Structure-Based Discovery of High-Affinity Small Molecule Ligands and Development of Tool Probes to Study the Role of Chitinase-3-Like Protein 1.

Chitinase-3-like-1 (CHI3L1), also known as YKL-40, is a glycoprotein linked to inflammation, fibrosis, and cancer. This study explored CHI3L1's interactions with various oligosaccharides using microscale thermophoresis (MST) and AlphaScreen (AS). These investigations guided the development of high-throughput screening assays to assess interference of small molecules in binding between CHI3L1 and biotinylated small molecules or heparan sulfate-based probes. Small molecule binders of YKL-40 were identified in our chitotriosidase inhibitors library with MST and confirmed through X-ray crystallography. Based on cocrystal structures of potent hit compounds with CHI3L1, small molecule probes 19 and 20 were designed for an AS assay. Structure-based optimization led to compounds 30 and 31 with nanomolar activities and drug-like properties. Additionally, an orthogonal AS assay using biotinylated heparan sulfate as a probe was developed. The compounds' affinity showed a significant correlation in both assays. These screening tools and compounds offer novel avenues for investigating the role of CHI3L1.

Novel orally bioavailable piperidine derivatives as extracellular arginase inhibitors developed by a ring expansion.

Arginase is a multifaced enzyme that plays an important role in health and disease being regarded as a therapeutic target for the treatment of various pathological states such as malignancies, asthma, and cardiovascular disease. The discovery of boronic acid-based arginase inhibitors in 1997 revolutionized attempts of medicinal chemistry focused on development of drugs targeting arginase. Unfortunately, these very polar compounds had limitations such as analysis and purification without chromophores, synthetically challenging space, and poor oral bioavailability. Herein, we present a novel class of boronic acid-based arginase inhibitors which are piperidine derivatives exhibiting a different pharmacological profile compared to our drug candidate in cancer immunotherapy -OATD-02 - dual ARG1/2 inhibitor with high intracellular activity. Compounds from this new series show low intracellular activity, hence they can inhibit mainly extracellular arginase, providing different therapeutic space compared to a dual intracellular ARG1/2 inhibitor. The disclosed series showed good inhibitory potential towards arginase enzyme in vitro (IC50 up to 160 nM), favorable pharmacokinetics in animal models, and encouraging preliminary in vitro and in vivo tolerability. Compounds from the new series have moderate-to-high oral bioavailability (up to 66 %) and moderate clearance in vivo. Herein we describe the development and optimization of the synthesis of the new class of boronic acid-based arginase inhibitors via a ring expansion approach starting from the inexpensive chirality source (d-hydroxyproline). This upgraded methodology facilitated a gram-scale delivery of the final compound and eliminated the need for costly and time-consuming chiral resolution.

Arginase 1/2 Inhibitor OATD-02: From Discovery to First-in-man Setup in Cancer Immunotherapy.

Pharmacologic inhibition of the controlling immunity pathway enzymes arginases 1 and 2 (ARG1 and ARG2) is a promising strategy for cancer immunotherapy. Here, we report the discovery and development of OATD-02, an orally bioavailable, potent arginases inhibitor. The unique pharmacologic properties of OATD-02 are evidenced by targeting intracellular ARG1 and ARG2, as well as long drug-target residence time, moderate to high volume of distribution, and low clearance, which may jointly provide a weapon against arginase-related tumor immunosuppression and ARG2-dependent tumor cell growth. OATD-02 monotherapy had an antitumor effect in multiple tumor models and enhanced an efficacy of the other immunomodulators. Completed nonclinical studies and human pharmacokinetic predictions indicate a feasible therapeutic window and allow for proposing a dose range for the first-in-human clinical study in patients with cancer. SIGNIFICANCE: We have developed an orally available, small-molecule intracellular arginase 1 and 2 inhibitor as a potential enhancer in cancer immunotherapy. Because of its favorable pharmacologic properties shown in nonclinical studies, OATD-02 abolishes tumor immunosuppression induced by both arginases, making it a promising drug candidate entering clinical trials.

OATD-02 Validates the Benefits of Pharmacological Inhibition of Arginase 1 and 2 in Cancer.

BACKGROUND: Arginases play essential roles in metabolic pathways, determining the fitness of both immune and tumour cells. Along with the previously validated role of ARG1 in cancer, the particular significance of ARG2 as a therapeutic target has emerged as its levels correlate with malignant phenotype and poor prognosis. These observations unveil arginases, and specifically ARG2, as well-validated and promising therapeutic targets. OATD-02, a new boronic acid derivative, is the only dual inhibitor, which can address the benefits of pharmacological inhibition of arginase 1 and 2 in cancer. METHODS: The inhibitory activity of OATD-02 was determined using recombinant ARG1 and ARG2, as well as in a cellular system using primary hepatocytes and macrophages. In vivo antitumor activity was determined in syngeneic models of colorectal and kidney carcinomas (CT26 and Renca, respectively), as well as in an ARG2-dependent xenograft model of leukaemia (K562). RESULTS: OATD-02 was shown to be a potent dual (ARG1/ARG2) arginase inhibitor with a cellular activity necessary for targeting ARG2. Compared to a reference inhibitor with predominant extracellular activity towards ARG1, we have shown improved and statistically significant antitumor efficacy in the CT26 model and an immunomodulatory effect reflected by Treg inhibition in the Renca model. Importantly, OATD-02 had a superior activity when combined with other immunotherapeutics. Finally, OATD-02 effectively inhibited the proliferation of human K562 leukemic cells both in vitro and in vivo. CONCLUSIONS: OATD-02 is a potent small-molecule arginase inhibitor with optimal drug-like properties, including PK/PD profile. Excellent activity against intracellular ARG2 significantly distinguishes OATD-02 from other arginase inhibitors. OATD-02 represents a very promising drug candidate for the combined treatment of tumours, and is the only pharmacological tool that can effectively address the benefits of ARG1/ARG2 inhibition. OATD-02 will enter clinical trials in cancer patients in 2022.

The Anti-Inflammatory Effect of Acidic Mammalian Chitinase Inhibitor OAT-177 in DSS-Induced Mouse Model of Colitis.

Inflammatory bowel diseases (IBD) are chronic and relapsing gastrointestinal disorders, where a significant proportion of patients are unresponsive or lose response to traditional and currently used therapies. In the current study, we propose a new concept for anti-inflammatory treatment based on a selective acidic mammalian chitinase (AMCase) inhibitor. The functions of chitinases remain unclear, but they have been shown to be implicated in the pathology of various inflammatory disorders regarding the lung (asthma, idiopathic pulmonary fibrosis) and gastrointestinal tract (IBD and colon cancer). The aim of the study is to investigate the impact of AMCase inhibitor (OAT-177) on the dextran sulfate sodium (DSS)-induced models of colitis. In the short-term therapeutic protocol, OAT-177 given intragastrically in a 30 mg/kg dose, twice daily, produced a significant (p < 0.001) anti-inflammatory effect, as shown by the macroscopic score. Additionally, OAT-177 significantly decreased TNF-α mRNA levels and MPO activity compared to DSS-only treated mice. Intraperitoneal administration of OAT-177 at a dose of 50 mg/kg caused statistically relevant reduction of the colon length. In the long-term therapeutic protocol, OAT-177 given intragastrically in a dose of 30 mg/kg, twice daily, significantly improved colon length and body weight compared to DSS-induced colitis. This is the first study proving that AMCase inhibitors may have therapeutic potential in the treatment of IBD.

Chitinases and Chitinase-Like Proteins as Therapeutic Targets in Inflammatory Diseases, with a Special Focus on Inflammatory Bowel Diseases.

Chitinases belong to the evolutionarily conserved glycosyl hydrolase family 18 (GH18). They catalyze degradation of chitin to N-acetylglucosamine by hydrolysis of the ß-(1-4)-glycosidic bonds. Although mammals do not synthesize chitin, they possess two enzymatically active chitinases, i.e., chitotriosidase (CHIT1) and acidic mammalian chitinase (AMCase), as well as several chitinase-like proteins (YKL-40, YKL-39, oviductin, and stabilin-interacting protein). The latter lack enzymatic activity but still display oligosaccharides-binding ability. The physiologic functions of chitinases are still unclear, but they have been shown to be involved in the pathogenesis of various human fibrotic and inflammatory disorders, particularly those of the lung (idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, sarcoidosis, and asthma) and the gastrointestinal tract (inflammatory bowel diseases (IBDs) and colon cancer). In this review, we summarize the current knowledge about chitinases, particularly in IBDs, and demonstrate that chitinases can serve as prognostic biomarkers of disease progression. Moreover, we suggest that the inhibition of chitinase activity may be considered as a novel therapeutic strategy for the treatment of IBDs.

Discovery of OATD-01, a First-in-Class Chitinase Inhibitor as Potential New Therapeutics for Idiopathic Pulmonary Fibrosis.

Chitotriosidase (CHIT1) and acidic mammalian chitinase (AMCase) are the enzymatically active chitinases that have been implicated in the pathology of chronic lung diseases such as asthma and interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF) and sarcoidosis. The clinical and preclinical data suggest that pharmacological inhibition of CHIT1 might represent a novel therapeutic approach in IPF. Structural modification of an advanced lead molecule 3 led to the identification of compound 9 (OATD-01), a highly active CHIT1 inhibitor with both an excellent PK profile in multiple species and selectivity against a panel of other off-targets. OATD-01 given orally once daily in a range of doses between 30 and 100 mg/kg showed significant antifibrotic efficacy in an animal model of bleomycin-induced pulmonary fibrosis. OATD-01 is the first-in-class CHIT1 inhibitor, currently completed phase 1b of clinical trials, to be a potential treatment for IPF.