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.

Inhibition of Macrophage-Specific CHIT1 as an Approach to Treat Airway Remodeling in Severe Asthma.

Chitotriosidase (CHIT1) is an enzyme produced by macrophages that regulates their differentiation and polarization. Lung macrophages have been implicated in asthma development; therefore, we asked whether pharmacological inhibition of macrophage-specific CHIT1 would have beneficial effects in asthma, as it has been shown previously in other lung disorders. CHIT1 expression was evaluated in the lung tissues of deceased individuals with severe, uncontrolled, steroid-naïve asthma. OATD-01, a chitinase inhibitor, was tested in a 7-week-long house dust mite (HDM) murine model of chronic asthma characterized by accumulation of CHIT1-expressing macrophages. CHIT1 is a dominant chitinase activated in fibrotic areas of the lungs of individuals with fatal asthma. OATD-01 given in a therapeutic treatment regimen inhibited both inflammatory and airway remodeling features of asthma in the HDM model. These changes were accompanied by a significant and dose-dependent decrease in chitinolytic activity in BAL fluid and plasma, confirming in vivo target engagement. Both IL-13 expression and TGFß1 levels in BAL fluid were decreased and a significant reduction in subepithelial airway fibrosis and airway wall thickness was observed. These results suggest that pharmacological chitinase inhibition offers protection against the development of fibrotic airway remodeling in severe asthma.

Inhibition of CHIT1 as a novel therapeutic approach in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and eventually fatal lung disease with a complex etiology. Approved drugs, nintedanib and pirfenidone, modify disease progression, but IPF remains incurable and there is an urgent need for new therapies. We identified chitotriosidase (CHIT1) as new driver of fibrosis in IPF and a novel therapeutic target. We demonstrate that CHIT1 activity and expression are significantly increased in serum (3-fold) and induced sputum (4-fold) from IPF patients. In the lungs CHIT1 is expressed in a distinct subpopulation of profibrotic, disease-specific macrophages, which are only present in patients with ILDs and CHIT1 is one of the defining markers of this fibrosis-associated gene cluster. To define CHIT1 role in fibrosis, we used the therapeutic protocol of the bleomycin-induced pulmonary fibrosis mouse model. We demonstrate that in the context of chitinase induction and the macrophage-specific expression of CHIT1, this model recapitulates lung fibrosis in ILDs. Genetic inactivation of Chit1 attenuated bleomycin-induced fibrosis (decreasing the Ashcroft scoring by 28%) and decreased expression of profibrotic factors in lung tissues. Pharmacological inhibition of chitinases by OATD-01 reduced fibrosis and soluble collagen concentration. OATD-01 exhibited anti-fibrotic activity comparable to pirfenidone resulting in the reduction of the Ashcroft score by 32% and 31%, respectively. These studies provide a preclinical proof-of-concept for the antifibrotic effects of OATD-01 and establish CHIT1 as a potential new therapeutic target for IPF.

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.

Benzoxazepine-Derived Selective, Orally Bioavailable Inhibitor of Human Acidic Mammalian Chitinase.

Human acidic mammalian chitinase (hAMCase) is one of two true chitinases in humans, the function of which remains elusive. In addition to the defense against highly antigenic chitin and chitin-containing pathogens in the gastric and intestinal contents, AMCase has been implicated in asthma, allergic inflammation, and ocular pathologies. Potent and selective small-molecule inhibitors of this enzyme have not been identified to date. Here we describe structural modifications of compound OAT-177, a previously developed inhibitor of mouse AMCase, leading to OAT-1441, which displays high activity and selectivity toward hAMCase. Significantly reduced off-target activity toward the human ether-à-go-go-related gene (hERG) and a good pharmacokinetic profile make OAT-1441 a potential candidate for further preclinical development as well as a useful tool compound to study the physiological role of hAMCase.

Cyclic derivatives of morphiceptin possess anti-transit effect in the gastrointestinal tract and alleviate abdominal pain in mice.

BACKGROUND: Irritable bowel syndrome (IBS) is a chronic condition with recurring gastrointestinal (GI) symptoms: altered motility and abdominal pain. As endogenous opioid system participates in pain perception and in the control of GI peristalsis, opioids have been proposed as a promising therapy in IBS. In a previous study, we observed that morphiceptin derivative, P-317 (Dmt-cyclo-(D-Lys-Phe-D-Pro-Asp)-NH2), presents promising features to be applied in IBS. In this project, we tested whether modifications in cyclic morphiceptin-based structure: fluorination (compound 1) or peptide bond reduction (compound 2) improve pharmacological effect. METHODS: We evaluated tested derivatives in the mouse GI system under physiological (GI transit) and pathophysiological (castor oil diarrhea, stress-induced hypermotility, visceral pain) conditions. RESULTS: Both compounds prolonged GI transit. Compound 1 and P-317 inhibited upper GI transit and motility of the colon; compound 2 remained inactive. Compound 1 and P-317 inhibited hypermotility in stressed mice and delayed the acute diarrhea in comparison to control. Only P-317 exerted antinociceptive effect. None of tested derivatives, similar to P-317, affected locomotor activity. CONCLUSIONS: Compound 1 is equally effective as P-317 in the mouse GI tract. The peptide bond reduction decreased the activity of compound 2. Fluorination appears to be an efficient way to increase the effects of morphiceptin analogs in the GI tract.

Development of Dual Chitinase Inhibitors as Potential New Treatment for Respiratory System Diseases.

Acidic mammalian chitinase (AMCase) and chitotriosidase-1 (CHIT1) are two enzymatically active proteins produced by mammals capable of cleaving the glycosidic bond in chitin. Based on the clinical findings and animal model studies, involvement of chitinases has been suggested in several respiratory system diseases including asthma, COPD, and idiopathic pulmonary fibrosis. Exploration of structure-activity relationships within the series of 1-(3-amino-1H-1,2,4-triazol-5-yl)-piperidin-4-amines, which was earlier identified as a scaffold of potent AMCase inhibitors, led us to discover highly active dual (i.e., AMCase and CHIT1) inhibitors with very good pharmacokinetic properties. Among them, compound 30 was shown to reduce the total number of cells in bronchoalveolar lavage fluid of mice challenged with house dust mite extract after oral administration (50 mg/kg, qd). In addition, affinity toward the hERG potassium channel of compound 30 was significantly reduced when compared to the earlier reported chitinase inhibitors.

Discovery of selective, orally bioavailable inhibitor of mouse chitotriosidase.

This article describes our work towards the identification of a potent and selective inhibitor of mouse chitotriosidase (mCHIT1). A series of small molecule inhibitors of mCHIT1 and mAMCase have been developed from early lead compound 1. Examination of synthetized analogues led to discovery of several novel highly potent compounds. Among them compound 9 (OAT-2068) displays a remarkable 143-fold mCHIT1 vs. mAMCase selectivity. To explain the observed SAR molecular docking experiments were performed, which were in line with the experimental data from the enzymatic assays. Inhibitor 9 (OAT-2068) was found to have an excellent pharmacokinetic profile. This, together with high activity and selectivity, makes the compound an ideal and unique tool for studying the role of CHIT1 in biological models.

Targeting Acidic Mammalian chitinase Is Effective in Animal Model of Asthma.

This article highlights our work toward the identification of a potent, selective, and efficacious acidic mammalian chitinase (AMCase) inhibitor. Rational design, guided by X-ray analysis of several inhibitors bound to human chitotriosidase (hCHIT1), led to the identification of compound 7f as a highly potent AMCase inhibitor (IC50 values of 14 and 19 nM against human and mouse enzyme, respectively) and selective (>150× against mCHIT1) with very good PK properties. This compound dosed once daily at 30 mg/kg po showed significant anti-inflammatory efficacy in HDM-induced allergic airway inflammation in mice, reducing inflammatory cell influx in the BALF and total IgE concentration in plasma, which correlated with decrease of chitinolytic activity. Therapeutic efficacy of compound 7f in the clinically relevant aeroallergen-induced acute asthma model in mice provides a rationale for developing AMCase inhibitor for the treatment of asthma.

Cyclic side-chain-linked opioid analogs utilizing cis- and trans-4-aminocyclohexyl-D-alanine.

Cyclization of linear sequences is a well recognized tool in opioid peptide chemistry for generating analogs with improved bioactivities. Cyclization can be achieved through various bridging bonds between peptide ends or side-chains. In our earlier paper we have reported the synthesis and biological activity of a cyclic peptide, Tyr-c[D-Lys-Phe-Phe-Asp]NH2 (1), which can be viewed as an analog of endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH2). Cyclization was achieved through an amide bond between side-chains of D-Lys and Asp residues. Here, to increase rigidity of the cyclic structure, we replaced d-Lys with cis- or trans-4-aminocyclohexyl-D-alanine (D-ACAla). Two sets of analogs incorporating either Tyr or Dmt (2',6'-dimethyltyrosine) residues in position 1 were synthesized. In the binding studies the analog incorporating Dmt and trans-D-ACAla showed high affinity for both, µ- and δ-opioid receptors (MOR and DOR, respectively) and moderate affinity for the κ-opioid receptor (KOR), while analog with Dmt and cis-D-ACAla was exceptionally MOR-selective. Conformational analyses by NMR and molecular docking studies have been performed to investigate the molecular structural features responsible for the noteworthy MOR selectivity.

Antinociceptive and antidepressant-like action of endomorphin-2 analogs with proline surrogates in position 2.

In our efforts to develop new candidate drugs with antinociceptive and/or antidepressant-like activity, two novel endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH2) analogs, containing proline surrogates in position 2 were synthesized using commercially available racemic trans-4-phenylpyrrolidine-3-carboxylic acid (4-Ph-ß-Pro). The obtained mixture of two diastereoisomeric peptides (2a and 2b) was separated by HPLC and both enantiopure analogs were used in the in vitro and in vivo studies. To assign the absolute configuration to the 4-Ph-ß-Pro residues in both peptides, the stereoselective synthesis of (3R,4S)-4-phenylpyrrolidine-3-carboxylic acid was performed and this enantiomer was introduced into position 2 of EM-2 sequence. Based on the HPLC retention times we were able to assign the absolute configuration of 4-Ph-ß-Pro residues in both peptide analogs. Analog 2a incorporating (3R,4S)-4-Ph-ß-Pro residue produced strong analgesia in mice after intracerebroventricular (icv) administration which was antagonized by the µ-opioid receptor (MOR) antagonist, ß-funaltrexamine (ß-FNA). This analog also influenced an emotion-related behavior of mice, decreasing immobility time in the forced swimming and tail suspension tests, without affecting locomotor activity. The antidepressant-like effect was reversed by the δ-selective antagonist, naltrindole (NLT) and κ-selective nor-binaltorphimine (nor-BNI). Thus, the experiments with selective opioid receptor antagonists revealed that analgesic action of analog 2a was mediated through the MOR, while the δ- and κ-receptors (DOR and KOR, respectively) were engaged in the antidepressant-like activity. Analog 2b with (3S,4R)-4-Ph-ß-Pro in position 2 showed no antinociceptive or antidepressant-like activity in animal studies.

Novel glycosylated endomorphin-2 analog produces potent centrally-mediated antinociception in mice after peripheral administration.

We report the synthesis and pharmacological characterization of a novel glycosylated analog of a potent and selective endogenous µ-opioid receptor (MOP) agonist, endomorphin-2 (Tyr-Pro-Phe-Phe-NH2, EM-2), obtained by the introduction in position 3 of the tyrosine residue possessing the glucose moiety attached to the phenolic function via a ß-glycosidic bond. The improved blood-brain barrier permeability and enhanced antinociceptive effect of the novel glycosylated analog suggest that it may be a promising template for design of potent analgesics. Furthermore, the described methodology may be useful for increasing the bioavailability and delivery of opioid peptides to the CNS.

Synthesis of quaternary α-amino acid-based arginase inhibitors via the Ugi reaction.

The Ugi reaction has been successfully applied to the synthesis of novel arginase inhibitors. In an effort to decrease conformational flexibility of the previously reported series of 2-amino-6-boronohexanoic acid (ABH) analogs 1, we designed and synthesized a series of compounds, 2, in which a piperidine ring is linked directly to a quaternary amino acid center. Further improvement of in vitro activity was achieved by adding two carbon bridge in the piperidine ring, that is, tropane analogs 11. These improvements in activity are rationalized by X-ray crystallography analysis, which show that the tropane ring nitrogen atom moves into direct contact with Asp202 (arginase II numbering). The synthetic routes described here enabled the design of novel arginase inhibitors with improved potency and markedly different physico-chemical properties compared to ABH. Compound 11c represents the most in vitro active arginase inhibitor reported to date.