Discovery of Pyrazolo[1,5-a]pyrimidine derivative as a potent and selective PI3Kγ/δ dual inhibitor.

Phosphoinositol 3-kinases (PI3Ks) γ and δ are primarily expressed in leukocytes and play crucial roles in regulation of the immune system. Dual inhibition of PI3Kγ/δ has emerged as an effective approach to regulate the tumor microenvironment. Here, we report the exploration of structure-activity relationship optimization which led to the discovery of a potent PI3Kγ/δ dual inhibitor 15u (IHMT-PI3K-455). 15u exhibits strong potency in biochemical and cellular assays and it repolarizes M2 phenotype toward M1 phenotype in THP-1 and BMDM macrophages. In addition, it shows suitable in vivo properties as demonstrated through pharmacokinetic studies in rats and pharmacodynamics properties in a MC38 xenograft model.

Discovery of a highly potent pan-RAF inhibitor IHMT-RAF-128 for cancer treatment.

Although rat sarcoma viral oncogene homolog (RAS) mutations occur in about 30% of solid tumors, targeting RAS mutations other than KRAS-G12C is still challenging. As an alternative approach, developing inhibitors targeting RAF, the downstream effector of RAS signaling, is currently one of the main strategies for cancer therapy. Selective v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-V600E inhibitors Vemurafenib, Encorafenib, and Dabrafenib have been approved by FDA and received remarkable clinical responses, but these drugs are ineffective against RAS mutant tumors due to limited inhibition on dimerized RAF. In this study, we developed a highly potent pan-RAF inhibitor, IHMT-RAF-128, which exhibited similarly high efficacies in inhibiting both partners of the RAF dimer, and showed potent anti-tumor efficacy against a variety of cancer cells harboring either RAF or RAS mutations, especially Adagrasib and Sotorasib (AMG510) resistant-KRAS-G12C secondary mutations, such as KRAS-G12C-Y96C and KRAS-G12C-H95Q. In addition, IHMT-RAF-128 showed excellent pharmacokinetic profile (PK), and the bioavailability in mice and rats were 63.9%, and 144.1%, respectively. Furthermore, IHMT-RAF-128 exhibited potent anti-tumor efficacy on xenograft mouse tumor models in a dose-dependent manner without any obvious toxicities. Together, these results support further investigation of IHMT-RAF-128 as a potential clinical drug candidate for the treatment of cancer patients with RAF or RAS mutations.

Discovery of IHMT-MST1-39 as a novel MST1 kinase inhibitor and AMPK activator for the treatment of diabetes mellitus.

Insulin-producing pancreatic ß cell death is the fundamental cause of type 1 diabetes (T1D) and a contributing factor to type 2 diabetes (T2D). Moreover, metabolic disorder is another hallmark of T2D. Mammalian sterile 20-like kinase 1 (MST1) contributes to the progression of diabetes mellitus through apoptosis induction and acceleration of pancreatic ß cell dysfunction. AMP-activated protein kinase (AMPK) is an energy sensing kinase and its activation has been suggested as a treatment option for metabolic diseases. Thus, pharmacological inhibition of MST1 and activation of AMPK simultaneously represents a promising approach for diabetes therapy. Here, we discovered a novel selective MST1 kinase inhibitor IHMT-MST1-39, which exhibits anti-apoptosis efficacy and improves the survival of pancreatic ß cells under diabetogenic conditions, as well as primary pancreatic islets in an ex vivo disease model. Mechanistically, IHMT-MST1-39 activated AMPK signaling pathway in hepatocytes in vitro, combination of IHMT-MST1-39 and metformin synergistically prevented hyperglycemia and significantly ameliorated glucose tolerance and insulin resistance in diabetic mice. Taken together, IHMT-MST1-39 is a promising anti-diabetic candidate as a single agent or in combination therapy for both T1D and T2D.

Magnetosome-inspired synthesis of soft ferrimagnetic nanoparticles for magnetic tumor targeting.

Magnetic targeting is one of the most promising approaches for improving the targeting efficiency by which magnetic drug carriers are directed using external magnetic fields to reach their targets. As a natural magnetic nanoparticle (MNP) of biological origin, the magnetosome is a special "organelle" formed by biomineralization in magnetotactic bacteria (MTB) and is essential for MTB magnetic navigation to respond to geomagnetic fields. The magnetic targeting of magnetosomes, however, can be hindered by the aggregation and precipitation of magnetosomes in water and biological fluid environments due to the strong magnetic attraction between particles. In this study, we constructed a magnetosome-like nanoreactor by introducing MTB Mms6 protein into a reverse micelle system. MNPs synthesized by thermal decomposition exhibit the same crystal morphology and magnetism (high saturation magnetization and low coercivity) as natural magnetosomes but have a smaller particle size. The DSPE-mPEG-coated magnetosome-like MNPs exhibit good monodispersion, penetrating the lesion area of a tumor mouse model to achieve magnetic enrichment by an order of magnitude more than in the control groups, demonstrating great prospects for biomedical magnetic targeting applications.

Discovery of IHMT-MST1-58 as a Novel, Potent, and Selective MST1 Inhibitor for the Treatment of Type 1/2 Diabetes.

The critical pathogenesis of type 1 diabetes (T1D)/type 2 diabetes (T2D) is the physical status, mass, and function of pancreatic ß cells. Mammalian STE20-like protein 1 kinase (MST1) plays vital roles in the apoptosis and insulin secretion of ß cells. Here, we discovered a novel, potent, and selective MST1 inhibitor 19 (IC50 = 23 nM), which inhibited the phosphorylation of MST1-protected ß cells from the damage of inflammatory cytokines in vitro. In vivo, it displayed acceptable pharmacokinetic properties in different species. In the STZ-induced T1D/T2D mouse models, both monotherapy of 19 and in combination with metformin led to the decline of fasting blood glucose and showed protective effect of ß cells. In addition, the combination of 19 and metformin decreased the hemoglobin A1c level. Together, our study suggested that 19 might be a useful pharmacological tool to study MST1-mediated physiology and pathology as well as a potential drug candidate for diabetes.

Heat shock proteins: Biological functions, pathological roles, and therapeutic opportunities.

The heat shock proteins (HSPs) are ubiquitous and conserved protein families in both prokaryotic and eukaryotic organisms, and they maintain cellular proteostasis and protect cells from stresses. HSP protein families are classified based on their molecular weights, mainly including large HSPs, HSP90, HSP70, HSP60, HSP40, and small HSPs. They function as molecular chaperons in cells and work as an integrated network, participating in the folding of newly synthesized polypeptides, refolding metastable proteins, protein complex assembly, dissociating protein aggregate dissociation, and the degradation of misfolded proteins. In addition to their chaperone functions, they also play important roles in cell signaling transduction, cell cycle, and apoptosis regulation. Therefore, malfunction of HSPs is related with many diseases, including cancers, neurodegeneration, and other diseases. In this review, we describe the current understandings about the molecular mechanisms of the major HSP families including HSP90/HSP70/HSP60/HSP110 and small HSPs, how the HSPs keep the protein proteostasis and response to stresses, and we also discuss their roles in diseases and the recent exploration of HSP related therapy and diagnosis to modulate diseases. These research advances offer new prospects of HSPs as potential targets for therapeutic intervention.

Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib-induced KIT mutations in gastrointestinal stromal tumours.

Drug resistance remains a major challenge in the clinical treatment of gastrointestinal stromal tumours (GISTs). While acquired on-target mutations of mast/stem cell growth factor receptor (KIT) kinase is the major resistance mechanism, activation of alternative signalling pathways may also play a role. Although several second- and third-generation KIT kinase inhibitors have been developed that could overcome some of the KIT mutations conferring resistance, the low clinical responses and narrow safety window have limited their broad application. The present study revealed that nintedanib not only overcame resistance induced by a panel of KIT primary and secondary mutations, but also overcame ERK-reactivation-mediated resistance caused by the upregulation of fibroblast growth factor (FGF) activity. In preclinical models of GISTs, nintedanib significantly inhibited the proliferation of imatinib-resistant cells, including GIST-5R, GIST-T1/T670I and GIST patient-derived primary cells. In addition, it also exhibited dose-dependent inhibition of ERK phosphorylation upon FGF ligand stimulation. In vivo antitumour activity was also observed in several xenograft GIST models. Considering the well-documented safety and pharmacokinetic profiles of nintedanib, this finding provides evidence for the repurposing of nintedanib as a new therapy for the treatment of GIST patients with de novo or acquired resistance to imatinib.

Inhibition of the deubiquitinating enzyme USP47 as a novel targeted therapy for hematologic malignancies expressing mutant EZH2.

Activating mutations in EZH2, the catalytic component of PRC2, promote cell proliferation, tumorigenesis, and metastasis through enzymatic or non-enzymatic activity. The EZH2-Y641 gain-of-function mutation is one of the most significant in diffuse large B-cell lymphoma (DLBCL). Although EZH2 kinase inhibitors, such as EPZ-6438, provide clinical benefit, certain cancer cells are resistant to the enzymatic inhibition of EZH2 because of the inability to functionally target mutant EZH2, or because of cells' dependence on the non-histone methyltransferase activity of EZH2. Consequently, destroying mutant EZH2 protein may be more effective in targeting EZH2 mutant cancers that are dependent on the non-catalytic activity of EZH2. Here, using extensive selectivity profiling, combined with genetic and animal model studies, we identified USP47 as a novel regulator of mutant EZH2. Inhibition of USP47 would be anticipated to block the function of mutated EZH2 through induction of EZH2 degradation by promoting its ubiquitination. Moreover, targeting of USP47 leads to death of mutant EZH2-positive cells in vitro and in vivo. Taken together, we propose targeting USP47 with a small molecule inhibitor as a novel potential therapy for DLBCL and other hematologic malignancies characterized by mutant EZH2 expression.

Osimertinib successfully combats EGFR-negative glioblastoma cells by inhibiting the MAPK pathway.

Glioblastoma (GBM) patients have extremely poor prognoses, and currently no effective treatment available including surgery, radiation, and chemotherapy. MAPK-interacting kinases (MNK1/2) as the downstream of the MAPK-signaling pathway regulate protein synthesis in normal and tumor cells. Research has shown that targeting MNKs may be an effective strategy to treat GBM. In this study we investigated the antitumor activity of osimertinib, an FDA-approved epidermal growth factor receptor (EGFR) inhibitor, against patient-derived primary GBM cells. Using high-throughput screening approach, we screened the entire panel of FDA-approved drugs against primary cancer cells derived from glioblastoma patients, found that osimertinib (3 µM) suppressed the proliferation of a subset (10/22) of EGFR-negative GBM cells (>50% growth inhibition). We detected the gene expression difference between osimertinib-sensitive and -resistant cells, found that osimertinib-sensitive GBM cells displayed activated MAPK-signaling pathway. We further showed that osimertinib potently inhibited the MNK kinase activities with IC50 values of 324 nM and 48.6 nM, respectively, against MNK1 and MNK2 kinases; osimertinib (0.3-3 µM) dose-dependently suppressed the phosphorylation of eukaryotic translation initiation factor 4E (eIF4E). In GBM patient-derived xenografts mice, oral administration of osimertinib (40 mg· kg-1 ·d-1, for 18 days) significantly suppressed the tumor growth (TGI = 74.5%) and inhibited eIF4E phosphorylation in tumor cells. Given the fact that osimertinib could cross the blood-brain barrier and its toxicity was well tolerated in patients, our results suggest that osimertinib could be a new and effective drug candidate for the EGFR-negative GBM patients.

Discovery of (S)-2-(1-(4-Amino-3-(3-fluoro-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propyl)-3-cyclopropyl-5-fluoroquinazolin-4(3H)-one (IHMT-PI3Kδ-372) as a Potent and Selective PI3Kδ Inhibitor for the Treatment of Chronic Obstructive Pulmonary Disease.

Accumulated pieces of evidence have shown that PI3Kδ plays a critical role in chronic obstructive pulmonary disease (COPD). Using a fragment-hybrid approach, we discovered a potent and selective PI3Kδ inhibitor (S)-18. In the biochemical assay, (S)-18 inhibits PI3Kδ (IC50 = 14 nM) with high selectivity over other class I PI3Ks (56∼83 fold). (S)-18 also achieves good selectivity over other protein kinases in the kinome (S-score (35) = 0.015). In the cell, (S)-18 selectively and potently inhibits the PI3Kδ-mediated phosphorylation of AKT T308 but not other class I PI3K-mediated signaling. Additionally, (S)-18 exhibits no apparent inhibitory effect on CYP isoforms except for a moderate effect on CYP2C9. Furthermore, it shows no apparent inhibitory activity against hERG (IC50 > 10 µM). In vivo, (S)-18 displays favorable PK properties for inhaled delivery and improves lung function in a rodent model of pulmonary inflammation. These results suggest that (S)-18 might be a new potential therapeutic candidate for COPD.

Discovery of a highly selective VEGFR2 kinase inhibitor CHMFL-VEGFR2-002 as a novel anti-angiogenesis agent.

Angiogenesis is an essential process in tumor growth, invasion and metastasis. VEGF receptor 2 (VEGFR2) inhibitors targeting tumor angiogenic pathway have been widely used in the clinical cancer treatment. However, most of currently used VEGFR2 kinase inhibitors are multi-target inhibitors which might result in target-associated side effects and therefore limited clinical toleration. Highly selective VEGFR inhibitors are still highly demanded from both basic research and clinical application point of view. Here we report the discovery and characterization of a novel VEGFR2 inhibitor (CHMFL-VEGFR2-002), which exhibited high selectivity among structurally closed kinases including PDGFRs, FGFRs, CSF1R, etc. CHMFL-VEGFR2-002 displayed potent inhibitory activity against VEGFR2 kinase in the biochemical assay (IC50 = 66 nmol/L) and VEGFR2 autophosphorylation in cells (EC50s ∼100 nmol/L) as well as potent anti-proliferation effect against VEGFR2 transformed BaF3 cells (GI50 = 150 nmol/L). In addition, CHMFL-VEGFR2-002 also displayed good anti-angiogenesis efficacy in vitro and exhibited good in vivo PK (pharmacokinetics) profile with bioavailability over 49% and anti-angiogenesis efficacy in both zebrafish and mouse models without apparent toxicity. These results suggest that CHMFL-VEGFR2-002 might be a useful research tool for dissecting new functions of VEGFR2 kinase as well as a potential anti-angiogenetic agent for the cancer therapy.

Discovery of 6'-chloro-N-methyl-5'-(phenylsulfonamido)-[3,3'-bipyridine]-5-carboxamide (CHMFL-PI4K-127) as a novel Plasmodium falciparum PI(4)K inhibitor with potent antimalarial activity against both blood and liver stages of Plasmodium.

Starting from a bipyridine-sulfonamide scaffold, medicinal chemistry optimization leads to the discovery of a novel Plasmodium falciparum PI4K kinase (PfPI4K) inhibitor compound 15g (CHMFL-PI4K-127, IC50: 0.9 nM), which exhibits potent activity against 3D7 Plasmodium falciparum (P. falciparum) (EC50: 25.1 nM). CHMFL-PI4K-127 displays high selectivity against PfPI4K over human lipid and protein kinase. In addition, it exhibits EC50 values of 23-47 nM against a panel of the drug-resistant strains of P. falciparum. In vivo, the inhibitor demonstrates the favorable pharmacokinetic properties in both rats and mice. Furthermore, oral administration of CHMFL-PI4K-127 exhibits the antimalaria efficacy in both blood stage (80 mg/kg) and liver stage (1 mg/kg) of Plasmodium in infected rodent model. The results suggest that CHMFL-PI4K-127 might be a new potential drug candidate for malaria.

Low dose of emetine as potential anti-SARS-CoV-2 virus therapy: preclinical in vitro inhibition and in vivo pharmacokinetic evidences.

The global pandemic of COVID-19 has attracted extensive drug searching interets for the new coronavirus SARS-CoV-2. Although currently several of clinically used "old" drugs have been repurposed to this new disease for the urgent clinical investigation, there is still great demand for more effective therapies for the anti-infections. Here we report the discovery that an "old" drug Emetine could potently inhibit SARS-CoV-2 virus replication and displayed virus entry blocking effect in Vero cells at low dose. In addition, Emetine could significantly reduce the lipopolysaccharide (LPS) induced interleukin-6 (IL-6) protein level and moderately reduce the tumor necrosis factor (TNF-α) protein level in the M1 polarized THP-1 macrophages. In vivo animal pharmacokinetics (PK) study revealed that Emetine was enriched in the lung tissue and had a long retention time (over 12 h). With 1 mg/kg single oral dose, the effective concentration of Emetine in lung was up to 1.8 µM (mice) and 1.6 µM (rats) at 12 h, which is over 200-fold higher than the EC50 of the drug. The potent in vitro antiviral replication efficacy and the high enrichment in target tissue, combining with the well documented safety profiles in human indicate that low dose of Emetine might be a potentially effective anti-SARS-CoV-2 infection therapy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s43556-020-00018-9.

Discovery of a novel and highly selective CDK9 kinase inhibitor (JSH-009) with potent antitumor efficacy in preclinical acute myeloid leukemia models.

Acute myeloid leukemia (AML) is reported to be vulnerable to transcription disruption due to transcriptional addiction. Cyclin-dependent kinase 9 (CDK9), which regulates transcriptional elongation, has attracted extensive attention as a drug target. Although several inhibitors, such as alvocidib and dinaciclib, have shown potent therapeutic effects in clinical trials on AML, the lack of high selectivity for CDK9 and other CDKs has limited their optimal clinical efficacy. Therefore, developing highly selective CDK9 inhibitors is still imperative for the efficacy and safety profile in treating AML. Here, we report a novel highly selective CDK9 inhibitor, JSH-009, which exhibited high potency against CDK9 and displayed great selectivity over 468 kinases/mutants. It also demonstrates impressive in vitro and in vivo antileukemic efficacy in preclinical models of AML, which makes JSH-009 a useful pharmacological tool for elucidating CDK9-mediated transcription and a novel therapeutic candidate for AML.

Discovery of 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)acetamide (CHMFL-KIT-64) as a Novel Orally Available Potent Inhibitor against Broad-Spectrum Mutants of c-KIT Kinase for Gastrointestinal Stromal Tumors.

Starting from our previously developed c-KIT kinase inhibitor CHMFL-KIT-8140, through a type II kinase inhibitor binding element hybrid design approach, we discovered a novel c-KIT kinase inhibitor compound 18 (CHMFL-KIT-64), which is potent against c-KIT wt and a broad spectrum of drug-resistant mutants with improved bioavailability. 18 exhibits single-digit nM potency against c-KIT kinase and c-KIT T670I mutants in the biochemical assay and displays great potencies against most of the gain-of-function mutations in the juxtamembrane domain, drug-resistant mutations in the ATP binding pocket (except V654A), and activation loops (except D816V). In addition, 18 exhibits a good in vivo pharmacokinetic (PK) profile in different species including mice, rats, and dogs. It also displays good in vivo antitumor efficacy in the c-KIT T670I, D820G, and Y823D mutant-mediated mice models as well as in the c-KIT wt patient primary cells which are known to be imatinib-resistant. The potent activity against a broad spectrum of clinically important c-KIT mutants combining the good in vivo PK/pharmacodynamic properties of 18 indicates that it might be a new potential therapeutic candidate for gastrointestinal stromal tumors.

Discovery of ( E)- N1-(3-Fluorophenyl)- N3-(3-(2-(pyridin-2-yl)vinyl)-1 H-indazol-6-yl)malonamide (CHMFL-KIT-033) as a Novel c-KIT T670I Mutant Selective Kinase Inhibitor for Gastrointestinal Stromal Tumors (GISTs).

Gain-of-function mutations of c-KIT kinase play crucial pathological roles for the gastrointestinal stromal tumors (GISTs). Despite the success of imatinib as the first-line treatment of GISTs, dozens of drug-acquired resistant mutations emerge, and c-KIT T670I is one of the most common mutants among them. Although several kinase inhibitors are capable of overcoming the T670I mutant, none of them can achieve the selectivity over the c-KIT wild-type (wt), which also plays important roles in a variety of physiological functions such as hematopoiesis. Starting from axitinib, through fragment hybrid type II kinase inhibitor design approach, we have discovered a novel inhibitor 24, which not only exhibits potent activity to c-KIT T670I mutant but also achieves 12-fold selectivity over c-KIT wt. Compound 24 displays good antiproliferative effects against c-KIT T670I mutant-driven GIST cell lines (GIST-T1/T670I and GIST-5R) and also exhibits suitable in vivo pharmacokinetic profiles as well as dose-dependent antitumor efficacy. This study provides a proof of concept for developing a c-KIT mutant selective inhibitor that theoretically can render a better therapeutic window.

Discovery and characterization of a novel highly potent and selective type II native and drug-resistant V299L mutant BCR-ABL inhibitor (CHMFL-ABL-039) for Chronic Myeloid Leukemia (CML).

BCR fused ABL kinase is the critical driving oncogene for chronic myeloid leukemia (CML) and has been extensively studied as the drug discovery target in the past decade. The successful introduction of tyrosine kinase inhibitors (TKI) such as Imatinib, Dasatinib and Bosutinib has greatly improved the CML patient survival rate. However, upon the chronic treatment, a variety of TKI resistant mutants, such as the V299L mutant which has been found in more and more patients with the high-throughput sequencing technology, are observed, although the incidence is still considered rare compared to the more prevalent gatekeeper T315I mutant. However, with the progress of the precision medicine concept, the rare mutation (or the orphan drug target) has attracted more and more attention. Here we report a novel type II BCR-ABL kinase inhibitor, CHMFL-ABL-039, which not only displayed great potency (IC50: 7.9 nM) and selectivity (S score (1) = 0.02) against native ABL kinase among other kinases in the kinome, but also exhibited great potency (IC50: 27.9 nM) and selectivity against Imatinib-resistant V299L mutant among other frequently observed ABL kinase mutants. CHMFL-ABL-039 has demonstrated greater efficacies than Imatinib regarding to the anti-proliferation, inhibition of the signaling pathway, arrest of cell cycle progression, induction of apoptosis in vitro and suppression of the tumor progression in vivo in the native and V299L mutated BCR-ABL kinase-driven cells/xenograft models. It would be a useful pharmacological tool to study the TKI resistant ABL V299L mutant-mediated pathology and provide a potential precise treatment approach for this orphan CML subtype in the precision medicine era.

Repurposing cabozantinib to GISTs: Overcoming multiple imatinib-resistant cKIT mutations including gatekeeper and activation loop mutants in GISTs preclinical models.

Despite of the great success of imatinib as the first-line treatment for GISTs, the majority of patients will develop drug-acquired resistance due to secondary mutations in the cKIT kinase. Sunitinib and regorafenib have been approved as the second and third line therapies to overcome some of these drug-resistance mutations; however, their limited clinical response, toxicity and resistance of the activation loop mutants still makes new therapies bearing different cKIT mutants activity spectrum profile highly demanded. Through a drug repositioning approach, we found that cabozantinib exhibited higher potency than imatinib against primary gain-of-function mutations of cKIT. Moreover, cabozantinib was able to overcome cKIT gatekeeper T670I mutation and the activation loop mutations that are resistant to imatinib or sunitinib. Cabozantinib demonstrated good efficacy in vitro and in vivo in the cKIT mutant-driven preclinical models of GISTs while displaying a long-lasting effect after treatment withdrawal. Furthermore, it also exhibited dose-dependent anti-proliferative efficacy in the GIST patient derived primary cells. Considering clinical safety and PK profile of cabozantinib, this report provides the basis for the future clinical applications of cabozantinib as an alternative anti-GISTs therapy in precision medicine.