Active Small-molecule Inhibitor Research Articles

Old and New Biologics and Small Molecules in Inflammatory Bowel Disease: Anti Integrins

Recently, novel biologics or small molecular drugs have been introduced for overcoming the unmet needs associated with anti-tumor necrosis factor α agents for inflammtory bowel disease (IBD) treatment. Among these novel drugs, anti integrin agents block leukocyte trafficking to the intestine by blocking the interaction between integrin and cell adhesion molecules. Vedolizumab (anti-α4β7) is most widely used anti-integrin approved in both ulcerative colitis and Crohn's disease .It has been shown to be effective in both induction and maintenance therapy with a favorable safety profile due to gut selectivity. Several models incorporating clinical, genetic, immune and gut microbial markers to predict response to vedolizumab in IBD have been developed. Etrolizumab (anti-β7) blocks leukocyte trafficking via α4β7 and cell adhesion via αEβ7 integrins. In addition, the introduction of subcutaneous vedolizumab showed similar efficacy and safety with improved patients' convenience. Other investigational anti-integrin therapies include abrilumab (anti-α4β7 IgG2), PN-943 (orally administered and gut-restricted α4β7 antagonist peptide), AJM300 (orally active small molecule inhibitor of α4), and ontamalimab (anti-MAdCAM-1 IgG).

The galectin-3 inhibitor selvigaltin reduces liver inflammation and fibrosis in a high fat diet rabbit model of metabolic-associated steatohepatitis.

Galectin-3 is a pro-fibrotic β-galactoside binding lectin highly expressed in fibrotic liver and implicated in hepatic fibrosis. Selvigaltin (previously known as GB1211) is a novel orally active galectin-3 small molecule inhibitor that has high affinity for galectin-3 (human KD = 25nM; rabbit KD = 12nM) and high oral bioavailability in rabbits and man. In this study the efficacy of selvigaltin was investigated in a high fat diet (HFD) rabbit model of metabolic-associated steatohepatitis (MASH). Male New Zealand White rabbits were individually caged under standard conditions in a temperature and humidity-controlled room on a 12h light/darkness cycle. After 1week of regular diet (RD), rabbits were randomly assigned for 8 or 12weeks to different groups: RD/vehicle, RD/selvigaltin, HFD (8weeks), HFD/vehicle and HFD/selvigaltin (0.3, 1.0, 5.0 or 30mg/kg selvigaltin with vehicle/selvigaltin p.o. dosed therapeutically q.d. 5days per week from week 9 or 12). Liver inflammation, steatosis, ballooning, and fibrosis was measured via blood metabolic markers, histomorphological evaluation [Oil Red O, Giemsa, Masson's trichome, picrosirius red (PSR) and second harmonic generation (SHG)], and mRNA and protein expression. Steatosis, inflammation, ballooning, and fibrosis were all increased from RD to HFD/vehicle groups. Selvigaltin demonstrated target engagement by significantly decreasing galectin-3 levels in the liver as measured via immunohistochemistry and mRNA analysis. Selvigaltin dose-dependently reduced biomarkers of liver function (AST, ALT, bilirubin), inflammation (cells foci), and fibrosis (PSR, SHG), as well as decreasing the mRNA and protein expression of several key inflammation and fibrosis biomarkers (e.g., IL6, TGFβ3, SNAI2, collagen). Doses of 1.0 or 5.0mg/kg demonstrated consistent efficacy across most biological endpoints supporting the current clinical doses of selvigaltin being investigated in liver disease. Selvigaltin significantly reduced hepatic inflammation and fibrosis in an HFD rabbit model of MASH following therapeutic dosing for 4 weeks in a dose-dependent manner. These data support the human selvigaltin dose of 100mg b.i.d. that has been shown to reduce key liver biomarkers during a clinical study in liver cirrhosis.

HBCVTr: an end-to-end transformer with a deep neural network hybrid model for anti-HBV and HCV activity predictor from SMILES

Hepatitis B and C viruses (HBV and HCV) are significant causes of chronic liver diseases, with approximately 350 million infections globally. To accelerate the finding of effective treatment options, we introduce HBCVTr, a novel ligand-based drug design (LBDD) method for predicting the inhibitory activity of small molecules against HBV and HCV. HBCVTr employs a hybrid model consisting of double encoders of transformers and a deep neural network to learn the relationship between small molecules’ simplified molecular-input line-entry system (SMILES) and their antiviral activity against HBV or HCV. The prediction accuracy of HBCVTr has surpassed baseline machine learning models and existing methods, with R-squared values of 0.641 and 0.721 for the HBV and HCV test sets, respectively. The trained models were successfully applied to virtual screening against 10 million compounds within 240 h, leading to the discovery of the top novel inhibitor candidates, including IJN04 for HBV and IJN12 and IJN19 for HCV. Molecular docking and dynamics simulations identified IJN04, IJN12, and IJN19 target proteins as the HBV core antigen, HCV NS5B RNA-dependent RNA polymerase, and HCV NS3/4A serine protease, respectively. Overall, HBCVTr offers a new and rapid drug discovery and development screening method targeting HBV and HCV.

Mechanistic and thermodynamic characterization of antiviral inhibitors targeting nucleocapsid N-terminal domain of SARS-CoV-2

The nucleocapsid (N) protein of SARS-CoV-2 plays a pivotal role in encapsulating the viral genome. Developing antiviral treatments for SARS-CoV-2 is imperative due to the diminishing immunity of the available vaccines. This study targets the RNA-binding site located in the N-terminal domain (NTD) of the N-protein to identify the potential antiviral molecules against SARS-CoV-2. A structure-based repurposing approach identified the twelve high-affinity molecules from FDA-approved drugs, natural products, and the LOPAC1280 compound libraries that precisely bind to the RNA binding site within the NTD. The interaction of these potential antiviral agents with the purified NTD protein was thermodynamically characterized using isothermal titration calorimetry (ITC). A fluorescence-based plate assay to assess the RNA binding inhibitory activity of small molecules against the NTD has been employed, and the selected compounds exhibited significant RNA binding inhibition with calculated IC50 values ranging from 8.8 μM to 15.7 μM. Furthermore, the antiviral efficacy of these compounds was evaluated using in vitro cell-based assays targeting the replication of SARS-CoV-2. Remarkably, two compounds, Telmisartan and BMS-189453, displayed potential antiviral activity against SARS-CoV-2, with EC50 values of approximately 1.02 μM and 0.98 μM, and a notable selective index of >98 and > 102, respectively. This study gives valuable insight into developing therapeutic interventions against SARS-CoV-2 by targeting the N-protein, a significant effort given the global public health concern posed due to the virus re-emergence and long COVID-19 disease.

Targeting of STAT5 using the small molecule topotecan hydrochloride suppresses acute myeloid leukemia progression.

Acute myeloid leukemia (AML) is a common type of acute leukemia in adults and relapse is one of the main reasons for treatment failure. FLT3‑ITD mutations are associated with poor prognosis, short disease‑free progression survival and high relapse rates in patients with AML. STAT5 is activated by FLT3‑ITD and drives the pathogenesis of AML. STAT5 activation is usually a hallmark of hematologic malignancies and occurs in ~70% of patients with AML. Moreover, STAT5 is a key molecule which regulates hematopoiesis, and its high expression is closely associated with drug resistance, thus direct targeting of STAT5 for AML is of great clinical value. The present study introduces a new small‑molecule inhibitor that targets STAT5, presenting a promising approach for AML therapy. A high throughput fluorescence polarization (FP) screening system for STAT5 was designed and established, and used to screen an existing compound library to obtain the highly active small molecule inhibitor, topotecan hydrochloride. Topotecan hydrochloride was demonstrated to be an effective inhibitor of STAT5 by molecular docking prediction and cellular thermal shift assay. Topotecan hydrochloride bound to STAT5, inhibiting its dimerization, phosphorylation and transcription of specific target genes. The compound exhibits cellular activity at the nanomolar level and significantly inhibits the proliferation of human AML cell lines and FLT3‑ITD+ AML cells. Furthermore, topotecan hydrochloride has the potential to exert an anti‑tumor effect invivo. Overall, topotecan hydrochloride offers a new opportunity for the treatment of AML and other hematologic malignancies by directly targeting STAT5.

A phase 2 study of alrizomadlin (APG-115) in combination with pembrolizumab in patients with unresectable or metastatic cutaneous melanoma that has failed immuno-oncologic (IO) drugs.

9559 Background: Alrizomadlin is a novel, orally active small-molecule MDM2 inhibitor that activates p53-mediated apoptosis in tumor cells, also functions as a host immunomodulator, and may restore antitumor activity in patients with cancer that has progressed on PD-1/PD-L1 inhibitors. Alrizomadlin is currently being investigated for treatment of patients with unresectable or metastatic cutaneous melanoma that progressed on prior immunotherapy. Methods: This multicenter clinical trial was conducted in the United States and Australia in patients with unresectable or metastatic cutaneous melanoma that had progressed on PD-1/PD-L1 immunotherapy. Eligible patients had an ECOG performance status of 0-2. Alrizomadlin 150 mg was administered orally every other day for 2 consecutive weeks, with 1 week off, and pembrolizumab 200 mg intravenously for 30 minutes on Day 1 of a 21-day cycle. Results: As of December 12, 2022, preliminary and interim results are reported for 31 patients with cutaneous melanoma, of whom 21 were male and 10 female, with a median (range) age of 65 (27-84) years. Treatment-related adverse events (TRAE) of any grade ( > 10%) for either alrizomadlin or pembrolizumab were reported in 30/31 (96.8%) patients. These AEs included nausea (71%), vomiting (38.7%), fatigue (35.5%), thrombocytopenia (32.3%), diarrhea (25.8%), neutropenia (19.4%), decreased appetite (16.1%), and decreased leukocyte count (12.9%). A total of 4/31 (12.9%) patients reported treatment-related serious AEs, including anemia, thrombocytopenia, deep vein thrombosis, joint effusion, pulmonary embolism, and vomiting. In 26 efficacy-evaluable patients, the confirmed overall response rate (ORR; partial response [PR] + complete response [CR]) was 23.1%, including 2 patients reporting CRs and 4 reporting PRs based on RECIST v.1.1. The initial analysis indicated that the ORR observed in patients whose disease had failed IO treatment was primarily due to alrizomadlin combined with pembrolizumab, not the delayed effect of prior immunotherapy. Conclusions: Alrizomadlin combined with pembrolizumab is well tolerated and demonstrates clinical efficacy in these patients with highly refractory unresectable or metastatic cutaneous melanoma that had progressed on PD-1/PD-L1 immunotherapy. Internal study identifier: APG-115-US002. Clinical trial information: NCT03611868 .

A phase 1a/1b study of aurora kinase A inhibitor VIC-1911 as monotherapy and in combination with sotorasib for the treatment of KRAS G12C-mutant non–small-cell lung cancer.

TPS9140 Background: Activity of direct covalent KRASG12C inhibitors in NSCLC is limited by mutational and adaptive resistance. AURKA has roles in centrosome maturation and cytokinesis. AURKA amplification/overexpression is reported in multiple tumors, including NSCLC, and AURKA signaling may mediate adaptive resistance to KRAS inhibition. VIC-1911 is a highly selective, orally active small molecule inhibitor of AURKA. VIC-1911 demonstrated monotherapy activity in vitro in KRASG12C-mutant human NSCLC cells with intrinsic and acquired resistance to the KRASG12C inhibitor sotorasib, and combination VIC-1911 and sotorasib showed synergy in the same cell lines. Combination VIC-1911 and sotorasib delayed the emergence of adaptive resistance in vitro. These findings suggest that 1) AURKA activation is present in both intrinsic and acquired resistance to sotorasib in KRASG12C-mutant NSCLC and 2) the combination of VIC-1911 and sotorasib is a potential therapeutic approach for KRASG12C-mutant NSCLC with intrinsic or acquired resistance to sotorasib monotherapy. In addition, in vivo data suggest both sotorasib and adagrasib are synergistic in combination with VIC-1911 in human KRASG12C-mutant NSCLC cell line xenograft models. Methods: A non-randomized, open-label Phase 1a/1b study of VIC-1911, an aurora kinase A inhibitor, administered as monotherapy and in combination with sotorasib for the treatment of advanced KRASG12C-mutant NSCLC is in progress. Participants ≥18 years, with locally advanced or metastatic KRASG12C-mutant NSCLC, prior treatment with at least 1 line of PD-1/PD-L1 inhibitor therapy with or without platinum-based chemotherapy and have adequate organ function are eligible. Both KRASG12C inhibitor naïve and experienced patients are eligible. VIC-1911 monotherapy is given at oral doses of 25 – 90 mg twice daily in dose escalation and at the VIC-1911 maximum tolerated dose (MTD) in the expansion cohort in patients who are refractory/relapsed on prior KRASG12C inhibitor therapy. The combination regimen includes dose escalation with VIC-1911 at oral doses of 75, 150 or 200 mg twice daily on days 1-4, 8-11 and 15-18 every 28-day cycle plus sotorasib 960 mg daily in patients who are refractory/relapsed or naïve to prior KRASG12C inhibitor therapy. In dose escalation, a 3+3 schema is followed with dose limiting toxicity (DLT) criteria for toxicity and a sample size ≤ 36. For expansion cohorts, the sample size is based on Simon 2-stage optimal design, with a sample size ≤ 104. VIC-1911 and sotorasib dose modification criteria are provided. Supportive medications are allowed and early stopping rules for toxicity will be followed. VIC-1911 pharmacokinetics will be assessed. Pharmacodynamics will be determined from ctDNA and tumor biomarker analysis. Clinical trial information: NCT05374538 .

Biosensor-based active ingredient recognition system for screening TNF-α inhibitors from lotus leaves.

Erhuangquzhi granules (EQG) have been clinically proven to be effective in nonalcoholic steatohepatitis (NASH) treatment. However, the active components and molecular mechanisms remain unknown. This study aimed to screen active components targeting tumor necrosis factor α (TNF-α) in EQG for the treatment of NASH by a surface plasmon resonance (SPR) biosensor-based active ingredient recognition system (SPR-AIRS). The amine-coupling method was used to immobilize recombinant TNF-α protein on an SPR chip, the specificity of the TNF-α-immobilized chip was validated, and nine medicinal herbs in EQG were prescreened. Nuciferine (NF), lirinidine (ID), and O-nornuciferine (NNF) from lotus leaves were found and identified as TNF-α ligands by UPLC‒MS/MS, and the affinity constants of NF, ID, and NNF to TNF-α were determined by SPR experiments (Kd = 61.19, 31.02, and 20.71µM, respectively). NF, ID, and NNF inhibited TNF-α-induced apoptosis in L929 cells, the levels of secreted IL-6 and IL-1β were reduced, and the phosphorylation of IKKβ and IκB was inhibited in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In conclusion, a class of new active small-molecule TNF-α inhibitors was discovered, which also provides a valuable reference for the material basis and mechanism of EQG action in NASH treatment.

Factor XI inhibitors: cardiovascular perspectives.

Anticoagulants are the cornerstone for prevention and treatment of thrombosis but are not completely effective, and concerns about the risk of bleeding continue to limit their uptake. Animal studies and experience from patients with genetic coagulation factor XI deficiency suggesting that this factor is more important for thrombosis than for haemostasis raises the potential for drugs that target factor XI to provide safer anticoagulation. Multiple factor XI inhibitors are currently under evaluation in clinical trials, including parenterally administered antisense oligonucleotides, monoclonal antibodies, and orally active small-molecule inhibitors. Promising results of phase 2 trials in patients undergoing major orthopaedic surgery, and in those with end-stage kidney disease, atrial fibrillation and acute coronary syndromes have led to large phase 3 trials that are currently ongoing. We here review premises for the use of these agents, results so far accrued, ongoing studies, and perspectives for future patient care.

Development of an Orally Active Small-Molecule Inhibitor of Receptor Activator of Nuclear Factor-κB Ligand.

Receptor activator of nuclear factor-κB (RANK) and its ligand, RANKL, play pivotal roles in bone remodeling. The monoclonal antibody denosumab successfully inhibited the maturation of osteoclasts (OCs) by binding to RANKL in the clinic. We continued our efforts to develop small-molecule inhibitors of RANKL. In this work, 41 β-carboline derivatives were synthesized based on previously synthesized compound Y1599 to improve its drug-like properties. Compound Y1693 was identified as a potent RANKL inhibitor that improved absorption-distribution-metabolism-excretion properties and effectively prevented RANKL-induced osteoclastogenesis and bone resorption. Furthermore, Y1693 also suppressed the expression of OC marker genes. Moreover, Y1693 demonstrated good tolerability and efficacy in an orally administered mouse model of osteoporosis as well as the ability to rescue alveolar bone loss in vivo caused by periodontal disease. Collectively, the above findings may provide a valuable direction for the development of novel antiresorptive therapies that target RANKL.

CFI-402257, a TTK inhibitor, effectively suppresses hepatocellular carcinoma

Deregulation of cell cycle is a typical feature of cancer cells. Normal cells rely on the strictly coordinated spindle assembly checkpoint (SAC) to maintain the genome integrity and survive. However, cancer cells could bypass this checkpoint mechanism. In this study, we showed the clinical relevance of threonine tyrosine kinase (TTK) protein kinase, a central regulator of the SAC, in hepatocellular carcinoma (HCC) and its potential as therapeutic target. Here, we reported that a newly developed, orally active small molecule inhibitor targeting TTK (CFI-402257) effectively suppressed HCC growth and induced highly aneuploid HCC cells, DNA damage, and micronuclei formation. We identified that CFI-402257 also induced cytosolic DNA, senescence-like response, and activated DDX41-STING cytosolic DNA sensing pathway to produce senescence-associated secretory phenotypes (SASPs) in HCC cells. These SASPs subsequently led to recruitment of different subsets of immune cells (natural killer cells, CD4+ T cells, and CD8+ T cells) for tumor clearance. Our mass cytometry data illustrated the dynamic changes in the tumor-infiltrating immune populations after treatment with CFI-402257. Further, CFI-402257 improved survival in HCC-bearing mice treated with anti-PD-1, suggesting the possibility of combination treatment with immune checkpoint inhibitors in HCC patients. In summary, our study characterized CFI-402257 as a potential therapeutic for HCC, both used as a single agent and in combination therapy.

A small-molecule Skp1 inhibitor elicits cell death by p53-dependent mechanism

SummarySkp1 overexpression promotes tumor growth, whereas reduced Skp1 activity is also linked with genomic instability and neoplastic transformation. This highlights the need to gain better understanding of Skp1 biology in cancer settings. To this context, potent and cellularly active small-molecule Skp1 inhibitors may be of great value. Using a hypothesis-driven, structure-guided approach, we herein identify Z0933M as a potent Skp1 inhibitor with KD ∼0.054 μM. Z0933M occupies a hydrophobic hotspot (P1) – encompassing an aromatic cage of two phenylalanines (F101 and F139) – alongside C-terminal extension of Skp1 and, thus, hampers its ability to interact with F-box proteins, a prerequisite step to constitute intact and active SCF E3 ligase(s) complexes. In cellulo, Z0933M disrupted SCF E3 ligase(s) functioning, recapitulated previously reported effects of Skp1-reduced activity, and elicited cell death by a p53-dependent mechanism. We propose Z0933M as valuable tool for future efforts toward probing Skp1 cancer biology, with implications for cancer therapy.

Preclinical anti-tumor activity of small-molecule oral PD-L1 checkpoint inhibitors.

e14558 Background: The PD-1/PD-L1 checkpoint axis is a validated target in oncology, and immunotherapy with antibody approaches have proven efficacy across various tumor types. Oral small-molecule inhibitors of PD-L1 may offer advantages over antibody approaches by enabling tunable on-target engagement and the potential for better tissue penetration and improved efficacy. Here we report the preclinical in vitro activity of small-molecule inhibitors of PD-L1 possessing a novel mechanism of action, favorable pharmacokinetics, and demonstrated in vivo efficacy in a mouse colon adenocarcinoma model. Methods: In vitro activity was assessed in a Jurkat T cell NFAT reporter assay and PD-L1 reduction was confirmed in CHO-K1 cells expressing human PD-L1 (CHO-K1-hPD-L1) and in peripheral blood mononuclear cells (PBMCs) from healthy donors. T cell activation assays were conducted with PBMCs from healthy donors. Non-specific cytokine release was evaluated in human whole blood. Pharmacokinetic (PK) evaluations were conducted in rodents and non-human primates (NHP). In vivo efficacy was evaluated in an MC38 tumor humanized PD-L1 and PD-1 mouse model. Results: Small-molecule inhibitors of PD-L1 were able to disrupt PD-1:PD-L1 and PD-L1:CD80 interactions through the reduction of PD-L1 expression on the cell surface via a novel internalization mechanism, resulting in PD-L1 degradation (EC50s ranged from 1.9 – 24 nM in CHO-K1-hPD-L1 and primary human myeloid cells). Lead PD-L1 inhibitors mediated potent activation of T cells in a NFAT reporter assay (EC50s of 13 and 18 nM) as well as dose responsive elevations in IL-2 production in human PBMCs stimulated with staphylococcal enterotoxin B. Compound treatment did not elicit non-specific cytokine release in human whole blood, supportive of favorable immune safety. PK profiles showed low systemic clearance in rodents and NHP. In an MC38 tumor model, once daily oral administration of compounds at 3, 10, and 30 mg/kg resulted in dose responsive tumor reduction that was associated with T cell activation and infiltration into tumors. Conclusions: Oral small-molecule PD-L1 inhibitors possessing a novel mechanism of action and the ability to mediate T cell activation in primary human immune cell types were identified. These compounds display in vivo anti-tumor efficacy comparable to anti-PD-L1 antibody and possess favorable preclinical profiles for further development, with the potential for all-oral treatment regimens for oncology.

The p97 Inhibitor UPCDC-30245 Blocks Endo-Lysosomal Degradation.

The diverse modes of action of small molecule inhibitors provide versatile tools to investigate basic biology and develop therapeutics. However, it remains a challenging task to evaluate their exact mechanisms of action. We identified two classes of inhibitors for the p97 ATPase: ATP competitive and allosteric. We showed that the allosteric p97 inhibitor, UPCDC-30245, does not affect two well-known cellular functions of p97, endoplasmic-reticulum-associated protein degradation and the unfolded protein response pathway; instead, it strongly increases the lipidated form of microtubule-associated proteins 1A/1B light chain 3B (LC3-II), suggesting an alteration of autophagic pathways. To evaluate the molecular mechanism, we performed proteomic analysis of UPCDC-30245 treated cells. Our results revealed that UPCDC-30245 blocks endo-lysosomal degradation by inhibiting the formation of early endosome and reducing the acidity of the lysosome, an effect not observed with the potent p97 inhibitor CB-5083. This unique effect allows us to demonstrate UPCDC-30245 exhibits antiviral effects against coronavirus by blocking viral entry.

Decisive role of water and protein dynamics in residence time of p38\u03b1 MAP kinase inhibitors

Target residence time plays a crucial role in the pharmacological activity of small molecule inhibitors. Little is known, however, about the underlying causes of inhibitor residence time at the molecular level, which complicates drug optimization processes. Here, we employ all-atom molecular dynamics simulations (~400 μs in total) to gain insight into the binding modes of two structurally similar p38α MAPK inhibitors (type I and type I½) with short and long residence times that otherwise show nearly identical inhibitory activities in the low nanomolar IC50 range. Our results highlight the importance of protein conformational stability and solvent exposure, buried surface area of the ligand and binding site resolvation energy for residence time. These findings are further confirmed by simulations with a structurally diverse short residence time inhibitor SB203580. In summary, our data provide guidance in compound design when aiming for inhibitors with improved target residence time.

Exploring sodium glucose cotransporter (SGLT2) inhibitors with machine learning approach: A novel hope in anti-diabetes drug discovery

Conventional anti-diabetes agents exhibit some undesirable side effects. Recently, lactic acidosis and/or bladder cancer were also reported with the use of these agents. Hence, there is an urgent need for alternative anti-diabetes in order to reduce/avoid the unwanted effects. In this scenario sodium glucose cotransporter 2 (SGLT2) inhibitors has already been established as an important class of anti-diabetic drug. The search for new generation SGLT2 inhibitors with high affinity is still an ongoing process. Here, we aim to develop computational models to predict the SGLT2 inhibitory activity of small molecules based on chemical structures. This work provides in-silico analysis to propose possible fragment/fingerprint identification recommended for SGLT2 inhibitors. Up-to-our knowledge, this study is an initiative to propose fingerprints responsible for SGLT2 inhibition.Furthermore, we used nine different algorithms to build machine learning (ML) models that could be used to prioritize compounds as SGLT2 inhibitors from large libraries. The best performing ML models were applied to virtually screen a large collection of FDA approved drugs. The best predicted compounds have been recommended to be biologically investigated in future in order to identify next generation SGLT2 inhibitors with different chemical structure.

Discovery and Mechanism of Action of Small Molecule Inhibitors of Ceramidases.

Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to produce pro-proliferative sphingosine-1-phosphate. Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases. However, due to their hydrophobic nature, no specific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug-like inhibitors of alkaline ceramidase 3 (ACER3). In particular, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts.

Discovery and Mechanism of Action of Small Molecule Inhibitors of Ceramidases**

AbstractSphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro‐apoptotic lipid catabolized by ceramidase enzymes to produce pro‐proliferative sphingosine‐1‐phosphate. Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases. However, due to their hydrophobic nature, no specific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug‐like inhibitors of alkaline ceramidase 3 (ACER3). In particular, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non‐lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts.

Anti-Integrins for the Treatment of Inflammatory Bowel Disease: Current Evidence and Perspectives.

Leukocyte trafficking to the gastrointestinal tract is recognized to play a role in the pathogenesis of inflammatory bowel disease (IBD). Integrins are expressed on immune cells and interact with cell adhesion molecules (CAM) to mediate leukocyte trafficking. Blockade of the gut-tropic integrin α4β7 and its subunits has been exploited as a therapeutic target in IBD. Natalizumab (anti-α4) is approved for moderate to severe Crohn’s disease (CD), but its use is limited due to potential risk of progressive multifocal leukoencephalopathy. Vedolizumab (anti-α4β7) is approved for the treatment of ulcerative colitis (UC) and CD. It is the most widely used anti-integrin therapy in IBD and has been shown to be effective in both induction and maintenance therapy, with a favorable safety profile. Several models incorporating clinical, genetic, immune, gut microbial, and vitamin D markers to predict response to vedolizumab in IBD have been developed. Etrolizumab (anti-β7) blocks leukocyte trafficking via α4β7 and cell adhesion via αEβ7 integrins. Large phase 3 clinical trials evaluating efficacy of etrolizumab in the induction and maintenance of patients with IBD are underway. Other investigational anti-integrin therapies include abrilumab (anti-α4β7 IgG2), PN-943 (orally administered and gut-restricted α4β7 antagonist peptide), AJM300 (orally active small molecule inhibitor of α4), and ontamalimab (anti-MAdCAM-1 IgG).

Abstract 1257: HM43239, a novel FLT3 inhibitor, has the potential to inhibit mutations resistant to FLT3 inhibitors

Abstract Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 (FLT3) mutations is associated with poor prognosis with a high risk of relapse after therapy and reduces overall survival. Approximately 30 % of AML patients carry FLT3 internal tandem duplication (ITD) or tyrosine kinase domain (TKD) mutations. Sustained FLT3 inhibition can result in the emergence of resistance-conferring genetic alteration in TKD domain, usually at residues D835 and F691. Therefore, acquired TKD mutations has become a critical therapeutic target in AML therapy. On the other hand, SYK is one of kinases deeply implicated in many hematologic malignancies and highly activated in FLT3 mutation AML. SYK overexpression is known to promote over transformation of FLT3 driven AML and induce resistance to FLT3 targeted therapy. In this study, we characterized HM43239, a novel FLT3 inhibitor with SYK inhibitory activity, and assessed its potential as a novel therapeutic agent to overcome the resistances against AML therapy using current FLT3 inhibitors. HM43239 is an orally active small molecule inhibitor and it exhibited sub-nanomolar potency on binding affinity for FLT3 wild type, ITD, TKDs and ITD/TKDs mutants. HM43239 potently inhibited phosphorylation of FLT3 and its downstream such as p-STAT3/p-STAT5 dose dependently in both MOLM-14 cells harboring FLT3 ITD/F691L and FLT3 ITD/D835Y. In KG-1a cells, HM43239 potently inhibited phosphorylation of SYK, STAT3 and STAT5. Moreover, it inhibited the proliferation and induced the apoptosis of leukemic stem cell (LSC) marker-expressing KG-1a cells (CD34+/CD38- cells), suggesting the possibility of targeting LSC. Furthermore, HM43239 showed to be exhibited good inhibitory activity against FLT3 mutated AML cell lines and effectively regress the tumors in MOLM-14 cells expressing FLT3 ITD/F691L or FLT3 ITD/D835Y xenograft mice models. On the other hand, it was confirmed that HM43239 strongly inhibited the phosphorylation of SYK in the medium of co-culture of stromal cells and AML cells. As a result, HM43239 alone more effectively induced tumor regression and prolonged the survival duration of animals than an approved FLT3 inhibitor (e.g. gilteritinib) in resistant FLT3 ITD/D835Y or ITD/F691L mutated MOLM-14 xenograft mice models. These results suggest that HM43239 could overcome the resistance induced by bone marrow microenvironment in AML patients. Taken together, HM43239 showed strong anticancer activity through various in vitro and in vivo preclinical models of AML, implicating the mechanism of overcoming resistance and preventing relapse. The effect of HM43239 in human would be demonstrated in ongoing Phase I/II clinical trials (NCT03850574) to develop promising therapeutics for patients with AML. Citation Format: Inhwan Bae, Jaeyul Choi, Jiyoung Song, Joo-Yun Byun, Eunyoung Lee, Taehun Song, Yu-Yon Kim, Yesol Bak, Young Hoon Kim, Young Gil Ahn, Kwee Hyun Suh. HM43239, a novel FLT3 inhibitor, has the potential to inhibit mutations resistant to FLT3 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 1257.