Search

Acute lung injury (ALI) is an acute, progressive hypoxic respiratory failure that could develop into acute respiratory distress syndrome (ARDS) with very high mortality rate. ALI is believed to be caused by uncontrolled inflammation, and multiple types of immune cells, especially neutrophils, are critically involved in the development of ALI. The treatment for ALI/ARDS is very limited, a better understanding of the pathogenesis and new therapies are urgently needed. Here we discover that GPR84, a medium chain fatty acid receptor, plays critical roles in ALI development by regulating neutrophil functions. GPR84 is highly upregulated in the cells isolated from the bronchoalveolar lavage fluid of LPS-induced ALI mice. GPR84 deficiency or blockage significantly ameliorated ALI mice lung inflammation by reducing neutrophils infiltration and oxidative stress. Further studies reveal that activation of GPR84 strongly induced reactive oxygen species production from neutrophils by stimulating Lyn, AKT and ERK1/2 activation and the assembly of the NADPH oxidase. These results reveal an important role of GPR84 in neutrophil functions and lung inflammation and strongly suggest that GPR84 is a potential drug target for ALI.

The putative medium-chain free fatty acid receptor GPR84 is a G protein-coupled receptor primarily expressed in myeloid cells that constitute the innate immune system, including neutrophils, monocytes, and macrophages in the periphery and microglia in the brain. The fact that GPR84 expression in leukocytes is remarkably increased under acute inflammatory stimuli such as lipopolysaccharide (LPS) and TNFα suggests that it may play a role in the development of inflammatory and fibrotic diseases. Here we demonstrate that GPR84 is highly upregulated in inflamed colon tissues of active ulcerative colitis (UC) patients and dextran sulfate sodium (DSS)-induced colitis mice. Infiltrating GPR84+ macrophages are significantly increased in the colonic mucosa of both the UC patients and the mice with colitis. Consistently, GPR84−/− mice are resistant to the development of colitis induced by DSS. GPR84 activation imposes pro-inflammatory properties in colonic macrophages through enhancing NLRP3 inflammasome activation, while the loss of GPR84 prevents the M1 polarization and properties of proinflammatory macrophages. CLH536, a novel GPR84 antagonist discovered by us, suppresses colitis by reducing the polarization and function of pro-inflammatory macrophages. These results define a unique role of GPR84 in innate immune cells and intestinal inflammation, and suggest that GPR84 may serve as a potential drug target for the treatment of UC.

The farnesoid X receptor (FXR) plays an important role in the regulation of bile acid, lipid, and glucose homeostasis. Recent findings have shown that the inhibition of FXR is beneficial to improvement of related metabolic diseases and cholestasis. In the present work, 9,11-seco-cholesterol derivatives were designed and synthesized by cleaving the C ring of cholesterol and were identified as novel structures of FXR antagonists. Compound

GPR84 is a proinflammatory G protein-coupled receptor associated with several inflammatory and fibrotic diseases. GPR84 antagonists have been evaluated in clinical trials to treat ulcerative colitis, idiopathic pulmonary fibrosis, and nonalcoholic steatohepatitis. However, the variety of potent and selective GPR84 antagonists is still limited. Through high-throughput screening, a novel phosphodiester compound hit

HDAC inhibitors (HDACis) have been intensively studied for their roles and potential as drug targets in T-cell lymphomas and other hematologic malignancies. Bisthianostat is a novel bisthiazole-based pan-HDACi evolved from natural HDACi largazole. Here, we report the preclinical study of bisthianostat alone and in combination with bortezomib in the treatment of multiple myeloma (MM), as well as preliminary first-in-human findings from an ongoing phase 1a study. Bisthianostat dose dependently induced acetylation of tubulin and H3 and increased PARP cleavage and apoptosis in RPMI-8226 cells. In RPMI-8226 and MM.1S cell xenograft mouse models, oral administration of bisthianostat (50, 75, 100 mg·kg

Background: Multiple myeloma (MM) is the second most common hematological malignancy. This disease remains incurable as nearly all patients will relapse and become refractory to established MM therapy. Thus, new treatment option for relapsed or refractory (R/R) MM is needed, particularly those with different mechanisms of action. One such approach is to inhibit histone deacetylase (HDAC) and produce synergistic anti-myeloma activity via mechanisms of epigenetic modulations. In 2015, panobinostat was approved by US FDA as the first HDACi to treat R/R MM in combination with bortezomib and dexamethasone. Bisthianostat is a novel bisthiazole-based HDACi evolved from the thiazole-thiazoline cap group in natural product Largazole (Nan et al., ACS Med Chem Lett. 2014). It is orally available and displayed inhibition against a series of MM cell lines. Here we presented preliminary in-human findings from CH-020PI study, an ongoing phase 1 study of bisthianostat. (Trial registered at ClinicalTrial.gov: NCT03618602) Methods: CH-020PI is a first-in-human study to investigate the safety, tolerability, pharmacokinetics, and efficacy of bisthianostat in R/R MM patients. It is a single center, open-label, single arm, dose escalating phase I study. A standard 3+3 cohort design with 100mg as the starting dose was used to determine the maximum tolerated dose of bisthianostat. This study comprised two phases: a pharmacokinetics phase and an expansion phase. In the pharmacokinetics phase, a single-dose of bisthianostat was administered on day 1, and then multiple-dose was administered on a twice-weekly schedule for 4 consecutive weeks. Patients in the expansion phase received continuous bisthianostat twice weekly until progressive disease or unacceptable toxicities. Results: Until 30 June 2019, 8 patients were enrolled at 3 dose levels from 100 to 400mg. The median age at enrollment was 62 years (range, 51-70 years). The median number of previous lines of therapy was 5 (range, 2-6). Per protocol, all of 8 patients were evaluable for pharmacokinetics, toxicities and efficacy. In the pharmacokinetic evaluation, for all the 8 patients tested at day 1, the peak concentration of bisthianostat was reached within 2.3 hours; half life time were around 4 hours; bisthianostat uptake represented by AUClast were in good proportion to the level of dose as 100, 200 and 400mg, respectively. Similar results were observed at day 28. Any grade hematological treatment-related adverse events (AEs) occurred in 4 of 8 patients (50%), while grade 3/4 hematological AEs occurred in 2 (25%) patients. Any grade non-hematological treatment-emergent AEs were observed in 3 (37.5%) patients; no grade 3/4 non-hematological AEs were reported. No patient discontinued the treatment of bisthianostat due to AEs. Except patient 007 (200mg cohort) experienced a grade 2 nausea, no patients experienced diarrhea, nausea, or vomiting. It is worthy to note that gastrointestinal toxicity is common with the use of panobinostat, a FDA-approved HDAC inhibitor. Overall single-agent efficacy was modest, and stable disease (SD) was observed in 4 (50%) patients. At the time of data cut-off for statistical analysis, no dose-limiting toxicity has been observed. Conclusions: Bisthianostat proved to be well absorbed and tolerated. It exhibited modest anti-tumor efficacy in our cohort of heavily pretreated patients with R/R MM. This phase I clinical trial is currently ongoing, and future trials should compare different doses and schedules of the combination in order to optimize the treatment tolerability and enhance its efficacy. Disclosures No relevant conflicts of interest to declare.

Retigabine, an activator of KCNQ2-5 channels, is currently used to treat partial-onset seizures. The aim of this study was to explore the possibility that structure modification of retigabine could lead to novel inhibitors of KCNQ2 channels, which were valuable tools for KCNQ channel studies. A series of retigabine derivatives was designed and synthesized. KCNQ2 channels were expressed in CHO cells. KCNQ2 currents were recorded using whole-cell voltage clamp technique. Test compound in extracellular solution was delivered to the recorded cell using an ALA 8 Channel Solution Exchange System. A total of 23 retigabine derivatives (HN31-HN410) were synthesized and tested electrophysiologically. Among the compounds, HN38 was the most potent inhibitor of KCNQ2 channels (its IC50 value=0.10±0.05 μmol/L), and was 7-fold more potent than the classical KCNQ inhibitor XE991. Further analysis revealed that HN38 (3 μmol/L) had no detectable effect on channel activation, but accelerated deactivation at hyperpolarizing voltages. In contrast, XE991 (3 μmol/L) did not affect the kinetics of channel activation and deactivation. The retigabine derivative HN38 is a potent KCNQ2 inhibitor, which differs from XE991 in its influence on the channel kinetics. Our study provides a new strategy for the design and development of potent KCNQ2 channel inhibitors.

Two divalent metal ions are commonly seen in the active site cavity of methionine aminopeptidase, and at least one of the metal ions is directly involved in catalysis. Although ample structural and functional information is available for dimetalated enzyme, methionine aminopeptidase likely functions as a monometalated enzyme under physiological conditions. Information on structure, as well as catalysis and inhibition, of the monometalated enzyme is lacking. By improving conditions of high throughput screening, we identified a unique inhibitor with specificity toward the monometalated enzyme. Kinetic characterization indicates a mutual exclusivity in binding between the inhibitor and the second metal ion at the active site. This is confirmed by X-ray structure, and this inhibitor coordinates with the first metal ion and occupies the space normally occupied by the second metal ion. Kinetic and structural analyses of the inhibition by this and other inhibitors provide insight in designing effective inhibitors of methionine aminopeptidase.

Twenty one chromogenic thiopeptolide substrates were designed and synthesized as the active site probes and analyzed with each S1 site of mutant residues and enzymes of wild-type MetAP1s. The preliminary enzymatic experiments indicate that cysteine 70 or 202, at either Escherichia coli or human MetAP1, played a crucial role in the methionine hydrolysis.

Methionine aminopeptidase (MetAP) catalyzes the removal of methionine from newly synthesized polypeptides. MetAP carries out this cleavage with high precision, and Met is the only natural amino acid residue at the N terminus that is accepted, although type I and type II MetAPs use two different sets of residues to form the hydrophobic S1 site. Characteristics of the S1 binding pocket in type I MetAP were investigated by systematic mutation of each of the seven S1 residues in Escherichia coli MetAP type I (EcMetAP1) and human MetAP type I (HsMetAP1). We found that Tyr-65 and Trp-221 in EcMetAP1, as well as the corresponding residues Phe-197 and Trp-352 in HsMetAP1, were essential for the hydrolysis of a thiopeptolide substrate, Met-S-Gly-Phe. Mutation of Phe-191 to Ala in HsMetAP1 caused inactivity in contrast to the full activity of EcMetAP1(Y62A), which may suggest a subtle difference between the two type I enzymes. The more striking finding is that mutation of Cys-70 in EcMetAP1 or Cys-202 in HsMetAP1 opens up the S1 pocket. The thiopeptolides Leu-S-Gly-Phe and Phe-S-Gly-Phe, with previously unacceptable Leu or Phe as the N-terminal residue, became efficient substrates of EcMetAP1(C70A) and HsMetAP1(C202A). The relaxed specificity shown in these S1 site mutants for the N-terminal residues was confirmed by hydrolysis of peptide substrates and inhibition by reaction products. The structural features at the enzyme active site will be useful information for designing specific MetAP inhibitors for therapeutic applications.

A series of pyridine-2-carboxylic acid derivatives were synthesized according to the leads from the screening, and potent inhibitors have been obtained by structural modification. They have shown submicromolar inhibition of the enzymes (for example, for 9n, IC(50) = 130 nM for EcMetAP1 and IC(50) = 380 nM for ScMetAP1). They represent small-molecule MetAP inhibitors with novel structures different from alkylating fumagillin derivatives and peptidic bestatin-based MetAP inhibitor.

Pharmacological augmentation of neuronal KCNQ muscarinic (M) currents by drugs such as retigabine (RTG) represents a first-in-class therapeutic to treat certain hyperexcitatory diseases by dampening neuronal firing. Whereas all five potassium channel subtypes (KCNQ1–KCNQ5) are found in the nervous system, KCNQ2 and KCNQ3 are the primary players that mediate M currents. We investigated the plasticity of subtype selectivity by two M current effective drugs, retigabine and zinc pyrithione (ZnPy). Retigabine is more effective on KCNQ3 than KCNQ2, whereas ZnPy is more effective on KCNQ2 with no detectable effect on KCNQ3. In neurons, activation of muscarinic receptor signaling desensitizes effects by retigabine but not ZnPy. Importantly, reduction of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) causes KCNQ3 to become sensitive to ZnPy but lose sensitivity to retigabine. The dynamic shift of pharmacological selectivity caused by PIP 2 may be induced orthogonally by voltage-sensitive phosphatase, or conversely, abolished by mutating a PIP 2 site within the S4–S5 linker of KCNQ3. Therefore, whereas drug-channel binding is a prerequisite, the drug selectivity on M current is dynamic and may be regulated by receptor signaling pathways via PIP 2 .

A series of 2,4,6-trihydroxychalcone derivatives were synthesized and identified as reversible and competitive protein tyrosine phosphatase (PTP) 1B inhibitors with IC₅₀ values in the micromolar range. Compound 4a had the greatest in vitro inhibition activity against PTP1B (IC₅₀= 0.27 ± 0.01 μM) and the best selectivity (6.9-fold) for PTP1B relative to T-cell protein tyrosine phosphatases. The compounds identified herein provide a foundation on which to design specific inhibitors of PTP1B and other PTPs.

To screen the antimalarial compounds of daphnetin derivatives against Plasmodium falciparum in vitro.Plasmodium faciparum (FCC1) was cultured in vitro by a modified method of Trager and Jensen. Antimalarial compounds were screened by microscopy-based assay and microfluorimetric method.DA79 and DA78 showed potent antimalarial activity against Plasmodium falciparum cultured in vitro.Though the relationship between the structures of daphnetin derivatives and their antimalarial activities has not been clarified yet, this study may provide a new direction for discovery of more potential antimalarial compounds.

Tyrosine kinases have been strongly implicated as therapeutic targets that influence the angiogenic process in growing tumors. In this study, we revealed that TKI-31 is a potent broad spectrum tyrosine kinase inhibitor, which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor beta (PDGFRbeta) and also inhibits kinases of other class, such as c-Kit and c-Src on molecular base, but showed no activity against vascular endothelial growth factor receptor 1 (VEGFR1) and epidermal growth factor receptor (EGFR). TKI-31 inhibits VEGF-induced phosphorylation of VEGFR2 in endothelial cells as well as PDGF(BB)-induced phosphorylation in fibroblast cells, and leading to the inhibition of down-stream signaling triggered by these receptors such as PI3K/Akt/mTOR, MAPK42/44(ERK) and paxillin. TKI-31 also inhibited VEGF-induced endothelial cells proliferation, migration and their differentiation into capillary-like tube formation. Its anti-angiogenic property was further confirmed by the inhibition of neovascularization on CAM, in vivo. These results collectively highlight the therapeutic potential of this compound for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

A series of isoquinoline-1,3,4-trione derivatives were identified as novel and potent inhibitors of caspase-3 through structural modification of the original compound from high-throughput screening. Various analogues (2, 6, 9, 13, and 14) were synthesized and identified as caspase inhibitors, and the introduction of a 6-N-acyl group (compound 13) greatly improved their activity. Some of them showed low nanomolar potency against caspase-3 in vitro (for example, for 6k, IC50 = 40 nM) and significant protection against apoptosis in a model cell system. Additionally, compound 13f demonstrated a dose-dependent decrease in infarct volume in the transient MCA occlusion stroke model. The present small-molecule caspase-3 inhibitor with novel structures different from structures of known caspase inhibitors revealed a new direction for therapeutic strategies directed against diseases involving abnormally up-regulated apoptosis.

A trifunctional probe (binding element-photoreactive group-affinity tag) of natural product paeoniflorin was designed and synthesized based on the previous primary structure-activity relationship. This new probe is a potential tool for labeling, purification, and identification of the target proteins.

Tyrosine kinases are used as important biomarkers in many tumor types. Preclinical and clinical anti-tumor studies have shown that broadly acting tyrosine kinase inhibitors may be more useful than specific inhibitors, since the former might overcome redundancies and crosstalk in tumor cell growth signaling pathways. Here, we aim to identify a novel potent tyrosine kinase inhibitor. Computer modeling of the pyrido-pyrimidine class compound, TKI-28(6-(2,6-dichlorophenyl)-8-methyl-2-phenylamino-8H-pyrido[2,3-d]pyrimidine-7-one), predicted that the compound would dock well in the ATP pocket of the ErbB-2 tyrosine kinase, yielding a high binding affinity for ErbB receptors. Biochemical studies revealed that TKI-28 potently inhibited the activities of tyrosine kinases such as ErbB-2, EGFR, KDR, PDGFRbeta, c-kit and c-Src, but had little effect on Flt-1 in cell-free system. TKI-28 also efficiently blocked autophosphorylation of the listed receptor tyrosine kinases, and subsequently downregulated phosphorylation of many downstream signaling proteins at the cellular level. TKI-28 exhibited a more potent anti-proliferative activity against EGF- and neuregulin-stimulated SK-OV-3 cells versus serum-stimulated cells, accompanied by apparent induction of apoptosis. Finally, TKI-28 was found to possess anti-angiogenic effects, characterized by inhibition of cell proliferation driven by EGF, VEGF and PDGF, as well as decreased cell migration and tube formation in HMECs. These results collectively highlight the pharmacological characteristics of TKI-28 as a broad-spectrum tyrosine kinase inhibitor, suggesting that it has great potential as an anti-cancer and anti-angiogenesis agent.

Systematic SAR studies on the thiazole ring 5-substituent of TCAT derivatives revealed that the introduction of a β-alkoxy or an amino group enhanced the inhibitory activity significantly. The present compounds are representative of specific Co(II)-MetAP1 inhibitors. Before the physiologically relevant metal ions for MetAPs are established, these small molecular compounds could be used as tools for detailed biological studies.

Systematic SAR studies on the HTS hit pyridine-2-carboxylic acid thiazol-2-ylamide (PACT) analogues revealed that the scaffold of PCAT is indispensable for the inhibition of type I MetAP. For effective inhibition of the enzyme, the most suitable position to modify is the 3-position of the pyridine ring of PCAT, and the best substituents are those containing O or N atoms connected directly with the pyridine ring. These findings provide useful information for the design and discovery of more potent inhibitors of type I MetAPs.

Systematic SAR studies on the pyridine ring 3-substituent of PCAT, an inhibitor of EcMetAP1 and ScMetAP1, revealed that 3-substituents have different selectivity for EcMetAP1 and ScMetAP1. The selective inhibitors of type I MetAP are useful tools for investigating the detailed interactions between the enzymes and their inhibitors. In addition, these findings provide useful information for the design and discovery of more potent inhibitors of type I MetAPs.

To screen antifungal drug candidates using in vitro and in vivo assays based on type I methionine aminopeptidase from Saccharomyces cerevisiae (ScMetAP1).A colorimetric assay suitable for high throughput screening (HTS) using recombinant ScMetAP1 protein expressed in Escherichia coli was established for antifungal lead discovery. A series of pyridine-2-carboxylic acid derivatives were characterized and a chemical library of 12,800 pure organic compounds was screened with the in vitro ScMetAP1 assay. Active compounds from the in vitro assay were further evaluated by a growth inhibition assay on yeast strain with deletion of ScMetAP1 gene map1 in comparison with the wild-type yeast strain and the yeast strain with deletion of type II enzyme (ScMetAP2) gene map2.Active ScMetAP1 inhibitors were identified from HTS. Some of the pyridine-2-carboxylic acid derivatives (compound 2 and 3) had selective inhibition of the growth of map2 deletion yeast and weak inhibition on wild-type yeast growth, while no inhibition on map1 deletion yeast.ScMetAP1 is a novel potential target for developing antifungal drugs. The in vitro and in vivo ScMetAP1 assays can serve as tools in discovering antifungal drug candidates.

Methionine aminopeptidase (MetAP) carries out an essential posttranslational modification of nascent proteins by removing the initiator methionine and is recognized as a potential target for developing antibacterial, antifungal, and anticancer agents. We have established an Escherichia coli expression system for human type I MetAP (HsMetAP1) and characterized the full length HsMetAP1 and its N-terminal-truncated mutants HsMetAP1(Delta1-66) and HsMetAP1(Delta1-135) for hydrolysis of several thiopeptolide and peptide substrates and inhibition by a series of nonpeptidic inhibitors. Although the N-terminal extension with zinc finger motifs in HsMetAP1 is not required for enzyme activity, it has a significant impact on the interaction of the enzyme with substrates and inhibitors. In hydrolysis of the thiopeptolide substrates, a relaxation of stringent specificity for the terminal methionine was observed in the truncated mutants. However, this relaxation of specificity was not detectable in hydrolysis of tripeptide or tetrapeptide substrates. Several nonpeptidic inhibitors showed potent inhibition of the mutant HsMetAP1(Delta1-66) but exhibited only weak or no inhibition of the full length enzyme. With the recombinant HsMetAP1 available, we have identified several MetAP inhibitors with submicromolar inhibitory potencies against E. coli MetAP (EcMetAP1) that do not affect HsMetAP1. These results have demonstrated the possibility of developing MetAP inhibitors as antibacterial agents with minimum human toxicity. In addition, micromolar inhibitors of HsMetAP1 identified in this study can serve as tools for investigating the functions of HsMetAP1 in physiological and pathological processes.

Methionine aminopeptidases (MetAPs) have been studied in vitro as Co(II) enzymes, but their in vivo metal remains to be defined. While activation of Escherichia coli MetAP (EcMetAP1) by Co(II), Mn(II), and Zn(II) was detectable by a colorimetric Met–S–Gly–Phe assay, significant activation by Ni(II) was shown in a fluorescence Met-AMC assay, in addition to Co(II) and Mn(II) activation. When tested on the metal-substituted EcMetAP1s, a few inhibitors that we obtained recently from a random screening on Co-EcMetAP1 either became much weak or lost activity on Mn- or Zn-EcMetAP1, although they kept inhibitory activity on Ni-EcMetAP1. A couple of peptidic inhibitors and the methionine mimetic (3R)-amino-(2S)-hydroxyheptanoic acid (AHHpA, 6) maintained moderate activities on Co-, Mn-, Zn-, and Ni-EcMetAP1s. Our results clearly demonstrate that the metal-substitution has changed the enzyme specificity for substrates and inhibitors. Therapeutic applications call for inhibitors specific for MetAP with a physiologically relevant metal at its active site.

Background Methionine aminopeptidase is a potential target of future antibacterial and anticancer drugs. Structural analysis of complexes of the enzyme with its inhibitors provides valuable information for structure-based drug design efforts. Results Five new X-ray structures of such enzyme-inhibitor complexes were obtained. Analysis of these and other three similar structures reveals the adaptability of a surface-exposed loop bearing Y62, H63, G64 and Y65 (the YHGY loop) that is an integral part of the substrate and inhibitor binding pocket. This adaptability is important for accommodating inhibitors with variations in size. When compared with the human isozymes, this loop either becomes buried in the human type I enzyme due to an N-terminal extension that covers its position or is replaced by a unique insert in the human type II enzyme. Conclusion The adaptability of the YHGY loop in E. coli methionine aminopeptidase, and likely in other bacterial methionine aminopeptidases, enables the enzyme active pocket to accommodate inhibitors of differing size. The differences in this adaptable loop between the bacterial and human methionine aminopeptidases is a structural feature that can be exploited to design inhibitors of bacterial methionine aminopeptidases as therapeutic agents with minimal inhibition of the corresponding human enzymes.