used in research to induce skin inflammation in mice as a model for psoriasis. We observed that this inflammatory response was not strictly imiquimod dependent and we set out to establish which components drive the proinflammatory effects. To this end, we examined the induction response in a BALB/cJRj mouse model, in which 50 mg of cream is applied to 2 cm2 of skin (125 mg/kg imiquimod-5% W/V, and/or 625 mg/kg isostearic acid-25% W/V). Comparing cream formulations containing isostearic acid, imiquimod and the combination, we observed that isostearic acid causes skin inflammation within 2 days, whereas imiquimod requires up to 5 days for initial signs. Isostearic acid activated an inflammasome response, stimulated release of proinflammatory cytokines and upregulated the IL-23/17 axis. Animals treated with isostearic acid had enlarged livers (+ 40% weight), which was not observed with imiquimod alone. Imiquimod was readily metabolized and cleared from plasma and liver, but was maintained at high levels in the skin throughout the body (200 mM at area of application; 200 µM in untreated skin). Imiquimod application was associated with splenomegaly, cytokine induction/release and initial body weight loss over 3 days. Despite high imiquimod skin levels throughout the animal, inflammation was only apparent in the treated areas and was less severe than in isostearic acid groups. As the concentrations in these areas are well above the 10 µM required for TLR7 responses in vitro, there is an implication that skin inflammation following imiquimod is due to effects other than TLR7 agonism (e.g., adenosine receptor agonism). In brain, isostearic caused no major changes in cytokine expression while imiquimod alone sightly stimulated expression of IL-1ß and CCL9. However, the combination of both caused brain induction of CCL3, -9, CXCL10, -13, IL-1ß and TNFα. The implication of these data is that isostearic acid facilitates the entry of imiquimod or peripherally secreted cytokines into the brain. Our data suggest that psoriaform skin responses in mice are more driven by isostearic acid, than generally reported and that the dose and route used in the model, leads to profound systemic effects, which may complicate the interpretation of drug effects in this model.

Immune cell targeted fumaric esters support a role of GPR109A as a primary target of monomethyl fumarate in vivo.

Dimethyl fumarate (DMF) is approved as a treatment for multiple sclerosis (MS), however, its mode of action remains unclear. One hypothesis proposes that Michael addition to thiols by DMF, notably glutathione is immunomodulatory. The alternative proposes that monomethyl fumarate (MMF), the hydrolysis product of DMF, is a ligand to the fatty acid receptor GPR109A found in the lysosomes of immune cells. We prepared esters of MMF and macrolides derived from azithromycin, which were tropic to immune cells by virtue of lysosomal trapping. We tested the effects of these substances in an assay of response to Lipopolysaccharide (LPS) in freshly isolated human peripheral blood mononuclear cells (PBMCs). In this system, we observed that the 4'' ester of MMF (compound 2 and 3) reduced levels of Interleukins (IL)-1ß, IL-12 and tumor necrosis factor alpha (TNFα) significantly at a concentration of 1 µM, while DMF required about 25 µM for the same effect. The 2' esters of MMF (compound 1 and 2) were, like MMF itself, inactive in vitro. The 4'' ester formed glutathione conjugates rapidly while the 2' conjugates did not react with thiols but did hydrolyze slowly to release MMF in these cells. We then tested the substances in vivo using the imiquimod/isostearate model of psoriasis where the 2' ester was the most active at 0.06-0.12 mg/kg (approximately 0.1 µmol/kg), improving skin score, body weight and cytokine levels (TNFα, IL-17A, IL-17F, IL-6, IL-1ß, NLRP3 and IL-23A). In contrast, the thiol reactive 4'' ester was less active than the 2' ester while DMF was ca. 300-fold less active. The thiol reactive 4'' ester was not easily recovered from either plasma or organs while the 2' ester exhibited conventional uptake and elimination. The 2' ester also reduced levels of IL-6 in acute monosodium urate (MSU) induced inflammation. These data suggest that mechanisms that are relevant in vivo center on the release of MMF. Given that GPR109A is localized to the lysosome, and that lysosomal trapping increases 2' ester activity by > 300 fold, these data suggest that GPR109A may be the main target in vivo. In contrast, the effects associated with glutathione (GSH) conjugation in vitro are unlikely to be as effective in vivo due to the much lower dose in use which cannot titrate the more concentrated thiols. These data support the case for GPR109A modulation in autoimmune diseases.

Direct 3D printed biocompatible microfluidics: assessment of human mesenchymal stem cell differentiation and cytotoxic drug screening in a dynamic culture system.

BACKGROUND: In vivo-mimicking conditions are critical in in vitro cell analysis to obtain clinically relevant results. The required conditions, comparable to those prevalent in nature, can be provided by microfluidic dynamic cell cultures. Microfluidics can be used to fabricate and test the functionality and biocompatibility of newly developed nanosystems or to apply micro- and nanoelectromechanical systems embedded in a microfluidic system. However, the use of microfluidic systems is often hampered by their accessibility, acquisition cost, or customization, especially for scientists whose primary research focus is not microfluidics. RESULTS: Here we present a method for 3D printing that can be applied without special prior knowledge and sophisticated equipment to produce various ready-to-use microfluidic components with a size of 100 µm. Compared to other available methods, 3D printing using fused deposition modeling (FDM) offers several advantages, such as time-reduction and avoidance of sophisticated equipment (e.g., photolithography), as well as excellent biocompatibility and avoidance of toxic, leaching chemicals or post-processing (e.g., stereolithography). We further demonstrate the ease of use of the method for two relevant applications: a cytotoxicity screening system and an osteoblastic differentiation assay. To our knowledge, this is the first time an application including treatment, long-term cell culture and analysis on one chip has been demonstrated in a directly 3D-printed microfluidic chip. CONCLUSION: The direct 3D printing method is tested and validated for various microfluidic components that can be combined on a chip depending on the specific requirements of the experiment. The ease of use and production opens up the potential of microfluidics to a wide range of users, especially in biomedical research. Our demonstration of its use as a cytotoxicity screening system and as an assay for osteoblastic differentiation shows the methods potential in the development of novel biomedical applications. With the presented method, we aim to disseminate microfluidics as a standard method in biomedical research, thus improving the reproducibility and transferability of results to clinical applications.

2,2,2-Trifluoroethanol-mediated hydroarylation of fluorinated alkynes with indoles: Application to diindolylmethanes.

A new mild and practically simple alkyne hydroarylation protocol for the synthesis of 3-(indol-3-yl)-3-(trifluoromethyl)acrylic acid esters by the reaction of indole derivatives with ethyl/methyl 4,4,4-trifluoro-3-(indol-3-yl)but-2-enoates in trifluoroethanol was developed. This method has the following advantages: no catalyst, atom economy, high yields, broad substrate scope, and large-scale synthesis. The potential application of this protocol was further demonstrated by the synthesis of a variety of CF3 -substituted synthons and a new class of (un)symmetrical 3,3'-diindolylmethanes with a quaternary carbon core that might be biologically active.

Neuroprotective Effect of Luteolin-7-O-Glucoside against 6-OHDA-Induced Damage in Undifferentiated and RA-Differentiated SH-SY5Y Cells.

Luteolin is one of the most common flavonoids present in edible plants and its potential benefits to the central nervous system include decrease of microglia activation, neuronal damage and high antioxidant properties. The aim of this research was to evaluate the neuroprotective, antioxidant and anti-inflammatory activities of luteolin-7-O-glucoside (Lut7). Undifferentiated and retinoic acid (RA)-differentiated SH-SY5Y cells were pretreated with Lut7 and incubated with 6-hydroxydopamine (6-OHDA). Cytotoxic and neuroprotective effects were determined by MTT assay. Antioxidant capacity was determined by DPPH, FRAP, and ORAC assays. ROS production, mitochondrial membrane potential (ΔΨm), Caspase-3 activity, acetylcholinesterase inhibition (AChEI) and nuclear damage were also determined in SH-SY5Y cells. TNF-α, IL-6 and IL-10 release were evaluated in LPS-induced RAW264.7 cells by ELISA. In undifferentiated SH-SY5Y cells, Lut7 increased cell viability after 24 h, while in RA-differentiated SH-SY5Y cells, Lut7 increased cell viability after 24 and 48 h. Lut7 showed a high antioxidant activity when compared with synthetic antioxidants. In undifferentiated cells, Lut7 prevented mitochondrial membrane depolarization induced by 6-OHDA treatment, decreased Caspase-3 and AChE activity, and inhibited nuclear condensation and fragmentation. In LPS-stimulated RAW264.7 cells, Lut7 treatment reduced TNF-α levels and increased IL-10 levels after 3 and 24 h, respectively. In summary, the results suggest that Lut7 has neuroprotective effects, thus, further studies should be considered to validate its pharmacological potential in more complex models, aiming the treatment of neurodegenerative diseases.

Antiulcerogenic Potential of the Ethanolic Extract of Ceiba speciosa (A. St.-Hil.) Ravenna Evaluated by In Vitro and In Vivo Studies.

Gastrointestinal diseases, such as peptic ulcers, are caused by a damage in the gastric mucosa provoked by several factors. This stomach injury is regulated by many inflammatory mediators and is commonly treated with proton-pump inhibitors, histamine H2 receptor blockers and antacids. However, various medicinal plants have demonstrated positive effects on gastric ulcer treatment, including plants of the Ceiba genus. The aim of this study was to evaluate the antiulcer and anti-inflammatory activities of the stem bark ethanolic extract of Ceiba speciosa (A. St.-Hil.) Ravenna. We performed a preliminary quantification of phenolic compounds by high-performance liquid chromatography-diode array detection (HPLC-DAD), followed by the prospection of other chemical groups through nuclear magnetic resonance (NMR) spectroscopy. A set of in vitro assays was used to evaluate the extract potential regarding its antioxidant activity (DPPH: 19.83 ± 0.34 µg/mL; TPC: 307.20 ± 6.20 mg GAE/g of extract), effects on cell viability and on the release of TNF-α in whole human blood. Additionally, in vivo assays were performed to evaluate the leukocyte accumulation and total protein quantification in carrageenan-induced air pouch, as well as the antiulcerogenic effect of the extract on an ethanol-induced ulcer in rats. The extract contains flavonoids and phenolic compounds, as well as sugars and quinic acid derivatives exhibiting potent antioxidant activity and low toxicity. The extract reduced the release of TNF-α in human blood and inhibited the activity of p38α (1.66 µg/mL), JAK3 (5.25 µg/mL), and JNK3 (8.34 µg/mL). Moreover, it reduced the leukocyte recruitment on the pouch exudate and the formation of edema, reverting the effects caused by carrageenan. The extract presented a significant prevention of ulcer formation and a higher reduction than the reference drug, Omeprazole. Therefore, C. speciosa extract has demonstrated relevant therapeutic potential for the treatment of gastric diseases, deserving the continuation of further studies to unveil the mechanisms of action of plant bioactive ingredients.

In vitro and in vivo anti-inflammatory and anticoagulant activities of Myrciaria plinioides D. Legrand ethanol leaf extract.

Myrciaria plinioides D. Legrand (Myrtaceae) is a native plant of Southern Brazil, which have potential in the food industry due to its edible fruits. Many plants belonging to this genus have been used for a variety of illnesses, including inflammatory disorders due to antioxidant properties. However, therapeutic uses of M. plinioides have been poorly studied. The aim of study was to assess the anti-inflammatory and anticoagulant activities of the ethanol leaf extract of M. plinioides. In M. plinioides extract-treated RAW 264.7 cells, assessments of cell viability, TNF-α release and p38 MAPK pathway-dependent protein expression were detected. In addition, rat paw edema models were used to analyze the anti-inflammatory effect of the extract. Macrophages cell line treated with M. plinioides extract showed a slight decrease in cell viability. In LPS-stimulated macrophages treated with different concentrations of the extract for 24 h, TNF-α release was inhibited, while modulation of p38 signaling pathway and inhibition of NF-κB p65 protein expression were dose-dependent. In rats, the extract inhibited the formation of paw edema, while an inhibitory effect on trypsin-like enzymes derived from mast cells was seen. Furthermore, the extract presented anticoagulant activity via extrinsic pathway, being able to block specifically factor Xa and thrombin. The study suggests that extract possess potent anti-inflammatory and anticoagulant effects. M. plinioides present great biological potential as a source for the development of anti-inflammatory and anticoagulant drugs. Additional studies can be proposed to better elucidate the mechanism by which M. plinioides exerts its effects.

A Highly Selective In Vitro JNK3 Inhibitor, FMU200, Restores Mitochondrial Membrane Potential and Reduces Oxidative Stress and Apoptosis in SH-SY5Y Cells.

Current treatments for neurodegenerative diseases (ND) are symptomatic and do not affect disease progression. Slowing this progression remains a crucial unmet need for patients and their families. c-Jun N-terminal kinase 3 (JNK3) are related to several ND hallmarks including apoptosis, oxidative stress, excitotoxicity, mitochondrial dysfunction, and neuroinflammation. JNK inhibitors can play an important role in addressing neuroprotection. This research aims to evaluate the neuroprotective, anti-inflammatory, and antioxidant effects of a synthetic compound (FMU200) with known JNK3 inhibitory activity in SH-SY5Y and RAW264.7 cell lines. SH-SY5Y cells were pretreated with FMU200 and cell damage was induced by 6-hydroxydopamine (6-OHDA) or hydrogen peroxide (H2O2). Cell viability and neuroprotective effect were assessed with an MTT assay. Flow cytometric analysis was performed to evaluate cell apoptosis. The H2O2-induced reactive oxygen species (ROS) generation and mitochondrial membrane potential (ΔΨm) were evaluated by DCFDA and JC-1 assays, respectively. The anti-inflammatory effect was determined in LPS-induced RAW264.7 cells by ELISA assay. In undifferentiated SH-SY5Y cells, FMU200 decreased neurotoxicity induced by 6-OHDA in approximately 20%. In RA-differentiated cells, FMU200 diminished cell death in approximately 40% and 90% after 24 and 48 h treatment, respectively. FMU200 reduced both early and late apoptotic cells, decreased ROS levels, restored mitochondrial membrane potential, and downregulated JNK phosphorylation after H2O2 exposure. In LPS-stimulated RAW264.7 cells, FMU200 reduced TNF-α levels after a 3 h treatment. FMU200 protects neuroblastoma SH-SY5Y cells against 6-OHDA- and H2O2-induced apoptosis, which may result from suppressing the JNK pathways. Our findings show that FMU200 can be a useful candidate for the treatment of neurodegenerative disorders.

Current jakinibs for the treatment of rheumatoid arthritis: a systematic review.

OBJECTIVE: One-third of patients with severe rheumatoid arthritis (RA) do not achieve remission or low disease activity, or they have side effects from cDMARD and bDMARD. They will need a new treatment option such as the small molecule JAK inhibitors. In this systematic review, we evaluate the efficacy and safety data of the current jakinibs: tofacitinib, peficitinib, decernotinib, upadacitinib, baricitinib and filgotinib in patients in whom treatment with conventional or biological disease-modifying antirheumatic drugs (cDMARD and/or bDMARD) failed. METHODS: We searched for randomized controlled trials comparing efficacy and safety of jakinibs for RA treatment using the Web of Science, Scopus, PubMed, and clinicaltrials.gov databases with the terms: "rheumatoid arthritis" OR "arthritis rheumatoid" OR "RA" AND "inhibitor" OR "jak inhibitor" AND "clinical trial" OR "treatment" OR "therapy". RESULTS: All jakinibs achieved good results in ACR 20, 50, 70 and with CRP-DAS28 for LDA and remission, upadacitinib showed better results compared to the others. In ESR-DAS28 for remission, tofacitinib achieved the best result. Regarding the safety of all jakinibs, peficitinib, baricitinib and filgotinib did not register deaths in their studies unlike tofacitinib that presented 11 deaths. Despite all benefits of jakinibs, the use in patients with severe liver and kidney disease should be avoided. CONCLUSIONS: Jakinibs in monotherapy or in combination with methotrexate can be considered a viable alternative in the treatment of moderate-to-severe RA. Even after failures with combination of cDMARDS and bDMARDS, jakinibs demonstrated efficacy.

Design, Synthesis and Biological Evaluation of Novel Pyrazolo[1,2,4]triazolopyrimidine Derivatives as Potential Anticancer Agents.

Three novel pyrazolo-[4,3-e][1,2,4]triazolopyrimidine derivatives (1, 2, and 3) were designed, synthesized, and evaluated for their in vitro biological activity. All three compounds exhibited different levels of cytotoxicity against cervical and breast cancer cell lines. However, compound 1 showed the best antiproliferative activity against all tested tumor cell lines, including HCC1937 and HeLa cells, which express high levels of wild-type epidermal growth factor receptor (EGFR). Western blot analyses demonstrated that compound 1 inhibited the activation of EGFR, protein kinase B (Akt), and extracellular signal-regulated kinase (Erk)1/2 in breast and cervical cancer cells at concentrations of 7 and 11 µM, respectively. The results from docking experiments with EGFR suggested the binding of compound 1 at the ATP binding site of EGFR. Furthermore, the crystal structure of compound 3 (7-(4-bromophenyl)-9-(pyridin-4-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine) was determined by single crystal X-ray analysis. Our work represents a promising starting point for the development of a new series of compounds targeting EGFR.

Controlling the Covalent Reactivity of a Kinase Inhibitor with Light.

Covalent kinase inhibitors account for some of the most successful drugs that have recently entered the clinic and many others are in preclinical development. A common strategy is to target cysteines in the vicinity of the ATP binding site using an acrylamide electrophile. To increase the tissue selectivity of kinase inhibitors, it could be advantageous to control the reactivity of these electrophiles with light. Here, we introduce covalent inhibitors of the kinase JNK3 that function as photoswitchable affinity labels (PALs). Our lead compounds contain a diazocine photoswitch, are poor non-covalent inhibitors in the dark, and become effective covalent inhibitors after irradiation with visible light. Our proposed mode of action is supported by X-ray structures that explain why these compounds are unreactive in the dark and undergo proximity-based covalent attachment following exposure to light.

Candidate drugs against SARS-CoV-2 and COVID-19.

Outbreak and pandemic of coronavirus SARS-CoV-2 in 2019/2020 will challenge global health for the future. Because a vaccine against the virus will not be available in the near future, we herein try to offer a pharmacological strategy to combat the virus. There exists a number of candidate drugs that may inhibit infection with and replication of SARS-CoV-2. Such drugs comprise inhibitors of TMPRSS2 serine protease and inhibitors of angiotensin-converting enzyme 2 (ACE2). Blockade of ACE2, the host cell receptor for the S protein of SARS-CoV-2 and inhibition of TMPRSS2, which is required for S protein priming may prevent cell entry of SARS-CoV-2. Further, chloroquine and hydroxychloroquine, and off-label antiviral drugs, such as the nucleotide analogue remdesivir, HIV protease inhibitors lopinavir and ritonavir, broad-spectrum antiviral drugs arbidol and favipiravir as well as antiviral phytochemicals available to date may limit spread of SARS-CoV-2 and morbidity and mortality of COVID-19 pandemic.

Duplex Shiny app quantification of the sepsis biomarkers C-reactive protein and interleukin-6 in a fast quantum dot labeled lateral flow assay.

Fast point-of-care (POC) diagnostics represent an unmet medical need and include applications such as lateral flow assays (LFAs) for the diagnosis of sepsis and consequences of cytokine storms and for the treatment of COVID-19 and other systemic, inflammatory events not caused by infection. Because of the complex pathophysiology of sepsis, multiple biomarkers must be analyzed to compensate for the low sensitivity and specificity of single biomarker targets. Conventional LFAs, such as gold nanoparticle dyed assays, are limited to approximately five targets-the maximum number of test lines on an assay. To increase the information obtainable from each test line, we combined green and red emitting quantum dots (QDs) as labels for C-reactive protein (CRP) and interleukin-6 (IL-6) antibodies in an optical duplex immunoassay. CdSe-QDs with sharp and tunable emission bands were used to simultaneously quantify CRP and IL-6 in a single test line, by using a single UV-light source and two suitable emission filters for readout through a widely available BioImager device. For image and data processing, a customized software tool, the MultiFlow-Shiny app was used to accelerate and simplify the readout process. The app software provides advanced tools for image processing, including assisted extraction of line intensities, advanced background correction and an easy workflow for creation and handling of experimental data in quantitative LFAs. The results generated with our MultiFlow-Shiny app were superior to those generated with the popular software ImageJ and resulted in lower detection limits. Our assay is applicable for detecting clinically relevant ranges of both target proteins and therefore may serve as a powerful tool for POC diagnosis of inflammation and infectious events.