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Nitroreductases (NTRs), a family of flavin-containing enzymes, can be overexpressed in regions of tumor hypoxia, i.e., areas deprived of oxygen. The detection of NTR may be applied for monitoring the hypoxia level in tumors. To quantify NTR, novel sensors were designed based on the conjugation of pyridazino-1,3a,6a-triazapentalene to a para-nitrophenyl, directly or with an alkyne linker, resulting in two probes denoted as 1-NO2 and 2-NO2. Both probes had a weak fluorescence (form off), while their reduction by NTR led to the over 15-fold enhancement of fluorescence intensity (form on) regardless of the oxygen concentration in their environment. The detection limit was as low as ca. 20−30 ng/mL of NTR. Interestingly, the presence of human serum albumin significantly enhanced the observed fluorescence turned on by NTR in particular for the 2-NO2 probe. The in vitro response to both probes was evaluated on the highly metastatic human melanoma A2058 cell line, where NTR levels increased under hypoxic conditions. Their low toxicity, high photostability, and efficient uptake combined with a strong correlation between the enhancement of fluorescence and hypoxia in cells indicate the high potential of the 1-NO2 and 2-NO2 probes for the assessment of the hypoxic environment in biomedical research. The designed compounds allowed for a fast determination of the difference in NTR content of cells without the need for special sample preparation such as cell lysis. The changes can be monitored using a plate reader, flow cytometer, and fluorescence microscope, which that makes these probes a remarkably universal tool.

Background Stimulator of Interferon Genes (STING) is a major player in the activation of robust innate immune response leading to initiation and enhancement of tumor-specific adaptive immunity. Several clinical and pre-clinical programs have shown that activation of the STING pathway triggers immune-mediated antitumor response. Although vast majority of programs focus on development of analogues of the endogenous STING ligands, their chemical nature and stability often limit their use to local administration. Herein, we present recent results from the development of our selective non-nucleotide, non-macrocyclic, small molecule direct STING agonists, suitable for systemic administration, characterized by improved activity in human immune cells. Methods Binding to recombinant STING protein was examined using FTS, MST, FP and crystallography studies. Phenotypic screen was performed in THP-1 Dual reporter cells. Mouse bone marrow-derived dendritic cells (BMDC) were obtained from C57BL/6 mice and differentiated with mIL-4 and mGM-CSF. STING agonists were administered into BALB/c mice and cytokine release was measured in plasma. Additionally, mice were inoculated with CT26 murine colon carcinoma or EMT6 murine breast carcinoma cells and the compound was administered, followed by the regular tumor growth and body weight monitoring. Results Ryvu’s small-molecule agonists demonstrate strong binding affinity to recombinant STING proteins across all tested species. The compounds bind to all human STING protein variants and trigger pro-inflammatory cytokine release from human immune cells regardless of the STING haplotype. Moreover, new generation of developed agonists show significantly improved binding to human protein as well as in vitro activity on human cells. Systemic, intravenous in vivo administration leads to a dose-dependent upregulation of STING-dependent pro-inflammatory cytokines, which results in a dose-dependent antitumor efficacy observed in CT26 and EMT6 mouse cancer models, leading to complete tumor remissions in all treated animals. Furthermore, observed efficacy is accompanied by development of a lasting immunological response demonstrated by lack of tumor engraftment or a delayed tumor growth in cured animals challenged with repeated inoculation of cancer cells. Conclusions New generation Ryvu’s STING agonists are strong and selective activators of STING-dependent signaling in both mouse and human immune cells promoting anti-tumor immunity. Treatment with Ryvu’s small-molecule STING agonists leads to engagement of the immune system which results in a complete tumor remission and development of immunological memory of the cancer antigens. The compounds show good selectivity and ADME properties enabling development for systemic administration. In addition developed compounds maintain small functional handles amenable to linker attachment making the series suitable for versatile development as single agents, for combinations with immunotherapies or as targeted agents.

BackgroundHematopoietic progenitor kinase 1 (HPK1, MAP4K1) is emerging as a well-renowned, druggable target for T cell-based immunotherapies. HPK1 is a member of the serine/threonine MAP4K family, predominantly expressed in hematopoietic cell lineages and shown to be a negative regulator of the T cell receptor (TCR) signaling pathway. Upon TCR activation, HPK1 is recruited to the proximity of the cell membrane and phosphorylates an adaptor protein SLP-76 at the Ser376 residue which, in turn, abrogates TCR signaling. Other studies point to a potential role of HPK1 in T cell exhaustion as well as in functional re-programming of regulatory T cells. Moreover, mounting evidence suggest that HPK1 kinase activity suppresses the immune functions of a wide range of other immune cell subsets like B cells and dendritic cells. Taken together, these observations support small-molecule HPK1 inhibitors as an attractive modality in cancer immunotherapy either as single agents or in combination with immune checkpoint inhibitors.MethodsActivity of compounds against HPK1 and selected off- and anti-targets was assessed in biochemical assays. Phosphorylation of SLP-76 was measured either by flow cytometry or TR-FRET. Jurkat and primary T cells were activated and cultured in the presence of tested compounds and immunosuppressive agents. Impact on TCR selectivity and T cell function was measured by AlphaLISA and flow cytometry. Target engagement was measured in splenocytes of mice administered orally with tested compounds followed by IP injection of aCD3 antibody. Anti-tumor efficacy of HPK1 inhibitors was assessed in a syngeneic tumor model.ResultsRyvu's proprietary small molecule HPK1 inhibitors exhibit sub-nanomolar activity against human and mouse HPK1 proteins and good selectivity against other TCR pathway kinases. Tested compounds efficiently block phosphorylation of SLP-76 upon TCR engagement. TCR selectivity of Ryvu's inhibitors, measured as a ratio between CD69 and pSer376 SLP-76 inhibition, is on par or superior to reference molecules. Tested compounds are not only able to overcome PGE-2 induced resistance following TCR activation in human PBMCs, inducing elevated IL-2 release but also affect T cell function in co-culture assay. Developed molecules have favorable PK profiles, allowing for sustained target coverage in proposed dosing regimens and demonstrate efficacy in a mammary carcinoma syngeneic model.ConclusionsRyvu has developed potent and selective HPK1 inhibitors with favorable PK and PD profiles, whose activity in vitro translates to in vivo efficacy. Further preclinical work is warranted to select a lead candidate for IND-enabling studies and subsequently clinical studies across a variety of solid tumors.

Background: Hematopoietic progenitor kinase 1 (HPK1, MAP4K1) is a member of the serine/threonine MAP4K family predominantly expressed in hematopoietic cell lineages which plays a pivotal role in the negative regulation of the signaling cascade triggered by T cell receptor engagement. Inhibition of its activity promotes secretion of IL-2, T cell maturation, and proliferation. It has been also suggested that inhibition of HPK1 may hyperactivate both B cells and dendritic cells via hampering HPK1- mediated negative feedback mechanisms involved in BCR regulation and enhancement of antigen presenting capability of dendritic cells. Suppression of the TCR inhibitory mechanisms with small molecule HPK1 inhibitors might constitute a novel approach in tumor immunotherapy which enhances neoantigen recognition and boosts immune responses of T and B lymphocytes against cancer cells. Thus, small molecule HPK1 inhibitors may provide additional benefit for patients subjected to 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 selected anti- and off-targets and profiled against a broad kinase panel. Phosphorylation of serine 376 on SLP-76 adaptor protein and specific TCR activation-related markers upon HPK1 inhibition was monitored by either Western Blotting or flow cytometry in human and murine T-cells. IL-2 release was measured in human PBMCs and mouse splenic T cells. PBMC cells were activated and exposed to compounds in the presence of PGE-2, followed by IL-2 release measurement. Mice were challenged with the compounds and pharmacodynamic biomarkers were evaluated through flow cytometry and AlphaLisa. Results: Small molecule Ryvu HPK1 inhibitors block kinase activity of recombinant mouse and human protein with sub-nanomolar IC50 values. Ryvu compounds selectively engage downstream biomarkers in human and murine T cells. While inhibiting phosphorylation of serine 376, Ryvu HPK1 inhibitors do not affect activatory phosphorylation of specific phosphotyrosine residues of SLP-76 in human or mouse CD3+ T cells. Ryvu compounds overcome PGE-2 induced resistance following TCR activation in human PBMCs, and mouse CD3+ T cells, inducing IL-2 release. Ryvu compounds have good physicochemical properties and good overall selectivity. In vivo activity and target engagement have been confirmed in the model with anti-CD3 antibody infusion. Conclusion: Pharmacological inhibition of HPK1 kinase activity has strong potential to become a novel immunomodulatory approach for cancer treatment. Citation Format: Maciej Kujawa, Eliza Zimoląg, Michał Gałęzowski, Paweł Guzik, Agata Dudek, Andrzej Gondela, Marcin Nowogródzki, Marianna Girardi, Kostiantyn Krolenko, Marta Bugaj, Sylwia Sudoł, Agnieszka Gibas, Joanna Szeremeta-Spisak, Aneta Bobowska, Magdalena Zastawna, Przemysław Wyrębek, Nicolas Boutard, Aleksandra Więckowska, Wojciech Jasnosz, Wojciech Schonemann, Karol Zuchowicz, David Synak, Urszula Kulesza, Oleksandr Leventes, Mateusz Świrski, Sujit Sasmal, Karolina Gluza, Patryk Kret, Mateusz Ogórek, Kinga Michalik, Katarzyna Banaszak, Adrian Podkowa, Katarzyna Wnuk-Lipińska, Monika Dobrzańska, Peter Littlewood, Krzysztof Brzózka, Anna Bartosik, Stefan Chmielewski. Development and characterization of small molecule HPK1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1281.

Background. Stimulator of interferon genes, known as STING, is an intracellular sensor of nucleic acids and one of key regulators in activating the innate immune response. Employing synthetic STING agonists has been shown to promote immune-mediated antitumor response in preclinical animal models. Ryvu is developing small-molecule STING agonists suitable for systemic administration. Herein we present unpublished results from characterization of the new generation of our agonist series with significantly improved potency on human immune cells. Methods. Binding to recombinant STING protein was examined using Fluorescence Thermal Shift and Fluorescence Polarisation and was confirmed by X-ray crystallography. Primary screen was performed in THP-1 Dual reporter cells and selectivity was confirmed in THP-1 reporter cells with knocked out STING or expressing varying STING variants. T cell viability and proliferation was assessed by flow cytometry using activated, human T cells exposed to STING agonists. STING pathway activation pattern in cells treated with Ryvu's molecules was confirmed using Western blot analysis. BALB/c mice were injected with compounds and the levels of cytokine release were measured in the plasma. Mice were inoculated with CT26 or EMT6 tumor cells and the compound was administered intravenously followed by the regular monitoring of tumor growth. Results. New generation Ryvu STING agonists are strong binders of human STING protein. Ryvu's compounds show high cellular potency inducing cytokine production in human immune cells at low nM range. Moreover, high activity of developed agonists is maintained irrespective of the natural human STING variant as seen in THP-1 reporter cells as well as in human primary immune cells. High cellular potency of developed compounds also translates into efficacy observed in vivo, where systemic intravenous administration leads to significant tumor growth inhibition and complete tumor regressions in mouse syngeneic models. Conclusion. Ryvu has developed a new generation of potent, direct and selective small-molecule STING agonists. The compounds are characterized by drug-like properties and high in vitro potency on par or outperforming known references. Ryvu agonists are suitable for systemic administration and allow to achieve excellent antitumor efficacy. Taken together, the promising results suggest that the developed series holds high potential for improving immunotherapy in cancer patients. Citation Format: Maciej Krzysztof Rogacki, Stefan Chmielewski, Jolanta Mazurek, Magdalena Zawadzka, Katarzyna Wnuk-Lipińska, Kamil Kuś, Katarzyna Wójcik-Jaszczyńska, Aleksandra Poczkaj, Łukasz Dudek, Wojciech Schonemann, Urszula Głowniak-Kwitek, Marcin Leś, Marek Wronowski, Tushar Mahajan, Urszula Kulesza, Magdalena Zastawna, David Synak, Karol Zuchowicz, Karolina Gluza, Katarzyna Banaszak, Karolina Wiatrowska, Izabela Strojny, Mirosława Gładysz, Justyna Jabłońska, Ewelina Gabor-Worwa, Monika Dobrzańska, Raghuram Tangirala, Peter Littlewood, Krzysztof Brzózka. New generation of STING agonists: Development and characterization of a novel series of systemic immunomodulators with improved potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1280.

Background: Synthetic lethality is one of the most innovative approaches for selective targeting of cancer cells with defined genetic background. SMARCA2 and SMARCA4 are two mutually exclusive helicase/ATPase catalytic subunits belonging to SWI/SNF chromatin remodeling complex. It is estimated that one in five tumors possess a mutation in proteins of this complex. The Cancer Genome Atlas (TCGA) and the Catalog of Somatic Mutation in Cancer (Cosmic) shows that SMARCA4 is one of the most mutated genes in lung, colorectal, breast, melanoma and CNS tumors. Herein, we present development of a potent and selective SMARCA2 degrader, structurally unrelated to known chemotypes with first in class potential selectively targeting SMARCA4 mutant cells. Methods: ADP-Glo assay was used to identify SMARCA2 inhibitors through HTS. Binding was confirmed using MST, FTS and SPR methods. Biochemical and biophysical investigation guided rational optimization. Identified ligands were used to build a hybrid PROTAC by linking to a ligand for the E3 ligase. Western blotting was used to assess degradation efficiency and selectivity. Target engagement was confirmed using qPCR and AlphaLISA methods. On target activity was confirmed using SMARCA4 isogenic cells and a panel of SMARCA4 WT and mutant cell lines. Results: High throughput screening allowed identification of novel inhibitors of SMARCA2 ATPase activity. Medicinal chemistry efforts improved potency and affinity to the target over 100 fold. Target engagement was confirmed using biophysical methods and biomarker modulation. Proprietary ligand was used to build a hybrid PROTAC. Western blotting confirmed selective and long-lasting degradation of SMARCA2. Co-treatment with Epoxomicin confirmed proteasomal dependent degradation of targeted protein. Specific SMARCA2 depletion in SMARCA4 mutated cancer cell lines induced apoptosis, growth inhibition and cell death. Observed mechanism of action is consistent with a phenotype seen with perturbation through inhibition of ATPase activity of SMARCA2 and genetic knock-down. Conclusion: Treatment with Ryvu's PROTACs led to selective, proteasomal dependent degradation of SMARCA2 protein and in consequence to a targeted cell death of SMARCA4 mutated cancers. Fine-tuning of available compounds will allow for proof-of-concept experiments in animal models as a single agent or in combinations with radio- or immuno-therapies. Citation Format: Andrzej Mazan, Anna Wróbel, Agnieszka Dreas, Adam Radzimierski, Kinga Michalik, Anna Kowal, Katarzyna Wiklik, Katarzyna Wnuk-Lipińska, Paulina Niedziejko, Kamila Kozłowska, Magdalena Łośko, Joanna Szczęśniak, Agnieszka Sroka-Porada, Justyna Martyka, Urszula Kulesza, Karol Zuchowicz, Magdalena Zastawna, Jose Alvarez, Luigi Stasi, Peter Littlewood, Tomasz Rzymski, Krzysztof Brzózka. Development of novel, selective SMARCA2 (BRM) degraders for treatment of SMARCA4 (BRG1) mutated tumors [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 3656.

The immunosuppressive role of adenosine attracts attention of many researchers as an interesting target for anticancer therapy. High concentrations of adenosine detected in many tumor models affect several subpopulations of infiltrating immune cells e.g. T lymphocytes, dendritic cells and macrophages. In these cells, adenosine signals via either A2A or A2B receptor, therefore the highest efficiency in reversal of adenosine mediated immunosuppression should be achieved by dual antagonism to both receptors. Here, we present data for best-in-class dual A2A/A2B antagonist RVU330 that exerts nanomolar potency in tumor-like adenosine-rich environment. By head-to-head comparison of our in-house developed dual A2A/A2B antagonist to compounds currently pursued in clinical development, we have demonstrated the best-in-class characteristics of our molecule. RVU330 maintains sub- or low nanomolar antagonistic potency in a high adenosine conditions on both A2A and A2B receptor. Multilevel pharmacological characterization spans from receptor binding and cAMP accumulation (assays in cell lines overexpressing target receptors) to functional assays in primary human immune cells which demonstrate the reversibility of immune suppression induced by adenosine. Our compound shows superior activity in antagonizing adenosine-mediated immunosuppression in CD4+ and CD8+ T-cells (IL2 and TNFα production), monocyte derived dendritic cells - moDCs (IL12 and TNFα production, driven mainly by A2B receptors) and macrophages (suppression of VEGF release by M2). High activity in human whole blood assay demonstrates that low nanomolar concentration is sufficient to fully antagonize adenosine. Therapeutic efficacy of RVU330 has been confirmed in syngeneic in vivo mouse model. New best-in-class dual A2A/A2B adenosine receptor antagonists RVU330 combines two crucial activities which addresses distinct, cell type mediated mode of action of adenosine related immunosupression. The dual A2A/A2B antagonistic profile as well as the ability to inhibit both receptors at very high adenosine conditions are key features differentiating RVU330 from other adenosine receptor antagonist. The RVU330 is currently being evaluated in preclinical studies. Citation Format: Michał Gałęzowski, Katarzyna Dziedzic, Paulina Węgrzyn, Aniela Gołas, Magdalena Bońkowska, Karolina Grycuk, Mateusz Ogórek, Aneta Bobowska, Joanna Szeremeta-Spisak, Marcin Nowogródzki, Grzegorz Satała, Iwona Łozińska-Raj, Przemysław Wyrębek, Marcelina Dudek, Anita Janiga, Marek Wronowski, Magdalena Zastawna, Mateusz Świrski, Luigi Stasi, Peter Littlewood, Krzysztof Brzózka, Mateusz O. Nowak. In vivo and in vitro characterization of RVU330 best-in-class dual A2A/A2B adenosine receptor antagonist [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 5555.

Adenosine is important messenger molecule that is involved in signal transmission in central nervous, cardiovascular and immunological systems. The immunosuppressive role of adenosine attract attention of many researchers recently and reversal of that effect is addressed by several different approaches i.e.: inhibition of enzymes that are producing the adenosine in tumor microenvironment like CD39 and CD73 but also the antagonization of adenosine receptors mainly A2A and A2B subtypes. Here we present a novel series of unsurmountable, dual A2A/A2B antagonists that retain its nanomolar potency in tumor-like adenosine-rich environment. This advantageous profile comes mainly from long residence time in adenosine receptors which transfers into high receptor occupancy even after full clearance of the compound from plasma. We are demonstrating how our inhibitors restore adenosine depleted immune functionality in the series of functional in vitro assays in primary cells. This includes the cytokine release by activated CD4+ and CD8+ human T-lymphocytes (driven by A2A) and dendritic cells (driven by A2B). Most importantly presented compounds show improved in vivo pharmacological profile in comparison to adenosine inhibitors currently tested in clinical trials manifested by high inhibition of CREB phosphorylation in mouse model. Citation Format: Michal Galezowski, Paulina Węgrzyn, Aneta Bobowska, Katarzyna Dziedzic, Joanna Szeremeta-Spisak, Marcin Nowogrodzki, Grzegorz Satala, Alicja Obara, Iwona Lozinska-Raj, Marcelina Dudek, Anita Janiga, Jacek Reus, Marek Wronowski, Magdalena Zastawna, Grzegorz Statkiewicz, Maciej Rogacki, Magdalena Ziembik, Karolina Grycuk, Foteini Soukou, Kinga Michalik, Agnieszka Adamus, Karolina Wiatrowska, Natalia Lewandowska, Aniela Golas, Olga Haberkiewicz, Rod Porter, Krzysztof Brzozka, Mateusz Nowak. Novel dual A2A A2B adenosine receptor antagonists for cancer immunotherapy: in vitro and in vivo characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4135.

Over-activation of the serine synthesis pathway, upregulation of SHMT2 has been described in over 20% of solid tumors (e.g. breast, lung, colorectal cancers). Such cancer cells are highly dependent on serine. Serine hydroxymethyltransferase (SHMT) plays a key role in a so-called one-carbon pathway, a group of biochemical reactions involved in amino acid metabolism. SHMT catalyzes the conversion of serine to glycine and also plays a role in the folate (vitamin B9) cycle. Antagonists of folate metabolism or antifolates are an established chemotherapy in certain cancers. Folate antagonism disrupts cell division, DNA/RNA synthesis and protein synthesis. Pemetrexed (for non-small cell lung carcinoma, mesothelioma) and methotreaxate (for autoimmune conditions like rheumatoid arthritis and certain cancers) are two well established and effective antifolates. The main drawback with antifolates in cancer treatment, however, is the development of resistance. In this study we report development of a series of small molecule SHMT1/2 inhibitors. Synthetized compounds exert potency in SHMT1/2 biochemical assay as well as in cellular assay (measured by the C13 serine to glycine conversion) with the low nanomolar range. Therapeutic effect of the compounds was investigated in the panel of cancer cell lines with different genetic background as well as with different SHMT2 levels. We identified several cell lines in which tested compounds inhibited cancer cell grow with nM GI50 values. Taken together, presented data supports our rationale for using SHMT1/2 inhibitors as a novel and interesting approach for the cancer therapy. Citation Format: Anna Bartosik, Pawel Guzik, Marta Sowinska, Karolina Gluza, Marcin Krol, Anna Wrobel, Agnieszka Dreas, Faustyna Iwanska, Magdalena Zastawna, Urszula Kulesza, Nicolas Boutard, David Schultz, Justyna Wujkowska, Karolina Pyziak, Agnieszka Sroka-Porada, Agnieszka Przybylowicz, Agnieszka Adamus, Magdalena Sieprawska-Lupa, Przemyslaw Golik, Piotr Kowalczyk, Krzysztof Brzozka, Tomasz Rzymski, Mateusz Nowak. Discovery of novel SHMT small molecule inhibitors for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3516.