SKLB-14b, a novel oral microtubule-destabilizing agent based on hydroxamic acid with potent anti-tumor and anti-multidrug resistance activities.

A hydroxamic acid based microtubule-destabilizing agent (MDA) SKLB-14b was discovered in this study, which was derived from shortening the linker length of the HDAC6 and microtubule dual-target inhibitor SKLB-23bb. SKLB-14b exhibited low nanomolar IC50 values on a wide spectrum of human cancer cell lines including both sensitive and multidrug-resistant cell lines. Surprisingly, its anti-proliferative activity relied on the presence of the hydroxamic acid group but lost inhibitory activity against HDACs. SKLB-14b bound to the colchicine site of tubulin and could inhibit tubulin polymerization. It exhibited good metabolic stability in liver microsomes, no inhibitory effect on CYP450 isoenzymes and high oral bioavailability. In vivo experiments revealed that SKLB-14b was potent in both sensitive (A2780S, HCT116) and resistant (A2780/T) xenograft mice models. Furthermore, in the patient-derived tumor xenograft (PDX) models of osimertinib resistant non-small cell lung cancer (NSCLC), 50 mg/kg of SKLB-14b administered every twodays inhibited tumor growth by 70.6% without obvious toxicity, better than the 59.7% inhibition rate of paclitaxel. Mechanistically, we found that SKLB-14b exerted anti-tumor and anti-multidrug resistance effects in vitro and in vivo through cell cycle arrest and pro-apoptotic activities, as well as vascular disrupting activities. Therefore, we discovered that SKLB-14b, as a novel MDA based on hydroxamic acid, could serve as a potential drug candidate for cancer therapy which deserves further investigation.

Identification of the target protein and molecular mechanism of honokiol in anti-inflammatory action.

BACKGROUND: Searching the targets of natural products is very important for drug discovery and elucidating the mechanism of drug action and disease. Honokiol (HK), as the major active component of Magnolia officinalis Rehder & E.H.Wilson, has been widely used in medicine and cosmetics. Among its bioactivities, its anti-inflammatory activity is particularly impressive. However, the target protein of HK in anti-inflammatory action and its regulatory mechanism are unclear. PURPOSE: Here, we identified the target protein and molecular mechanism of the anti- inflammatory action of HK. METHODS: First, an LPS-induced septic shock model and DSS-induced ulcerative colitis model were used to assess the anti-inflammatory efficacy of HK. Second, the drug affinity responsive target stability, proteomics analysis, thermal shift assays and cellular thermal shift assays were used to identify and validate the target of HK. Finally, western blot, ELISA, LDH immunofluorescence staining, shRNA and LC/MS for L-leucine analysis were performed to determine the mechanism of the anti-inflammatory action of HK. RESULTS: This study revealed that HK significantly alleviated LPS-induced septic shock and DSS-induced ulcerative colitis in vivo, suggesting that HK has significant anti-inflammatory activity. HK treatment dramatically reduced IL-1ß release and caspase-1 activation at different time points, showing that HK could inhibit both NLRP3 inflammasome priming and activation processes in cells. HK also suppressed adaptor apoptosis speck-like protein oligomerization. Mechanistically, SLC3A2 was identified as a direct target of HK in THP-1 cells. HK downregulated SLC3A2 expression by promoting its degradation via proteasome-mediated proteolysis. Further study demonstrated that HK triggered SLC3A2 to suppress NLRP3 inflammasome activation by significantly reducing the content of L-leucine transported into cells and lysosomes to block the mTORC1 pathway. CONCLUSIONS: Our work identified HK as a promising anti-inflammatory drug candidate through the SLC3A2/L-leucine/mTORC1/NLRP3 pathways.

HDAC I/IIb selective inhibitor Purinostat Mesylate combined with GLS1 inhibition effectively eliminates CML stem cells.

Purinostat Mesylate (PM) is a novel highly selective and active HDAC I/IIb inhibitor, and the injectable formulation of PM (PMF) based on the compound prescription containing cyclodextrin completely can overcome PM's poor solubility and improves its stability and pharmacokinetic properties. Here, we showed that PM effectively repressed the survival of Ph+ leukemia cells and CD34+ leukemia cells from CML patients in vitro. In vivo studies demonstrated that PMF significantly prevented BCR-ABL(T315I) induced CML progression by restraining leukemia stem cells (LSCs), which are insensitive to chemotherapy and responsible for CML relapse. Mechanism studies revealed that targeting HDAC I/IIb repressed several important factors for LSCs survival including c-Myc, ß-Catenin, E2f, Ezh2, Alox5, and mTOR, as well as interrupted some critical biologic processes. Additionally, PMF increased glutamate metabolism in LSCs by increasing GLS1. The combination of PMF and glutaminase inhibitor BPTES synergistically eradicated LSCs by altering multiple key proteins and signaling pathways which are critical for LSC survival and self-renewal. Overall, our findings represent a new therapeutic strategy for eliminating LSCs by targeting HDAC I/IIb and glutaminolysis, which potentially provides a guidance for PMF clinical trials in the future for TKI resistance CML patients.

Therapeutic efficacy of an injectable formulation of purinostat mesylate in SU-DHL-6 tumour model.

Background: Previous studies have proven that Purinostat Mesylate (PM) is a new HDAC inhibitor and exhibits significant antitumor efficacy. However, the clinical application of PM was greatly limited by its poor solubility in water and low bioavailability.Objective:To increase the solubility of PM through pharmaceutical research, and prepare it into an injection that meets the needs of intravenous use to promote its clinical application.MethodsThe prepared PM/HP-ß-CD inclusion complex was studied by computer simulation, fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (1H-NMR spectroscopy), and scanning electron microscopy (SEM). Then, the antitumor effects of PM/HP-ß-CD inclusion complex were studied by in vitro cytotoxicity assay, apoptosis assay, pharmacokinetic study and in vivo antitumor assay.Results:Phase Solubility Analysis revealed that PM and HP-ß-CD were compatible and the solubility of PM increased almost 220 times, to 2.02 mg/mL. The interaction mechanism studies revealed that PM could be embedded into the cavity of HP-ß-CD through the side of the aminobenzene ring. Cell viability and apoptosis assays showed that PM/HP-ß-CD complex maintained the good anti-cancer activity of PM, and PM/HP-ß-CD complex has a better anti-tumor effect and lower toxicity than LBH589 and Hyper-CVAD/RTX in vivo. All the results suggest that HP-ß-CD can solve the problem of PM administration and provide a way for clinical application of PM.Conclusions: In this study, an injectable formulation of PM in HP-ß-CD (10% w/v) was prepared to improve its water solubility. Our research provides a way for clinical administration of PM, which has been under phase I clinical trial for the treatment of relapsed or refractory B-cell-related hematologic malignancies in China and the USA.KEY MESSAGESWe developed a preparation of Purinostat Mesylate that can be administered intravenously, reducing the toxicity associated with oral administration.This preparation has an outstanding therapeutic effect on SU-DHL-6 xenograft tumour, indicating its clinical value, which has been under phase I clinical trial for the treatment of relapsed or refractory B-cell-related haematologic malignancies in China and the USA.

Honokiol Ameliorates Post-Myocardial Infarction Heart Failure Through Ucp3-Mediated Reactive Oxygen Species Inhibition.

Post-myocardial infarction heart failure (post-MI HF) is one of the leading global causes of death, and current prevention and treatment methods still cannot avoid the increasing incidence. Honokiol (HK) has previously been reported to improve myocardial ischemia/reperfusion injury and reverse myocardial hypertrophy by activating Sirt1 and Sirt3. We suspect that HK may also have a therapeutic effect on post-MI HF. In this study, we aimed to investigate the efficacy and mechanism of HK in the treatment of post-MI HF. We found that HK inhibited myocardial reactive oxygen species (ROS) production, reduced myocardial fibrosis, and improved cardiac function in mice after MI. HK also reduced the abnormality of mitochondrial membrane potential (MMP) and apoptosis of cardiomyocytes caused by peroxide in neonatal cardiomyocytes. RNAseq results revealed that HK restored the transcriptome changes to a certain extent and significantly enhanced the expression of mitochondrial inner membrane uncoupling protein isoform 3 (Ucp3), a protein that inhibits the production of mitochondrial ROS, protects cardiomyocytes, and relieves heart failure after myocardial infarction (MI). In cardiomyocytes with impaired Ucp3 expression, HK cannot protect against the damage caused by peroxide. More importantly, in Ucp3 knockout mice, HK did not change the increase in the ROS level and cardiac function damage after MI. Taken together, our results suggest that HK can increase the expression of the cardioprotective protein Ucp3 and maintain MMP, thereby inhibiting the production of ROS after MI and ameliorating heart failure.

Corrigendum: Honokiol ameliorates post-myocardial infarction heart failure through Ucp3-mediated reactive oxygen species inhibition.

[This corrects the article DOI: 10.3389/fphar.2022.811682.].

Identification of a Novel 2,8-Diazaspiro[4.5]decan-1-one Derivative as a Potent and Selective Dual TYK2/JAK1 Inhibitor for the Treatment of Inflammatory Bowel Disease.

In this study, we described a series of 2,8-diazaspiro[4.5]decan-1-one derivatives as selective TYK2/JAK1 inhibitors. Systematic exploration of the structure-activity relationship through the introduction of spirocyclic scaffolds based on the reported selective TYK2 inhibitor 14l led to the discovery of the superior derivative compound 48. Compound 48 showed excellent potency on TYK2/JAK1 kinases with IC50 values of 6 and 37 nM, respectively, and exhibited more than 23-fold selectivity for JAK2. Compound 48 also demonstrated excellent metabolic stability and more potent anti-inflammatory efficacy than tofacitinib in acute ulcerative colitis models. Moreover, the excellent anti-inflammatory effect of compound 48 was mediated by regulating the expression of related TYK2/JAK1-regulated genes, as well as the formation of Th1, Th2, and Th17 cells. Taken together, these findings suggest that compound 48 is a selective dual TYK2/JAK inhibitor, deserving to be developed as a clinical candidate.

Structure-Based Design and Synthesis of N-Substituted 3-Amino-ß-Carboline Derivatives as Potent αß-Tubulin Degradation Agents.

So far, relatively few small molecules have been reported to promote tubulin degradation. Our previous studies have found that compound 2, a noncovalent colchicine-site ligand, was capable of promoting αß-tubulin degradation. To further improve its antiproliferative activity, 66 derivatives or analogues of 2 were designed and synthesized based on 2-tubulin cocrystal structure. Among them, 12b displayed nanomolar potency against a variety of tumor cells, including paclitaxel- and adriamycin-resistant cell lines. 12b binds to the colchicine site and promotes αß-tubulin degradation in a concentration-dependent manner via the ubiquitin-proteasome pathway. The X-ray crystal structure revealed that 12b binds in a similar manner as 2, but there is a slight conformation change of the B ring, which resulted in better interaction of 12b with surrounding residues. 12b effectively suppressed tumor growth at an i.v. dose of 40 mg/kg (3 times a week) on both A2780S (paclitaxel-sensitive) and A2780T (paclitaxel-resistant) ovarian xenograft models, with respective TGIs of 92.42 and 79.75% without obvious side effects, supporting its potential utility as a tumor-therapeutic compound.

A size-shrinkable matrix metallopeptidase-2-sensitive delivery nanosystem improves the penetration of human programmed death-ligand 1 siRNA into lung-tumor spheroids.

Given the maturation of small-interfering RNA (siRNA) techniques with nanotechnology, and because overexpression of human programmed death-ligand 1 (PD-L1) is crucial for T cell inactivation and immunosuppression of the tumor microenvironment, application of siRNA-PD-L1 has demonstrated positive progress in preclinical studies; however, the limited penetration of this compound into solid tumors remains a challenge. To decrease PD-L1 expression and increase the penetration efficacy of solid tumors, we synthesized a novel tumor-microenvironment-sensitive delivery polymer by conjugating hyaluronic acid (HA) to polyethyleneimine (PEI), with a matrix metalloproteinase-2 (MMP-2)-sensitive peptide acting as the linker (HA-P-PEI), for use in delivery of PD-L1-siRNA. Concurrent synthesis of a linker-less HA-PEI compound allowed confirmation that negatively charged siRNA can be complexed onto the positively charged HA-PEI and HA-P-PEI compounds to form nanoparticles with the same particle size and uniform distribution with serum stability. We found that the size of the HA-P-PEI/siRNA nanoparticles decreased to <10 nm upon addition of MMP-2, and that H1975 cells overexpressing CD44, PD-L1, and MMP-2 aided confirmation of the delivery efficacy of the HA-P-PEI/siRNA nanocomplexes. Additionally, the use of HA-P-PEI caused less cytotoxicity than PEI alone, demonstrating its high cellular uptake. Moreover, pretreatment with MMP-2 increased nanocomplex tumor permeability, and western blot showed that HA-P-PEI/PD-L1-siRNA efficiently downregulated the PD-L1 expression in H1975 cells. These results demonstrated a novel approach for siRNA delivery and tumor penetration for future clinical applications in cancer treatment.

Discovery of a Series of Hydroxamic Acid-Based Microtubule Destabilizing Agents with Potent Antitumor Activity.

Hydroxamic acid group is one of the characteristic pharmacophores of histone deacetylase (HDAC) inhibitors. But here, we discovered a series of hydroxamic acid-based microtubule destabilizing agents (MDAs), which were derived from shortening the length of the linker in HDAC6 inhibitor SKLB-23bb. Interestingly, the low nanomolar antiproliferative activity of these MDAs depended on the presence of hydroxamic acid groups, but their inhibitory effects on HDAC were lost. Among them, 12b showed favorable metabolism stability, high bioavailability, and potent antitumor activity in multidrug-resistant cell lines and A2780/T xenograft model. More importantly, in the patient-derived xenograft models of triple-negative breast cancer and osimertinib-resistant non-small-cell lung cancer, both 20 mg/kg oral and 10 mg/kg intravenous administration of 12b could induce more than 70% tumor inhibition without obvious toxicity. Overall, we discovered that 12b, as a novel MDA based on hydroxamic acid, could serve as a potential MDA for further investigation.

Antitumor Effects of Docetaxel in Truncated Basic Fibroblast Growth Factor- Functionalized Liposomes Delivered by d-α-tocopheryl Polyethylene Glycol 2000 Succinate.

BACKGROUND: PEGylation of stealth liposomes elevates their stability and prolongs plasma half-life. Stealth liposomes modified with targeting ligands are expected to be ideal drug delivery carriers. OBJECTIVE: To encapsulate docetaxel in tbFGF (truncated basic fibroblast growth factor)-functionalized liposomes with mPEG2000-VE (d-κ-tocopheryl polyethylene glycol succinate, TPGS2K) and measure their antitumor effects in vitro and in vivo. METHODS: TPGS2K and COOH-PEG2000-VE were synthesized, and tbFGF was conjugated to COOH-PEG2000-VE to prepare tbFGF-PEG2000-VE. Then, tbFGF-functionalized liposomes (DTX-tbFGF-LPs) were prepared by inserting tbFGF-PEG2000-VE into docetaxel liposomes comprising TPGS2K (DTX-PEG-LPs). The stabilities and drug release profiles of the formulation were evaluated. P-glycoprotein (P-gp) inhibition was measured by ATPase assay. MTT and cell uptake were measured with B16 cells. A B16 C57BL/6 mouse model was used to evaluate in vivo antitumor efficacy. RESULTS: Both DTX-PEG-LPs and DTX-tbFGF-LPs exhibited good stability and sustained drug release. MTT, flow cytometry, and fluorescence microscopy of B16 cells revealed higher antitumor activity and more efficient cell uptake for DTX-tbFGF-LPs compared with DTX-PEG-LPs and DTX-LPs. The P-gp ATPase assay showed that both PEG-LP and tbFGF-PEG-LP formulations inhibited P-gp pump activity in vitro. DTX-tbFGF-LPs had the highest antitumor efficacy and lowest toxicity in vivo. CONCLUSION: Truncated basic fibroblast growth factor-functionalized liposomes with TPGS2K as drug delivery nanocarriers were effective chemotherapy agents targeting FGFR-overexpressing tumors.

Anti-Tumor Study of H6, a 4-Substituted Coumarins Derivative, Loaded Biodegradable Self-Assembly Nano-Micelles In Vitro and In Vivo.

In our previous study, we identified a class of 4-substituted coumarins as a powerful microtubule inhibitors binding to the colchicine site of ß-tubulin. H6 showed potent anti-proliferative ability with IC50 values from 7 to 47 nM, and remarkable ability to reduce tumor growth in several xenograft models including taxol resistant tumor models. However, the extremely hydrophobicity limited its clinical application. In this study, to improve the anticancer activity and reduce the toxicity of H6, we successfully prepared MPEG-PCL with different proportions and H6-loaded polymeric micelles (H6/MPEG2kPCL2k micelles) by a simple thin-film hydration method. The prepared H6/MPEG-PCL micelles had a drug loading of 3.79 ± 0.001%, an encapsulation efficiency of 98.00 ± 0.41%, a mean particle size of 30.45 ± 0.18nm and a polydispersity index (PDI) of 0.096 ± 0.009. Computer simulation results revealed a good compatibility of H6 and MPEG2k-PCL2k copolymer. In in vitro release study and pharmacokinetic study showed H6 micelles can release H6 over an extended period. Furthermore, H6 micelles possessed comparative effect as free H6 in inhibiting cell growth, preventing cell migration, and inducing apoptosis. Mechanism study identified that H6 is a novel reversible microtubule inhibitor. In in vivo studies, H6 micelles exhibited tumor growth inhibition on two pulmonary metastatic tumor models (B16/F10 and 4T1). Importantly, H6 micelles significantly improved the solubility, reduced the toxicity, extended the half-life of drugs, and augmented the therapeutic window. All these results imply that H6 micelles have great potential for suppression of tumor metastasis.

Purinostat Mesylate Is a Uniquely Potent and Selective Inhibitor of HDACs for the Treatment of BCR-ABL-Induced B-Cell Acute Lymphoblastic Leukemia.

PURPOSE: This study was to perform preclinical evaluation of a novel class I and IIb HDAC-selective inhibitor, purinostat mesylate, for the treatment of Ph+ B-cell acute lymphoblastic leukemia (B-ALL). EXPERIMENTAL DESIGN: Biochemical assays were used to test enzymatic activity inhibition of purinostat mesylate. Ph+ leukemic cell lines and patient cells were used to evaluate purinostat mesylate activity in vitro. BL-2 secondary transplantation Ph+ B-ALL mouse model was used to validate its efficacy, mechanism, and pharmacokinetics properties in vivo. BCR-ABL(T315I)-induced primary B-ALL mouse model and PDX mouse model derived from relapsed Ph+ B-ALL patient post TKI treatment were used to determine the antitumor effect of purinostat mesylate for refractory or relapsed Ph+ B-ALL. Long-term toxicity and hERG blockade assays were used to safety evaluation of purinostat mesylate. RESULTS: Purinostat mesylate, a class I and IIb HDAC highly selective inhibitor, exhibited robust antitumor activity in hematologic cancers. Purinostat mesylate at low nanomolar concentration induced apoptosis, and downregulated BCR-ABL and c-MYC expression in Ph+ leukemia cell lines and primary Ph+ B-ALL cells from relapsed patients. Purinostat mesylate efficiently attenuated Ph+ B-ALL progression and significantly prolonged the survival both in BL-2 secondary transplantation model with clinical patient symptoms of Ph+ B-ALL, BCR-ABL(T315I)-induced primary B-ALL mouse model, and PDX model derived from patients with relapsed Ph+ B-ALL post TKI treatment. In addition, purinostat mesylate possesses favorable pharmacokinetics and low toxicity properties. CONCLUSIONS: Purinostat mesylate provides a new therapeutic strategy for patients with Ph+ B-ALL, including those who relapse after TKI treatment.