Transesterification of Indazole-3-carboxamide Synthetic Cannabinoids: Identification of Metabolite Biomarkers for Diagnosing Co-abuse of 5F-MDMB-PINACA and Alcohol.

Concurrent use of alcohol with synthetic cannabinoids (SCs) has been widely recorded among drug abusers. The susceptibilities of three indazole-3-carboxamide type SCs with methyl ester moiety, 5F-MDMB-PINACA, 5F-MMB-PINACA, and MMB-FUBINACA, to transesterification in the presence of ethanol warranted further investigation in view of probable augmented toxicity. In vitro metabolite identification experiments were first performed using human liver microsomes (HLMs) to characterize the novel metabolites of the three parent SCs in the presence of ethanol. Formation of transesterified metabolite, hydrolyzed metabolite, and several oxidative metabolites in HLM in the presence of alcohol was further determined for each parent SC and the respective ethyl ester analog, 5F-EDMB-PINACA, 5F-EMB-PINACA, and EMB-FUBINACA, to quantitatively elucidate transesterification and hydrolysis activities. Our results suggested that all three SCs undergo carboxylesterase-mediated transesterification to their respective ethyl ester analog in the presence of ethanol, which was incubation time- and ethanol concentration-dependent. Each ethyl ester metabolite was sequentially and readily metabolized to novel oxidative metabolites with the intact ethyl ester moiety and the same hydrolyzed metabolite as derived from its parent SC. A smaller extent of transesterification was non-enzymatically driven. Notably, we proposed 5F-EDMB-PINACA oxidative defluorination metabolite as the biomarker for diagnosing the potential co-abuse of 5F-MDMB-PINACA and alcohol. Due to the comparable pharmacological activities between each SC and its ethyl ester metabolite, augmented toxicity associated with co-abuse of SCs and alcohol is probable and deserves further investigation.

Metabolic profiles and screening tactics for MDMB-4en-PINACA in human urine and serum samples.

MDMB-4en-PINACA (Methyl 3,3-dimethyl-2-[1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido] butanoate) is a potent agonist of the CB1 receptor. In 2021, it was one of the most common synthetic cannabinoid receptor agonists (SCRAs) seized by the Beijing Drug Control Agency. MDMB-4en-PINACA can be hard to detect in biological specimens because of ester hydrolysis. In this work, a highly sensitive liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was developed for the detection of MDMB-4en-PINACA metabolites in urine, serum, and hair samples. Metabolites from authentic samples were compared with those from human liver microsomes (HLMs) in vitro and in zebrafish in vivo. A total of 75 metabolites, including 44 previously unreported metabolites, were identified from urine samples. We found that 11 metabolic pathways were involved in MDMB-4en-PINACA metabolism, including acetylation, a novel metabolic pathway for SCRAs. Our results revealed that ester hydrolysis and hydroxylation were to the major metabolic pathways involved in MDMB-4en-PINACA metabolism. Using serum samples, we detected 9 metabolites along with the parent drug. Only the parent drug was detected using hair samples. The existence of ADB-4en-PINACA makes the currently used biomarkers for MDMB-4enPINACA not very specific for the intake of MDMB-4en-PINACA. Therefore, based on the identified metabolites and their structural features, we propose more sensitive screening tactics for MDMB-4en-PINACA using urine and serum samples.

The lonidamine derivative H2-gamendazole reduces cyst formation in polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a debilitating renal neoplastic disorder with limited treatment options. It is characterized by the formation of large fluid-filled cysts that develop from kidney tubules through abnormal cell proliferation and cyst-filling fluid secretion driven by cAMP-dependent Cl- secretion. We tested the effectiveness of the indazole carboxylic acid H2-gamendazole (H2-GMZ), a derivative of lonidamine, to inhibit these processes using in vitro and in vivo models of ADPKD. H2-GMZ was effective in rapidly blocking forskolin-induced, Cl--mediated short-circuit currents in human ADPKD cells, and it significantly inhibited both cAMP- and epidermal growth factor-induced proliferation of ADPKD cells. Western blot analysis of H2-GMZ-treated ADPKD cells showed decreased phosphorylated ERK and decreased hyperphosphorylated retinoblastoma levels. H2-GMZ treatment also decreased ErbB2, Akt, and cyclin-dependent kinase 4, consistent with inhibition of heat shock protein 90, and it decreased levels of the cystic fibrosis transmembrane conductance regulator Cl- channel protein. H2-GMZ-treated ADPKD cultures contained a higher proportion of smaller cells with fewer and smaller lamellipodia and decreased cytoplasmic actin staining, and they were unable to accomplish wound closure even at low H2-GMZ concentrations, consistent with an alteration in the actin cytoskeleton and decreased cell motility. Experiments using mouse metanephric organ cultures showed that H2-GMZ inhibited cAMP-stimulated cyst growth and enlargement. In vivo, H2-GMZ was effective in slowing postnatal cyst formation and kidney enlargement in the Pkd1flox/flox: Pkhd1-Cre mouse model. Thus, H2-GMZ treatment decreases Cl- secretion, cell proliferation, cell motility, and cyst growth. These properties, along with its reported low toxicity, suggest that H2-GMZ might be an attractive candidate for treatment of ADPKD.NEW & NOTEWORTHY Autosomal dominant polycystic kidney disease (ADPKD) is a renal neoplastic disorder characterized by the formation of large fluid-filled cysts that develop from kidney tubules through abnormal cell proliferation and cyst-filling fluid secretion driven by cAMP-dependent Cl- secretion. This study shows that the lonidamine derivative H2-GMZ inhibits Cl- secretion, cell proliferation, and cyst growth, suggesting that it might have therapeutic value for the treatment of ADPKD.

Indazole-based microtubule-targeting agents as potential candidates for anticancer drugs discovery.

Tremendous research is focused on developing novel drug candidates targeting microtubules to inhibit their function in several cellular processes, including cell division. In this regard, several indazole derivatives were sought to target the colchicine binding site on the ß-tubulin, a crucial protein required to form microtubules, to develop microtubule targeting agents. Even though there are several reviews on the indazole-based compounds, none of them focused on using indazole scaffold to develop microtubule targeting agents. Therefore, this review aims to present the advances in research on compounds containing indazole scaffolds as microtubule targeting agents based on the articles published in the last two decades. Among the articles reviewed, we found that compounds 6 and 7 showed the lowest IC50 values of 0.6 âˆ¼ 0.9 nM in the cell line studies, making them the strongest indazole derivatives that target microtubules. The compounds 30, 31, 37 (IC50 = ∼ 1 nM) and compounds 8, 38 (IC50 = ∼ 2 nM) have proved to be potent microtubule inhibitors. The compounds 18, 31, 44, 45 also showed strong anticancer activity (IC50 = ∼ 8 nM). It is important to notice that except for compounds 9, 12, 13, 15, and SRF, the top activity compounds including 6, 7, 8, 10, 11, 30, 31, 37, 44, and 45 contain 3,4,5­trimethoxyphenyl substitution similar to that of colchicine. Therefore, it appears that the 3,4,5­trimethoxyphenyl substituent on the indazole scaffold is crucial for targeting CBS.

Discovery of Novel GR Ligands toward Druggable GR Antagonist Conformations Identified by MD Simulations and Markov State Model Analysis.

Binding of different ligands to glucocorticoid receptor (GR) may induce different conformational changes and even trigger completely opposite biological functions. To understand the allosteric communication within the GR ligand binding domain, the folding pathway of helix 12 (H12) induced by the binding of the agonist dexamethasone (DEX), antagonist RU486, and modulator AZD9567 are explored by molecular dynamics simulations and Markov state model analysis. The ligands can regulate the volume of the activation function-2 through the residues Phe737 and Gln738. Without ligand or with agonist binding, H12 swings from inward to outward to visit different folding positions. However, the binding of RU486 or AZD9567 perturbs the structural state, and the passive antagonist state appears more stable. Structure-based virtual screening and in vitro bioassays are used to discover novel GR ligands that bias the conformation equilibria toward the passive antagonist state. HP-19 exhibits the best anti-inflammatory activity (IC50 = 0.041 ± 0.011 µm) in nuclear factor-kappa B signaling pathway, which is comparable to that of DEX. HP-19 also does not induce adverse effect-related transactivation functions of GR. The novel ligands discovered here may serve as promising starting points for the development of GR modulators.

Entrectinib-A SARS-CoV-2 Inhibitor in Human Lung Tissue (HLT) Cells.

Since the start of the COVID-19 outbreak, pharmaceutical companies and research groups have focused on the development of vaccines and antiviral drugs against SARS-CoV-2. Here, we apply a drug repurposing strategy to identify drug candidates that are able to block the entrance of the virus into human cells. By combining virtual screening with in vitro pseudovirus assays and antiviral assays in Human Lung Tissue (HLT) cells, we identify entrectinib as a potential antiviral drug.

Physiologically-Based Pharmacokinetic Modelling of Entrectinib Parent and Active Metabolite to Support Regulatory Decision-Making.

BACKGROUND AND OBJECTIVE: Entrectinib is a selective inhibitor of ROS1/TRK/ALK kinases, recently approved for oncology indications. Entrectinib is predominantly cleared by cytochrome P450 (CYP) 3A4, and modulation of CYP3A enzyme activity profoundly alters the pharmacokinetics of both entrectinib and its active metabolite M5. We describe development of a combined physiologically based pharmacokinetic (PBPK) model for entrectinib and M5 to support dosing recommendations when entrectinib is co-administered with CYP3A4 inhibitors or inducers. METHODS: A PBPK model was established in Simcyp® Simulator. The initial model based on in vitro-in vivo extrapolation was refined using sensitivity analysis and non-linear mixed effects modeling to optimize parameter estimates and to improve model fit to data from a clinical drug-drug interaction study with the strong CYP3A4 inhibitor, itraconazole. The model was subsequently qualified against clinical data, and the final qualified model used to simulate the effects of moderate to strong CYP3A4 inhibitors and inducers on entrectinib and M5 pharmacokinetics. RESULTS: The final model showed good predictive performance for entrectinib and M5, meeting commonly used predictive performance acceptance criteria in each case. The model predicted that co-administration of various moderate CYP3A4 inhibitors (verapamil, erythromycin, clarithromycin, fluconazole, and diltiazem) would result in an average increase in entrectinib exposure between 2.2- and 3.1-fold, with corresponding average increases for M5 of approximately 2-fold. Co-administration of moderate CYP3A4 inducers (efavirenz, carbamazepine, phenytoin) was predicted to result in an average decrease in entrectinib exposure between 45 and 79%, with corresponding average decreases for M5 of approximately 50%. CONCLUSIONS: The model simulations were used to derive dosing recommendations for co-administering entrectinib with CYP3A4 inhibitors or inducers. PBPK modeling has been used in lieu of clinical studies to enable regulatory decision-making.

AKAP9 supports spermatogenesis through its effects on microtubule and actin cytoskeletons in the rat testis.

In mammalian testes, extensive remodeling of the microtubule (MT) and actin cytoskeletons takes place in Sertoli cells across the seminiferous epithelium to support spermatogenesis. However, the mechanism(s) involving regulatory and signaling proteins remains poorly understood. Herein, A-kinase anchoring protein 9 (AKAP9, a member of the AKAP multivalent scaffold protein family) was shown to be one of these crucial regulatory proteins in the rat testis. Earlier studies have shown that AKAP9 serves as a signaling platform by recruiting multiple signaling and regulatory proteins to create a large protein complex that binds to the Golgi and centrosome to facilitate the assembly of the MT-nucleating γ-tubulin ring complex to initiate MT polymerization. We further expanded our earlier studies based on a Sertoli cell-specific AKAP9 knockout mouse model to probe the function of AKAP9 by using the techniques of immunofluorescence analysis, RNA interference (RNAi), and biochemical assays on an in vitro primary Sertoli cell culture model, and an adjudin-based animal model. AKAP9 robustly expressed across the seminiferous epithelium in adult rat testes, colocalizing with MT-based tracks, and laid perpendicular across the seminiferous epithelium, and prominently expressed at the Sertoli-spermatid cell-cell anchoring junction (called apical ectoplasmic specialization [ES]) and at the Sertoli cell-cell interface (called basal ES, which together with tight junction [TJ] created the blood-testis barrier [BTB]) stage specifically. AKAP9 knockdown in Sertoli cells by RNAi was found to perturb the TJ-permeability barrier through disruptive changes in the distribution of BTB-associated proteins at the Sertoli cell cortical zone, mediated by a considerable loss of ability to induce both MT polymerization and actin filament bundling. A considerable decline in AKAP9 expression and a disruptive distribution of AKAP9 across the seminiferous tubules was also noted during adjudin-induced germ cell (GC) exfoliation in this animal model, illustrating AKAP9 is essential to maintain the homeostasis of cytoskeletons to maintain Sertoli and GC adhesion in the testis.

KRIBB11 Induces Apoptosis in A172 Glioblastoma Cells via MULE-Dependent Degradation of MCL-1.

KRIBB11, an HSF1 inhibitor, was shown to sensitize various types of cancer cells to treatment with several anticancer drugs. However, the exclusive effects of KRIBB11 in preventing the growth of glioblastoma cells and the related mechanisms have not been elucidated yet. Herein, we aimed to examine the potential of KRIBB11 as an anticancer agent for glioblastoma. Using MTT and colony formation assays and Western blotting for c-PARP, we demonstrated that KRIBB11 substantially inhibits the growth of A172 glioma cells by inducing apoptosis. At the molecular level, KRIBB11 decreased anti-apoptotic protein MCL-1 levels, which was attributable to the increase in MULE ubiquitin ligase levels. However, the constitutive activity of HSF1 in A172 cells was not influenced by the exclusive treatment with KRIBB11. Additionally, based on cycloheximide chase assay, we found that KRIBB11 markedly retarded the degradation of MULE. In conclusion, stabilization of MULE upon KRIBB11 treatment is apparently an essential step for degradation of MCL-1 and the subsequent induction of apoptosis in A172 cells. Our results have expanded the knowledge on molecular pathways controlled by KRIBB11 and could be potentially effective for developing an inhibitory therapeutic strategy for glioblastoma.

Fluorescence umpolung enables light-up sensing of N-acetyltransferases and nerve agents.

Intramolecular charge transfer (ICT) is a fundamental mechanism that enables the development of numerous fluorophores and probes for bioimaging and sensing. However, the electron-withdrawing targets (EWTs)-induced fluorescence quenching is a long-standing and unsolved issue in ICT fluorophores, and significantly limits the widespread applicability. Here we report a simple and generalizable structural-modification for completely overturning the intramolecular rotation driving energy, and thus fully reversing the ICT fluorophores' quenching mode into light-up mode. Specifically, the insertion of an indazole unit into ICT scaffold can fully amplify the intramolecular rotation in donor-indazole-π-acceptor fluorophores (fluorescence OFF), whereas efficiently suppressing the rotation in their EWT-substituted system (fluorescence ON). This molecular strategy is generalizable, yielding a palette of chromophores with fluorescence umpolung that spans visible and near-infrared range. This strategy expands the bio-analytical toolboxes and allows exploiting ICT fluorophores for light-up sensing of EWTs including N-acetyltransferases and nerve agents.

X-ray Structure-Guided Discovery of a Potent, Orally Bioavailable, Dual Human Indoleamine/Tryptophan 2,3-Dioxygenase (hIDO/hTDO) Inhibitor That Shows Activity in a Mouse Model of Parkinson's Disease.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan 2,3-dioxygenase (hTDO) have been closely linked to the pathogenesis of Parkinson's disease (PD); nevertheless, development of dual hIDO1 and hTDO inhibitors to evaluate their potential efficacy against PD is still lacking. Here, we report biochemical, biophysical, and computational analyses revealing that 1H-indazole-4-amines inhibit both hIDO1 and hTDO by a mechanism involving direct coordination with the heme ferrous and ferric states. Crystal structure-guided optimization led to 23, which manifested IC50 values of 0.64 and 0.04 µM to hIDO1 and hTDO, respectively, and had good pharmacokinetic properties and brain penetration in mice. 23 showed efficacy against the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse motor coordination deficits, comparable to Madopar, an anti-PD medicine. Further studies revealed that different from Madopar, 23 likely has specific anti-PD mechanisms involving lowering IDO1 expression, alleviating dopaminergic neurodegeneration, reducing inflammatory cytokines and quinolinic acid in mouse brain, and increasing kynurenic acid in mouse blood.

Discovery of Potent and Brain-Penetrant Tau Tubulin Kinase 1 (TTBK1) Inhibitors that Lower Tau Phosphorylation In Vivo.

Structural analysis of the known NIK inhibitor 3 bound to the kinase domain of TTBK1 led to the design and synthesis of a novel class of azaindazole TTBK1 inhibitors exemplified by 8 (cell IC50: 571 nM). Systematic optimization of this series of analogs led to the discovery of 31, a potent (cell IC50: 315 nM) and selective TTBK inhibitor with suitable CNS penetration (rat Kp,uu: 0.32) for in vivo proof of pharmacology studies. The ability of 31 to inhibit tau phosphorylation at the disease-relevant Ser 422 epitope was demonstrated in both a mouse hypothermia and a rat developmental model and provided evidence that modulation of this target may be relevant in the treatment of Alzheimer's disease and other tauopathies.

Hypoxia-Activated Prodrug Enabling Synchronous Chemotherapy and HIF-1α Downregulation for Tumor Treatment.

The overexpression of HIF-1α in solid tumors due to hypoxia is closely related to drug resistance and consequent treatment failure. Herein, we constructed a hypoxia-activated prodrug named as YC-Dox. This prodrug could be activated under hypoxic conditions and undergo self-immolation to release doxorubicin (Dox) and YC-1 hemisuccinate (YCH-1), which could execute chemotherapy and result in HIF-1α downregulation, respectively. This prodrug is capable of specifically releasing Dox and YCH-1 in response to hypoxia, leading to a substantial synergistic potency and a remarkable cytotoxic selectivity (>8-fold) for hypoxic cancer cells over normoxic healthy cells. The in vivo experiments reveal that this prodrug can selectively aim at hypoxic cancer cells and avoid undesired targeting of normal cells, leading to elevated therapeutic efficacy for tumor treatment and minimized adverse effects on normal tissues.

New synthetic cannabinoids carrying a cyclobutyl methyl side chain: Human Phase I metabolism and data on human cannabinoid receptor 1 binding and activation of Cumyl-CBMICA and Cumyl-CBMINACA.

Synthetic cannabinoids (SCs) represent a large group of new psychoactive substances (NPS), sustaining a high prevalence on the drug market since their first detection in 2008. Cumyl-CBMICA and Cumyl-CBMINACA, the first representatives of a new subclass of SCs characterized by a cyclobutyl methyl (CBM) moiety, were identified in July 2019 and February 2020. This work aimed at evaluating basic pharmacological characteristics and human Phase I metabolism of these compounds. Human Phase I metabolites were tentatively identified by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QToF-MS) of urine samples and confirmed by a pooled human liver microsome (pHLM) assay. The basic pharmacological evaluation was performed by applying a competitive ligand binding assay and a functional activation assay (GTPγS) using cell membranes carrying the human cannabinoid receptor 1 (hCB1 ). Investigation of the human Phase I metabolism resulted in the identification of specific urinary markers built by monohydroxylation or dihydroxylation. Although Cumyl-CBMICA was primarily hydroxylated at the indole ring, hydroxylation of Cumyl-CBMINACA mainly occurred at the CBM moiety. Both substances acted as agonists at the hCB1 receptor, although substantial differences could be observed. Cumyl-CBMINACA showed higher binding affinity (Ki = 1.32 vs. 29.3 nM), potency (EC50 = 55.4 vs. 497 nM), and efficacy (Emax = 207% vs. 168%) than its indole counterpart Cumyl-CBMICA. This study confirms that substitution of an indole by an indazole core tends to increase in vitro potency, which is potentially reflected by higher in vivo potency. The emergence and disappearance of SCs distributed via online shops carrying a CBM moiety once more demonstrate the "cat-and-mouse" game between manufacturers and legislation.

In Vivo Metabolites of AB-PINACA in Solid Tissues Obtained from Its Abuser: Comparison with In Vitro Experiment.

In this study, solid tissues such as the lung, liver, kidney and urine were highlighted to profile the AB-PINACA in vivo metabolites in a fatal abuse case, although such metabolite analysis is usually made with urine specimens. We compared the relative peak intensities of in vivo metabolites of AB-PINACA in lung, liver, kidney and urine specimens collected at the autopsy of its abuser with its in vitro metabolites in human hepatocytes. The metabolites of AB-PINACA in tissues were extracted after homogenization. The urine specimen and portions of the extracted metabolites from tissues were firstly hydrolyzed with ß-glucuronidase, and the metabolites were extracted. For in vitro experiment, AB-PINACA was incubated with human hepatocytes for 3 h to produce its metabolites. The identification of the in vivo and in vitro metabolites was performed using liquid chromatography (LC)-high-resolution Orbitrap-tandem mass spectrometry (MS-MS), and the relative intensities of these metabolites were measured using low resolution LC-quadrupole-ion trap-MS-MS. Thirteen metabolites of AB-PINACA were characterized in vivo in several human specimens and in in vitro human hepatocytes. They were produced by the terminal amide hydrolysis to carboxylic acid, hydroxylation, carbonyl formation and/or glucuronidation. The most detectable metabolite in the hepatocytes, lung or liver was the one produced by the terminal amide hydrolysis, whereas the top metabolite in the kidney or urine was the one produced by hydroxylation or carbonyl formation on the pentyl side chain after the terminal amide hydrolysis, respectively. At least 12 metabolites of AB-PINACA were detected in authentic human lung, liver or kidney specimen from a cadaver. It is concluded that the postmortem metabolite profiling of AB-PINACA can be fulfilled with solid tissues, and the lung and kidney were most recommendable especially when urine specimen is not available.

In Vitro Interaction of AB-FUBINACA with Human Cytochrome P450, UDP-Glucuronosyltransferase Enzymes and Drug Transporters.

AB-FUBINACA, a synthetic indazole carboxamide cannabinoid, has been used worldwide as a new psychoactive substance. Because drug abusers take various drugs concomitantly, it is necessary to explore potential AB-FUBINACA-induced drug-drug interactions caused by modulation of drug-metabolizing enzymes and transporters. In this study, the inhibitory effects of AB-FUBINACA on eight major human cytochrome P450s (CYPs) and six uridine 5'-diphospho-glucuronosyltransferases (UGTs) of human liver microsomes, and on eight clinically important transport activities including organic cation transporters (OCT)1 and OCT2, organic anion transporters (OAT)1 and OAT3, organic anion transporting polypeptide transporters (OATP)1B1 and OATP1B3, P-glycoprotein, and breast cancer resistance protein (BCRP) in transporter-overexpressing cells were investigated. AB-FUBINACA inhibited CYP2B6-mediated bupropion hydroxylation via mixed inhibition with Ki value of 15.0 µM and competitively inhibited CYP2C8-catalyzed amodiaquine N-de-ethylation, CYP2C9-catalyzed diclofenac 4'-hydroxylation, CYP2C19-catalyzed [S]-mephenytoin 4'-hydroxylation, and CYP2D6-catalyzed bufuralol 1'-hydroxylation with Ki values of 19.9, 13.1, 6.3, and 20.8 µM, respectively. AB-FUBINACA inhibited OCT2-mediated MPP+ uptake via mixed inhibition (Ki, 54.2 µM) and competitively inhibited OATP1B1-mediated estrone-3-sulfate uptake (Ki, 94.4 µM). However, AB-FUBINACA did not significantly inhibit CYP1A2, CYP2A6, CYP3A4, UGT1A1, UGT1A3, UGT1A4, UGT1A6, or UGT2B7 enzyme activities at concentrations up to 100 µM. AB-FUBINACA did not significantly inhibit the transport activities of OCT1, OAT1/3, OATP1B3, P-glycoprotein, or BCRP at concentrations up to 250 µM. As the pharmacokinetics of AB-FUBINACA in humans and animals remain unknown, it is necessary to clinically evaluate potential in vivo pharmacokinetic drug-drug interactions induced by AB-FUBINACA-mediated inhibition of CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, OCT2, and OATP1B1 activities.

Topical 'dual-soft' glucocorticoid receptor agonist for dermatology.

Steroidal glucocorticoids (GR agonists) have been widely used for the topical treatment of skin disorders, including atopic dermatitis. They are a very effective therapy, but they are associated with both unwanted local effects in the skin (skin thinning/atrophy) and systemic side effects. These effects can limit the long-term utility of potent steroids. Here we report on a topically delivered non-steroidal GR agonist, that has the potential to deliver high efficacy in the skin, but due to rapid metabolism in the blood & liver ("dual-soft") it should have greater systemic safety than existing treatments. In addition, compared to less selective steroidal GR agonists, the new non-steroidal Selective Glucocorticoid Agonists (SEGRAs) have the potential to avoid the skin atrophy observed with existing topical steroids. Due to its potential for reduced skin atrophy and low systemic exposure, LEO 134310 (17) may be suitable for long term topical treatment of skin diseases such as atopic dermatitis and psoriasis.

Theoretical Investigations on Interactions of Arylsulphonyl Indazole Derivatives as Potential Ligands of VEGFR2 Kinase.

Vascular endothelial growth factor receptor 2 (VEGFR2) is a key receptor in the angiogenesis process. The VEGFR2 expression is upregulated in many cancers so this receptor is an important target for anticancer agents. In the present paper, we analyse interactions of several dimeric indazoles, previously investigated for anticancer activity, with the amino acids present in the VEGFR2 binding pocket. Using the docking method and MD simulations as well as theoretical computations (SAPT0, PIEDA, semi-empirical PM7), we confirmed that these azoles can efficiently bind into the kinase pocket and their poses can be stabilised by the formation of hydrogen bonds, π-π stacking, π-cation, and hybrid interactions with some amino acids of the kinase cavity like Ala866, Lys868, Glu885, Thr916, Glu917, and Phe918.

Optimization of a series of potent, selective and orally bioavailable SYK inhibitors.

Spleen tyrosine kinase (SYK) is a non-receptor cytosolic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signaling, inhibition of SYK has been targeted in a variety of disease areas. Herein, we report the optimization of a series of potent and selective SYK inhibitors, focusing on improving metabolic stability, pharmacokinetics and hERG inhibition. As a result, we identified 30, which exhibited no hERG activity but unfortunately was poorly absorbed in rats and mice. We also identified a SYK chemical probe, 17, which exhibits excellent potency at SYK, and an adequate rodent PK profile to support in vivo efficacy/PD studies.

Structure-Based Drug Discovery of N-((R)-3-(7-Methyl-1H-indazol-5-yl)-1-oxo-1-(((S)-1-oxo-3-(piperidin-4-yl)-1-(4-(pyridin-4-yl)piperazin-1-yl)propan-2-yl)amino)propan-2-yl)-2'-oxo-1',2'-dihydrospiro[piperidine-4,4'-pyrido[2,3-d][1,3]oxazine]-1-carboxamide (HTL22562): A Calcitonin Gene-Related Peptide Receptor Antagonist for Acute Treatment of Migraine.

Structure-based drug design enabled the discovery of 8, HTL22562, a calcitonin gene-related peptide (CGRP) receptor antagonist. The structure of 8 complexed with the CGRP receptor was determined at a 1.6 Å resolution. Compound 8 is a highly potent, selective, metabolically stable, and soluble compound suitable for a range of administration routes that have the potential to provide rapid systemic exposures with resultant high levels of receptor coverage (e.g., subcutaneous). The low lipophilicity coupled with a low anticipated clinically efficacious plasma exposure for migraine also suggests a reduced potential for hepatotoxicity. These properties have led to 8 being selected as a clinical candidate for acute treatment of migraine.