Selective Wee1 Inhibitors Led to Antitumor Activity In Vitro and Correlated with Myelosuppression.

Wee1 is a tyrosine kinase that is highly expressed in several cancer types. Wee1 inhibition can lead to suppression of tumor cell proliferation and sensitization of cells to the effects of DNA-damaging agents. AZD1775 is a nonselective Wee1 inhibitor for which myelosuppression has been observed as a dose-limiting toxicity. We have applied structure-based drug design (SBDD) to rapidly generate highly selective Wee1 inhibitors that demonstrate better selectivity than AZD1775 against PLK1, which is known to cause myelosuppression (including thrombocytopenia) when inhibited. While selective Wee1 inhibitors described herein still achieved in vitro antitumor efficacy, thrombocytopenia was still observed in vitro.

Determining Clinical Priorities Using a Clinical Practice Guideline Deconstruction Tool: COVID-19 in Austere Operational Environments.

The Joint Trauma System (JTS) publishes Clinical Practice Guidelines (CPGs) used by military and civilian healthcare providers worldwide. With the expansion of CPG development in recent years, there was a need to collate, sort, and deconflict existing and new guidance using systematic methodology both within and across CPGs. This need became readily apparent at the start of the COVID-19 pandemic when guidelines were rapidly developed and fielded in deployed environments. To meet the needs of deploying units requesting immediate and concise guidance for managing COVID-19, JTS developed the CPG entitled Management of Covid-19 in Austere Operational Environments. By applying a deconstruction process to organize clinical recommendations across multiple categories, JTS was able to present clear clinical recommendations across "role of care" and "scope of practice." The use of a deconstruction process supported the rapid socialization of the CPG and may have improved clinical understanding among deployed medical teams.

Discovery of Covalent Bruton's Tyrosine Kinase Inhibitors with Decreased CYP2C8 Inhibitory Activity.

Bruton's tyrosine kinase (BTK) is a member of the Tec kinase family that is expressed in cells of hematopoietic lineage. Evidence has shown that inhibition of BTK has clinical benefit for the treatment of a wide array of autoimmune and inflammatory diseases. Previously we reported the discovery of a novel nicotinamide selectivity pocket (SP) series of potent and selective covalent irreversible BTK inhibitors. The top molecule 1 of that series strongly inhibited CYP2C8 (IC50 =100 nM), which was attributed to the bridged linker group. However, our effort on the linker replacement turned out to be fruitless. With the study of the X-ray crystal structure of compound 1, we envisioned the opportunity of removal of this liability via transposition of the linker moiety in 1 from C6 to C5 position of the pyridine core. With this strategy, our optimization led to the discovery of a novel series, in which the top molecule 18 A displayed reduced CYP inhibitory activity and good potency. To further explore this new series, different warheads besides acrylamide, for example cyanamide, were also tested. However, this effort didn't lead to the discovery of molecules with better potency than 18 A. The loss of potency in those molecules could be related to the reduced reactivity of the warhead or reversible binding mode. Further profiling of 18 A disclosed that it had a strong hERG (human Ether-a-go-go Related Gene) inhibition, which could be related to the phenoxyphenyl group.

Discovery of potent and selective reversible Bruton's tyrosine kinase inhibitors.

Bruton's tyrosine kinase (BTK) is a cytoplasmic, non-receptor tyrosine kinase member of the TEC family of tyrosine kinases. Pre-clinical and clinical data have shown that targeting BTK can be used for the treatment for B-cell disorders. Here we disclose the discovery of a novel imidazo[4,5-b]pyridine series of potent, selective reversible BTK inhibitors through a rational design approach. From a starting hit molecule 1, medicinal chemistry optimization led to the development of a lead compound 30, which exhibited 58 nM BTK inhibitory potency in human whole blood and high kinome selectivity. Additionally, the compound demonstrated favorable pharmacokinetics (PK), and showed potent dose-dependent efficacy in a rat CIA model.

Discovery of Evobrutinib: An Oral, Potent, and Highly Selective, Covalent Bruton's Tyrosine Kinase (BTK) Inhibitor for the Treatment of Immunological Diseases.

Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

Synthesis and SAR development of quinoline analogs as novel P2X7 receptor antagonists.

The P2X7 receptor (P2X7R) plays an important role in diverse conditions associated with tissue damage and inflammation, suggesting that the human P2X7R (hP2X7R) is an attractive therapeutic target. In the present study, the synthesis and structure-activity relationship (SAR) of a novel series of quinoline derivatives as P2X7R antagonists are described herein. These compounds exhibited mechanistic activity (YO PRO) in an engineered HEK293 expressing hP2X7R as well as a functional response (IL-1ß) in human THP-1 (hTHP-1) cellular assays. Compound 19 was identified as the most promising compound in this series with excellent cellular potency, low liver microsomal clearance, good permeability and low efflux ratio. In addition, this compound also displayed good pharmacokinetic properties and acceptable brain permeability (Kp,uu of 0.37).

Discovery of Affinity-Based Probes for Btk Occupancy Assays.

Bruton's tyrosine kinase (Btk) is an attractive target for the treatment of a wide array of B-cell malignancies and autoimmune diseases. Small-molecule covalent irreversible Btk inhibitors targeting Cys481 have been developed for the treatment of such diseases. In clinical trials, probe molecules are required in occupancy studies to measure the level of engagement of the protein by these covalent irreversible inhibitors. The result of this pharmacodynamic (PD) activity provides guidance for appropriate dosage selection to optimize inhibition of the drug target and correlation of target inhibition with disease treatment efficacy. This information is crucial for successful evaluation of drug candidates in clinical trials. Based on the pyridine carboxamide scaffold of a novel solvent-accessible pocket (SAP) series of covalent irreversible Btk inhibitors, we successfully developed a potent and selective affinity-based biotinylated probe 12 (2-[(4-{4-[5-(1-{5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentanoyl]piperazine-1-carbonyl}phenyl)amino]-6-[1-(prop-2-enoyl)piperidin-4-yl]pyridine-3-carboxamide). Compound 12 has been used in Btk occupancy assays for preclinical studies to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon.

Discovery of a novel series of pyridine and pyrimidine carboxamides as potent and selective covalent inhibitors of Btk.

Btk is an attractive target for the treatment of a range of Bcell malignancies as well as several autoimmune diseases such as murine lupus and rheumatoid arthritis. Several covalent irreversible inhibitors of Btk are currently in development including ibrutinib which was approved for treatment of B-cell malignancies. Herein, we describe our efforts using X-ray guided structure based design (SBD) to identify a novel chemical series of covalent Btk inhibitors. The resulting pyridine carboxamides were potent and selective inhibitors of Btk having excellent enzymatic and cellular inhibitory activity.

Optimization of the efflux ratio and permeability of covalent irreversible BTK inhibitors.

Bruton's tyrosine kinase (Btk) is a member of the Tec kinase family that is expressed in cells of hematopoietic lineage (e.g. B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible Btk inhibitors targeting Cys481 within the ATP-binding pocket have been applied in the treatment of B-cell malignancies. Starting from a fragment, we discovered a novel series of potent covalent irreversible Btk inhibitors that bear N-linked groups occupying the solvent accessible pocket (SAP) of the active site of the Btk kinase domain. The hit molecules, however, displayed high P-gp mediated efflux ratio (ER) and poor A-B permeability in Caco-2 assay. By decreasing tPSA, installing steric hindrance and adjusting clogP, one top molecule 9 was discovered, which showed a 99% decrease in efflux ratio and a 90-fold increase in A-B permeability compared to hit molecule 1.

Discovery of potent, highly selective covalent irreversible BTK inhibitors from a fragment hit.

Bruton's Tyrosine Kinase (BTK) is a member of the TEC kinase family that is expressed in cells of hematopoietic lineage (e.g., in B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible BTK inhibitor targeting Cys481 within the ATP-binding pocket, for example ibrutinib, has been applied in the treatment of B-cell malignancies. Starting from a fragment hit, we discovered a novel series of potent covalent irreversible BTK inhibitors that occupy selectivity pocket of the active site of the BTK kinase domain. Guided by X-ray structures and a fragment-based drug design (FBDD) approach, we generated molecules showing comparable cellular potency to ibrutinib and higher kinome selectivity against undesirable off-targets like EGFR.

Btk inhibition treats TLR7/IFN driven murine lupus.

Bruton's tyrosine kinase (Btk) is expressed in a variety of immune cells and previous work has demonstrated that blocking Btk is a promising strategy for treating autoimmune diseases. Herein, we utilized a tool Btk inhibitor, M7583, to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon. In BXSB-Yaa lupus mice, Btk inhibition reduced autoantibodies, nephritis, and mortality. In the pristane-induced DBA/1 lupus model, Btk inhibition suppressed arthritis, but autoantibodies and the IFN gene signature were not significantly affected; suggesting efficacy was mediated through inhibition of Fc receptors. In vitro studies using primary human macrophages revealed that Btk inhibition can block activation by immune complexes and TLR7 which contributes to tissue damage in SLE. Overall, our results provide translational insight into how Btk inhibition may provide benefit to a variety of SLE patients by affecting both BCR and FcR signaling.

LC-MS-Based Metabolomics Discovers Purine Endogenous Associations with Low-Dose Salbutamol in Urine Collected for Antidoping Tests.

Current antidoping analytical methods are tailored mainly to the targeting of known drugs and endogenous molecules. This causes difficulties in rapidly reacting to emerging threats, such as designer drugs, biological therapeutic agents, and technologies. Biomarkers are considered as a promising approach for the fight against these threats to sport. The main purpose of this study was to find surrogate biomarkers induced by the intake of small amounts of the model compound salbutamol and explore a sensitive approach to help screen for possible drug misuse. Urine samples (91) from athletes with detectable salbutamol (30) and negative samples (61) were analyzed using a UHPLC-MS. A third group (30) was created by spiking salbutamol into negative samples to eliminate confounding effects. Data were then analyzed in XCMS to extract metabolic features. Orthogonal partial least squares-discriminant analysis was performed to select features correlated with detectable salbutamol (p(corr) > 0.5) and ROC analysis was performed to measure the predictive potential of the markers. Univariate analysis including Mann-Whitney U test and Spearman's correlation was conducted on selected markers. A total of 7000 metabolic features were parsed, one feature identified as hypoxanthine increased with salbutamol (p < 0.001). The ROC curve of hypoxanthine returned an AUC of 0.79 (p < 0.001). Correlation with salbutamol (r = 0.415, p < 0.01, Spearman's correlation) showed hypoxanthine and purine metabolism have association with salbutamol administration. This surrogate discovery approach needs further PK studies but in the meantime can be used as an intelligence-based complementary approach for targeting of athletes to be further tested.

Crystallographic structure of a small molecule SIRT1 activator-enzyme complex.

SIRT1, the founding member of the mammalian family of seven NAD(+)-dependent sirtuins, is composed of 747 amino acids forming a catalytic domain and extended N- and C-terminal regions. We report the design and characterization of an engineered human SIRT1 construct (mini-hSIRT1) containing the minimal structural elements required for lysine deacetylation and catalytic activation by small molecule sirtuin-activating compounds (STACs). Using this construct, we solved the crystal structure of a mini-hSIRT1-STAC complex, which revealed the STAC-binding site within the N-terminal domain of hSIRT1. Together with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis using full-length hSIRT1, these data establish a specific STAC-binding site and identify key intermolecular interactions with hSIRT1. The determination of the interface governing the binding of STACs with human SIRT1 facilitates greater understanding of STAC activation of this enzyme, which holds significant promise as a therapeutic target for multiple human diseases.

Discovery of 6,7-dihydro-5H-pyrrolo[2,3-a]pyrimidines as orally available G protein-coupled receptor 119 agonists.

GPR119 is a 7-transmembrane receptor that is expressed in the enteroendocrine cells in the intestine and in the islets of Langerhans in the pancreas. Indolines and 6,7-dihydro-5H-pyrrolo[2,3-a]pyrimidines were discovered as G protein-coupled receptor 119 (GPR119) agonists, and lead optimization efforts led to the identification of 1-methylethyl 4-({7-[2-fluoro-4-(methylsulfonyl)phenyl]-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)-1-piperidinecarboxylate (GSK1104252A) (3), a potent and selective GPR119 agonist. Compound 3 showed excellent pharmacokinetic properties and sufficient selectivity with in vivo studies supporting a role for GPR119 in glucose homeostasis in the rodent. Thus, 3 appeared to modulate the enteroinsular axis, improve glycemic control, and strengthen previous suggestions that GPR119 agonists may have utility in the treatment of type 2 diabetes.

Building self-assembled molecular layers with axially substituted titanium phthalocyanines.

Adsorption on graphite (HOPG) by titanium phthalocyanine axially bonded to a catechol ligand (TiPcat), titanylphthalocyanine (TiOPc), and 1:1 mixtures of these are studied at the HOPG-octylbenzene interface. The surface structures of a two-component bilayer, and of the individual monolayers of TiOPc and TiPcat, were determined by scanning tunneling microscopy (STM). TiPcat self-segregates onto a monolayer of TiOPc when an equal molar mixture is used. The preferential formation of a TiOPc monolayer from a solution containing both molecules is attributed to the difference in adsorption energies between TiPcat and TiOPc on graphite. The transformation of the hexagonal lattice of the pure TiOPc monolayer into a pseudo-square lattice was induced by the adsorption of TiPcat molecules. DFT calculations of the catechol orientation are presented.

GRACE: public health recovery methods following an environmental disaster.

Different approaches are necessary when community-based participatory research (CBPR) of environmental illness is initiated after an environmental disaster within a community. Often such events are viewed as golden scientific opportunities to do epidemiological studies. However, the authors believe that in such circumstances, community engagement and empowerment needs to be integrated into the public health service efforts in order for both those and any science to be successful, with special care being taken to address the immediate health needs of the community first, rather than the pressing needs to answer important scientific questions. The authors will demonstrate how they have simultaneously provided valuable public health service, embedded generalizable scientific knowledge, and built a successful foundation for supplemental CBPR through their on-going recovery work after the chlorine gas disaster in Graniteville, South Carolina.

Sensitivity of lamprey ammocoetes to six chemicals.

As part of the ecological risk assessment for Portland Harbor Superfund site, a study was conducted to address the question of whether the use of surrogate species in the risk assessment would be protective of lamprey ammocoetes. The study evaluated the acute toxicity of six chemicals: pentachlorophenol, copper, diazinon, aniline, naphthalene, and lindane; these chemicals represent the toxic modes of action of oxidative phosphorylation uncoupler, gill dysfunction, acetylcholinesterase inhibitor, polar narcosis, narcosis, and central nervous system interference, respectively. Field-collected lamprey ammocoetes were exposed to each of the six chemicals in a definitive 96-h flow-through acute water-only toxicity test. LC(50)s were calculated for pentachlorophenol at 31 µg/l, copper at 46 µg/l, diazinon at 8.9 mg/l, and aniline at 430 mg/l. Species sensitivity distributions based on LC(50)s for aquatic organisms indicated that lamprey ammocoetes were relatively sensitive to pentachlorophenol (15th percentile). The sensitivity of lamprey ammocoetes to copper approximated the average of aquatic species tested (46th percentile). Lamprey ammocoetes were relatively insensitive to diazinon and aniline (72nd and 90th percentile, respectively). The 96-h LC(50) for naphthalene was estimated at 10 mg/l, based on 50% mortality in the highest concentration. Based on a comparison with LC(50)s for four other fish species, ranging from 2.0 to 6.6 mg/l, lamprey ammocoetes were relatively insensitive to naphthalene. A 96-h LC(50) could not be derived for lindane, with 12.5% mortality in the highest test concentration of 2.68 mg/l. LC(50)s for numerous other fish species ranged from 0.001 to 0.24 mg/l, indicating that lamprey ammocoetes were relatively insensitive to lindane. The study concluded that the use of surrogate species in the ecological risk assessment for Portland Harbor would be protective of lamprey ammocoetes.

FXR agonist activity of conformationally constrained analogs of GW 4064.

Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Replacement of the metabolically labile stilbene with either benzothiophene or naphthalene rings led to the identification of potent full agonists 2a and 2g.

Substituted isoxazole analogs of farnesoid X receptor (FXR) agonist GW4064.

Starting from the known FXR agonist GW 4064 1a, a series of alternately 3,5-substituted isoxazoles was prepared. Several of these analogs were potent full FXR agonists. A subset of this series, with a tether between the isoxazole ring and the 3-position aryl substituent, were equipotent FXR agonists to GW 4064 1a, with the 2,6-dimethyl phenol analog 1t having greater FRET FXR potency than GW 4064 1a.

Conformationally constrained farnesoid X receptor (FXR) agonists: Naphthoic acid-based analogs of GW 4064.

Starting from the known FXR agonist GW 4064 1a, a series of stilbene replacements were prepared. The 6-substituted 1-naphthoic acid 1b was an equipotent FXR agonist with improved developability parameters relative to 1a. Analog 1b also reduced the severity of cholestasis in the ANIT acute cholestatic rat model.