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Background: Hematopoietic progenitor kinase 1 (HPK1, MAP4K1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator of T cells and dendritic cells (DC). Alteration of ERK/MAPK pathway by HPK1 in T-cells and dendritic cells is an inhibitory mechanism that negatively regulates TCR-induced IL-2 gene transcription, T cell maturation and proliferation. Inhibiting kinase activity of HPK1 results in activation of antigen presenting properties of dendritic cells and stimulates maturation and proliferation of T cells. Therefore, small molecule inhibitors of HPK1 could serve as a novel agent to transform cold, resistant tumors into sensitive hot cancers and provide additional benefit in combination with existing immunotherapies. Methods: Inhibition of HPK1 was assessed by biochemical assay with recombinant human and mouse protein. Small molecule inhibitors were tested in biochemical assay on other MAP4Ks and in addition profiled against broad kinase panel. Phosphorylation of Serine 376 and Tyrosine 128 of SLP-76 adaptor protein upon HPK1 inhibition was monitored by Western Blotting in human and murine T-cells. IL-2 release was monitored in total human PBMC, human CD3+ T cells and mouse CD3+ splenocytes. Human CD3+ T cells were isolated from PBMC, activated with plate-bound anti-CD3/anti-CD28 and exposed to compounds in the presence of PGE-2, followed by IL-2 release measurement, viability and proliferation assessment using flow cytometry. Mouse CD3+ splenocytes were isolated from Balb/c mice, activated with plate-bound anti-CD3/anti-CD28 and exposed to compounds in the presence of PGE-2, followed by IL-2 release assessment. Results: Small molecule Ryvu HPK1 inhibitors block kinase activity of recombinant mouse and human protein with nanomolar IC50 values. Ryvu compounds show broad kinome selectivity. Ryvu HPK1 inhibitors selectively engage downstream biomarkers in human and murine T cells. While inhibiting phosphorylation of Serine 376, Ryvu compounds do not affect activatory phosphorylation of Tyrosine 128 of SLP-76 in human or mouse CD3+ T cells. Ryvu HPK1 inhibitors overcome PGE-2 induced resistance following TCR activation in human PBMCs, CD3+ T-cells and mouse CD3+ T cells, inducing IL-2 release. Compounds have good druglike physicochemical properties. Conclusion: Ryvu HPK1 inhibitors promote activation of in-vitro immunostimulatory properties of both mouse and human immune cells, overcoming immunosuppression. The chemical series has the potential to show anti-tumor efficacy in syngeneic animal models as a single agent or in combination with checkpoint inhibitors. Citation Format: Stefan Chmielewski, Maciej Kujawa, Eliza Zimoląg, Paweł Guzik, Agata Dudek, Grzegorz Topolnicki, Sylwia Sudoł, Agnieszka Gibas, Marta Bugaj, Kostiantyn Krolenko, Marcin Nowogródzki, Anita Janiga, Przemysław Wyrębek, Jakub Pięta, Aleksandra Brzdonkiewicz, Grzegorz Wilkowski, Marcin Walczak, Katarzyna Maciejewska, Adam Radzimierski, Wojciech Jasnosz, Tushar Mahajan, Roberta Bartolotta, Karolina Gluza, Patryk Kret, Ewelina Rutkowska, Kinga Michalik, Katarzyna Banaszak, Adrian Podkowa, Aniela Gołas, Katarzyna Wnuk-Lipińska, Charles Fabritius, Luigi Stasi, Peter Littlewood, Krzysztof Brzózka, Anna Bartosik, Monika Dobrzańska. Development and characterization of small molecule HPK1 inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1947.

A disturbance of glucose homeostasis leading to type 2 diabetes mellitus (T2DM) is one of the severe side effects that may occur during a prolonged use of many drugs currently available on the market. In this manuscript we describe the most common cases of drug-induced T2DM, discuss available pharmacotherapies and propose new ones. Among various pharmacotherapies of T2DM, incretin therapies have recently focused attention due to the newly determined crystal structure of incretin hormone receptor GLP1R. Incretin hormone receptors: GLP1R and GIPR together with the glucagon receptor GCGR regulate food intake and insulin and glucose secretion. Our study showed that incretin hormone receptors, named also gut hormone receptors as they are expressed in the gastrointestinal tract, could potentially act as unintended targets (off-targets) for orally administrated drugs. Such off-target interactions, depending on their effect on the receptor (stimulation or inhibition), could be beneficial, like in the case of incretin mimetics, or unwanted if they cause, e.g., decreased insulin secretion. In this in silico study we examined which well-known pharmaceuticals could potentially interact with gut hormone receptors in the off-target way. We observed that drugs with the strongest binding affinity for gut hormone receptors were also reported in the medical information resources as the least disturbing the glucose homeostasis among all drugs in their class. We suggested that those strongly binding molecules could potentially stimulate GIPR and GLP1R and/or inhibit GCGR which could lead to increased insulin secretion and decreased hepatic glucose production. Such positive effect on the glucose homeostasis could compensate for other, adverse effects of pharmacotherapy which lead to drug-induced T2DM. In addition, we also described several top hits as potential substitutes of peptidic incretin mimetics which were discovered in the drug repositioning screen using gut hormone receptors structures against the ZINC15 compounds subset.

New HPLC method was developed for determination of amlodipine and valsartan in their binary mixture as a part of routine control of combined formulations. The method was validated to meet official requirements including selectivity, stability, linearity, precision and accuracy. Chromatography was carried out using a LiChrospher RP-18 column, a mixture containing acetonitrile, phosphate buffer of pH 3.5 and methanol (45:45:10, v/v/v) and new fluorescence detection at 255 nm for excitation and 448 nm for emission. The effect of methanol content, pH of the buffer, flow rate, detection wavelengths and column temperature was estimated in robustness study, according to a plan defined by the Plackett-Burman design. For identification of significant effects, both graphical and statistical methods were used. Ro-bustness for dissolution test was checked estimating the effects of paddle speed, temperature and pH of dissolution medium. The method was proved to complying with all official guidelines. Therefore, it is suitable for determination of amlodipine and valsartan in their binary mixtures for different analytical and pharmaceutical purposes.

D-Serine is an endogenous NMDA receptor co-agonist that activates synaptic NMDA receptors modulating neuronal networks in the cerebral cortex and plays a key role in long-term potentiation of synaptic transmission. D-serine is associated with NMDA receptor neurotoxicity and neurodegeneration and elevated D-serine concentrations have been associated with Alzheimer’s and Parkinsons’ diseases and amyotrophic lateral sclerosis. Previous studies have demonstrated that the ketamine metabolites (rac)-dehydronorketamine and (2S,6S)-hydroxynorketamine decrease intracellular D-serine concentrations in a concentration dependent manner in PC-12 cells. In the current study, PC-12 cells were incubated with a series of ketamine metabolites and the IC50 values associated with attenuated intracellular D-serine concentrations were determined. The results demonstrate that structural and stereochemical features of the studied compounds contribute to the magnitude of the inhibitory effect with (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine displaying the most potent inhibition with IC50 values of 0.18 ± 0.04 nM and 0.68 ± 0.09 nM. The data was utilized to construct a preliminary 3D-QSAR/pharmacophore model for use in the design of new and more efficient modulators of D-serine.

The β₂-adrenergic receptor (β₂-AR) agonist [(3)H]-(R,R')-methoxyfenoterol was employed as the marker ligand in displacement studies measuring the binding affinities (Ki values) of the stereoisomers of a series of 4'-methoxyfenoterol analogs in which the length of the alkyl substituent at α' position was varied from 0 to 3 carbon atoms. The binding affinities of the compounds were additionally determined using the inverse agonist [(3)H]-CGP-12177 as the marker ligand and the ability of the compounds to stimulate cAMP accumulation, measured as EC₅₀ values, were determined in HEK293 cells expressing the β₂-AR. The data indicate that the highest binding affinities and functional activities were produced by methyl and ethyl substituents at the α' position. The results also indicate that the Ki values obtained using [(3)H]-(R,R')-methoxyfenoterol as the marker ligand modeled the EC₅₀ values obtained from cAMP stimulation better than the data obtained using [(3)H]-CGP-12177 as the marker ligand. The data from this study was combined with data from previous studies and processed using the Comparative Molecular Field Analysis approach to produce a CoMFA model reflecting the binding to the β₂-AR conformation probed by [(3)H]-(R,R')-4'-methoxyfenoterol. The CoMFA model of the agonist-stabilized β₂-AR suggests that the binding of the fenoterol analogs to an agonist-stabilized conformation of the β₂-AR is governed to a greater extend by steric effects than binding to the [(3)H]-CGP-12177-stabilized conformation(s) in which electrostatic interactions play a more predominate role.

The aim of the present study was to use the analysis of surface water chemistry to understand vegetation succession pathways in terrestrializing polyhumic lakes. We hypothesized that Sphagnum mire development was accompanied by a decrease in the mineral content in water. A total of 111 vegetation plots along 23 transects were analysed in 11 lakes and adjacent peat lands in the Wigry National Park (NE Poland). The vegetation of the lake-mire systems forms distinct zones: (1) nymphaeid-, bladderwort- and bryophyte-dominated aquatic vegetation; (2) sedge-dominated edge of the Sphagnumcarpet; (3) quaking, extremely poor fen with various Cyperaceae; (4) non-quaking, Eriophorum vaginatum-dominated bog-like vegetation and (5) pine woodland. Surface water corrected conductivity (ECcorr.), pH, COD-KMnO4 and Ca2+, Mg2+, Fetot. and SiO2 were measured along the transects. The environmental gradients best explaining the observed pattern were pH (with the highest values in the lake and the lowest in the bog-like vegetation) and COD-KMnO4 (showing an inverse direction). At least in some Sphagnum-mires conditions were more minerotrophic than in the lakes. The process of humic lake overgrowing by Sphagnum-mires in NE Poland results in pine woodlands on mineralised peat. The climate conditions in NE Poland, combined with evapotranspiration accelerated by encroaching trees, do not seem to support the development of ombrotrophic bogs.