Development of an analytical platform for the affinity screening of natural extracts by SEC-MS towards PPARα and PPARγ receptors.

BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) belong to the superfamily of nuclear receptors and represent the targets for the therapeutical treatment of type 2 diabetes, dyslipidemia and hyperglycemia associated with metabolic syndrome. Some medicinal plants have been traditionally used to treat this kind of metabolic diseases. Today only few drugs targeting PPARs have been approved and for this reason, the rapid identification of novel ligands and/or chemical scaffolds starting from natural extracts would benefit of a selective affinity ligand fishing assay. RESULTS: In this paper we describe the development of a new ligand fishing assay based on size exclusion chromatography (SEC) coupled to LC-MS for the analysis of complex samples such as botanical extracts. The known PPARα and PPARγ ligands, WY-14643 and rosiglitazone respectively, were used for system development and evaluation. The system has found application on an Allium lusitanicum methanolic extract, containing saponins, a class of chemical compounds which have attracted interest as PPARs ligands because of their hypolipidemic and insulin-like properties. SIGNIFICANCE: A new SEC-AS-MS method has been developed for the affinity screening of PPARα and PPARγ ligands. The system proved to be highly specific and will be used to improve the throughput for the identification of new selective metabolites from natural souces targeting PPARα and PPARγ.

Therapeutic Drug Monitoring of Pazopanib in Renal Cell Carcinoma and Soft Tissue Sarcoma: A Systematic Review.

BACKGROUND: Pazopanib, an anti-angiogenic multitarget tyrosine kinase inhibitor, has been approved for the treatment of metastatic renal cell carcinoma and soft tissue sarcoma. However, its recommended dose does not always produce consistent outcomes, with some patients experiencing adverse effects or toxicity. This variability is due to differences in the systemic exposure to pazopanib. This review aimed to establish whether sufficient evidence exists for the routine or selective therapeutic drug monitoring of pazopanib in adult patients with approved indications. METHODS: A systematic search of the PubMed and Web of Science databases using search terms related to pazopanib and therapeutic drug monitoring yielded 186 and 275 articles, respectively. Ten articles associated with treatment outcomes or toxicity due to drug exposure were selected for review. RESULTS: The included studies were evaluated to determine the significance of the relationship between drug exposure/Ctrough and treatment outcomes and between drug exposure and toxicity. A relationship between exposure and treatment outcomes was observed in 5 studies, whereas the trend was nonsignificant in 4 studies. A relationship between exposure and toxicity was observed in 6 studies, whereas 2 studies did not find a significant relationship; significance was not reported in 3 studies. CONCLUSIONS: Sufficient evidence supports the therapeutic drug monitoring of pazopanib in adult patients to improve its efficacy and/or safety in the approved indications.

Comparison of feline and human immunodeficiency virus reverse transcriptase enzymes through chemical screening and computational analysis.

Feline immunodeficiency virus (FIV) is a common infection found in domesticated and wild cats worldwide. Despite the wealth of therapeutic understanding of the disease in humans, considerably less information exists regarding the treatment of the disease in felines. Current treatment relies on drugs developed for the related human immunodeficiency virus (HIV) and includes compounds of the popular non-nucleotide reverse transcriptase (NNRTI) class. This is despite FIV-RT being only 67% similar to HIV-1 RT at the enzyme level, increasing to 88% for the allosteric pocket targeted by NNRTIs. The goal of this project was to try to quantify how well the more extensive pharmacological knowledge available for human disease translates to felines. To this end we screened known NNRTIs and 10 diverse pyrimidine analogs identified virtually. We use this chemo-centric probe approach to (a) assess the similarity between the two related RT targets based on the observed experimental inhibition values, (b) try to identify more potent inhibitors at FIV, and (c) gain a better appreciation of the structure-activity relationships (SAR). We found the correlation between IC50s at the two targets to be strong (r2 = 0.87) and identified compound 1 as the most potent inhibitor of FIV with IC50 of 0.030 µM ± 0.009. This compared to FIV IC50 values of 0.22 ± 0.17 µM, 0.040 ± 0.010 µM and >160 µM for known anti HIV-1 RT drugs Efavirenz, Rilpivirine, and Nevirapine, respectively. This knowledge, along with an understanding of the structural origin that give rise to any differences could improve the way HIV drugs are repurposed for FIV.

A comparative study of two thienopyrimidine Schiff base probes for sequential monitoring of Ga3+ and Pd2.

Two Schiff base probes (S1 and S2) were prepared and synthesized by incorporating thienopyrimidine into salicylaldehyde or 3-ethoxysalicylaldehyde individually, with the aim of detecting Ga3+ and Pd2+ sequentially. Upon chelation with Ga3+, S1 and S2 exhibited fluorescence enhancement in DMSO/H2O buffer. Both S1-Ga3+ and S2-Ga3+ were quenched by Pd2+. The limit of detection for S1 in response to Ga3+ and Pd2+ was 2.86 × 10-7 and 4.4 × 10-9 M, respectively. For S2, the limit of detection for Ga3+ and Pd2+ was 4.15 × 10-8 and 3.0 × 10-9 M, respectively. Furthermore, the complexation ratios of both S1 and S2 with Ga3+ and Pd2+ were determined to be 1:2 through Job's plots, ESI-MS analysis, and theoretical calculations. Two molecular logic gates were constructed, leveraging the response behaviors of S1 and S2. Moreover, the potential utility of S1 and S2 for monitoring Ga3+ and Pd2+ in domestic water was verified.

Structure-Based Design of Novel Thiazolone[3,2-a]pyrimidine Derivatives as Potent RNase H Inhibitors for HIV Therapy.

Ribonuclease H (RNase H) was identified as an important target for HIV therapy. Currently, no RNase H inhibitors have reached clinical status. Herein, a series of novel thiazolone[3,2-a]pyrimidine-containing RNase H inhibitors were developed, based on the hit compound 10i, identified from screening our in-house compound library. Some of these derivatives exhibited low micromolar inhibitory activity. Among them, compound 12b was identified as the most potent inhibitor of RNase H (IC50 = 2.98 µM). The experiment of magnesium ion coordination was performed to verify that this ligand could coordinate with magnesium ions, indicating its binding ability to the catalytic site of RNase H. Docking studies revealed the main interactions of this ligand with RNase H. A quantitative structure activity relationship (QSAR) was also conducted to disclose several predictive mathematic models. A molecular dynamics simulation was also conducted to determine the stability of the complex. Taken together, thiazolone[3,2-a]pyrimidine can be regarded as a potential scaffold for the further development of RNase H inhibitors.

Ruxolitinib 1.5% Topical Cream for the Treatment of Pediatric Alopecia Areata.

Alopecia areata (AA) is a common autoimmune disorder. Although its pathogenesis is not fully understood, AA involves CD8 T cell-mediated destruction of the hair follicle. Several treatment options exist; however, there is minimal evidence in the pediatric population. Currently, there are no curative treatments for AA. The literature suggests that Janus kinase (JAK) inhibitors may be an effective treat-ment for AA, but evidence in pediatric patients is limited. Here, we report a case of severe pediatric AA treated with topical ruxolitinib, a JAK inhibitor. J Drugs Dermatol. 2024;23(5):378-379. doi:10.36849/JDD.7782.

QbD Enabled Development and Evaluation of Pazopanib Loaded Nanoliposomes for PDAC Treatment.

Pancreatic ductal adenocarcinoma (PDAC) is one of the highly fatal types of cancer with high mortality/incidence. Considering the crucial role of vascular endothelial growth factor (VEGF) in PDAC progression, its inhibition can be a viable strategy for the treatment. Pazopanib, a second-generation VEGF inhibitor, is approved for the treatment of various oncological conditions. However, due to associated limitations like low oral bioavailability (14-39%), high inter/intra-subject variability, stability issues, etc., high doses (800 mg) are required, which further lead to non-specific toxicities and also contribute toward cancer resistance. Thus, to overcome these challenges, pazopanib-loaded PEGylated nanoliposomes were developed and evaluated against pancreatic cancer cell lines. The nanoliposomes were prepared by thin-film hydration method, followed by characterization and stability studies. This QbD-enabled process design successfully led to the development of a suitable pazopanib liposomal formulation with desirable properties. The % entrapment of PZP-loaded non-PEGylated and PEGylated nanoliposomes was found to be 75.2% and 84.9%, respectively, whereas their particle size was found to be 129.7 nm and 182.0 nm, respectively. The developed liposomal formulations exhibited a prolonged release and showed desirable physicochemical properties. Furthermore, these liposomal formulations were also assessed for in vitro cell lines, such as cell cytotoxicity assay and cell uptake. These studies confirm the effectiveness of developed liposomal formulations against pancreatic cancer cell lines. The outcomes of this work provide encouraging results and a way forward to thoroughly investigate its potential for PDAC treatment.

RuBi-Ruxolitinib: A Photoreleasable Antitumor JAK Inhibitor.

We describe the synthesis and biological testing of ruthenium-bipyridine ruxolitinib (RuBiRuxo), a photoreleasable form of ruxolitinib, a JAK inhibitor used as an antitumoral agent in cutaneous T-cell lymphomas (CTCL). This novel caged compound is synthesized efficiently, is stable in aqueous solution at room temperature, and is photoreleased rapidly by visible light. Irradiation of RuBiRuxo reduces cell proliferation and induces apoptosis in a light- and time-dependent manner in a CTCL cell line. This effect is specific and is mediated by a decreased phosphorylation of STAT proteins. Our results demonstrate the potential of ruthenium-based photocompounds and light-based therapeutic approaches for the potential treatment of cutaneous lymphomas and other pathologies.

In silico study and in vitro antileishmanial and antitrypanosomal evaluation of azopyrazoles and azopyrimidines.

Hydrazones 1-6, azo-pyrazoles 7-9 and azo-pyrimidines 10-15 are compounds that exhibit antibacterial activity. The mode of action and structures of these derivatives have been previously confirmed as antibacterial. In this investigation, biological screening and molecular docking studies were performed for derivatives 1-15, with compounds 2, 7, 8, 14 and 15 yielding the best energy scores (from -20.7986 to -10.5302 kcal/mol). Drug-likeness and in silico ADME prediction for the most potent derivatives, 2, 7, 8, 14 and 15, were predicted (from 84.46 to 96.85%). The latter compounds showed good recorded physicochemical properties and pharmacokinetics. Compound 8 demonstrated the strongest inhibition, which was similar to the positive control (eflornithine) against Trypanosoma brucei brucei (WT), with an EC50 of 25.12 and 22.52µM, respectively. Moreover, compound 14 exhibited the best activity against Leishmania mexicana promastigotes and Leishmania major promastigotes (EC50 =46.85; 40.78µM, respectively).

Molecular docking approach for the design and synthesis of new pyrazolopyrimidine analogs of roscovitine as potential CDK2 inhibitors endowed with pronounced anticancer activity.

Cyclin-dependent kinase 2 (CDK2) is a vital protein for controlling cell cycle progression that is critically associated with various malignancies and its inhibition could offer a convenient therapeutic approach in designing anticancer remedies. Consequently, this study aimed to design and synthesize new CDK2 inhibitors featuring roscovitine as a template model. The purine ring of roscovitine was bioisosterically replaced with the pyrazolo[3,4-d]pyrimidine scaffold, in addition to some modifications in the side chains. A preliminary molecular docking study for the target chemotypes in the CDK2 binding domain revealed their ability to accomplish similar binding patterns and interactions to that of the lead compound roscovitine. Afterwards, synthesis of the new derivatives was accomplished. Then, the initial anticancer screening at a single dose by the NCI revealed that compounds 7a, 9c, 11c, 17a and 17b achieved the highest GI% values reaching up to 150 % indicating their remarkable activity. These derivatives were subsequently selected to undertake five-dose testing, where compounds 7a, 9c, 11c and 17a unveiled the most pronounced activity against almost the full panel with GI50 ranges; 1.41-28.2, 0.116-2.39, 0.578-60.6 and 1.75-42.4 µM, respectively and full panel GI50 (MG-MID); 8.24, 0.6, 2.46 and 6.84 µM, respectively. CDK2 inhibition assay presented compounds 7a and 9c as the most potent inhibitors with IC50 values of 0.262 and 0.281 µM, respectively which are nearly 2.4 folds higher than the reference ligand roscovitine (IC50 = 0.641 µM). Besides, flow cytometric analysis on the most susceptible and safe cell lines depicted that 7a caused cell cycle arrest at G1/S phase in renal cancer cell line (RXF393) while 9c led to cell growth arrest at S phase in breast cancer cell line (T-47D) along with pronounced apoptotic induction in the mentioned cell lines. These findings afforded new anticancer pyrazolo[3,4-d]pyrimidine, roscovitine analogs, acting via CDK2 inhibition.

Development of a simple polymer-based sensor for detection of the Pirimicarb pesticide.

In this study, a sensitive and selective fluorescent chemosensor was developed for the determination of pirimicarb pesticide by adopting the surface molecular imprinting approach. The magnetic molecularly imprinted polymer (MIP) nanocomposite was prepared using pirimicarb as the template molecule, CuFe2O4 nanoparticles, and graphene quantum dots as a fluorophore (MIP-CuFe2O4/GQDs). It was then characterized using X-ray diffraction (XRD) technique, Fourier transforms infrared (FT-IR) spectroscopy, scanning electron microscope (SEM), and transmission electron microscopy (TEM). The response surface methodology (RSM) was also employed to optimize and estimate the effective parameters of pirimicarb adsorption by this polymer. According to the experimental results, the average particle size and imprinting factor (IF) of this polymer are 53.61 nm and 2.48, respectively. Moreover, this polymer has an excellent ability to adsorb pirimicarb with a removal percentage of 99.92 at pH = 7.54, initial pirimicarb concentration = 10.17 mg/L, polymer dosage = 840 mg/L, and contact time = 6.15 min. The detection of pirimicarb was performed by fluorescence spectroscopy at a concentration range of 0-50 mg/L, and a sensitivity of 15.808 a.u/mg and a limit of detection of 1.79 mg/L were obtained. Real samples with RSD less than 2 were measured using this chemosensor. Besides, the proposed chemosensor demonstrated remarkable selectivity by checking some other insecticides with similar and different molecular structures to pirimicarb, such as diazinon, deltamethrin, and chlorpyrifos.

Enhancement of rAAV titers via inhibition of the interferon signaling cascade in transfected HEK293 suspension cultures.

The production of recombinant adeno-associated virus (rAAV) for gene therapy applications relies on the use of various host cell lines, with suspension-grown HEK293 cells being the preferred expression system due to their satisfactory rAAV yields in transient transfections. As the field of gene therapy continues to expand, there is a growing demand for efficient rAAV production, which has prompted efforts to optimize HEK293 cell line productivity through engineering. In contrast to other cell lines like CHO cells, the transcriptome of HEK293 cells during rAAV production has remained largely unexplored in terms of identifying molecular components that can enhance yields. In our previous research, we analyzed global regulatory pathways and mRNA expression patterns associated with increased rAAV production in HEK293 cells. Our data revealed substantial variations in the expression patterns between cell lines with low (LP) and high-production (HP) rates. Moving to a deeper layer for a more detailed analysis of inflammation-related transcriptome data, we detected an increased expression of interferon-related genes in low-producing cell lines. Following upon these results, we investigated the use of Ruxolitinib, an interferon pathway inhibitor, during the transient production of rAAV in HEK293 cells as potential media additive to boost rAAV titers. Indeed, we find a two-fold increase in rAAV titers compared to the control when the interferon pathways were inhibited. In essence, this work offers a rational design approach for optimization of HEK293 cell line productivity and potential engineering targets, ultimately paving the way for more cost-efficient and readily available gene therapies for patients.

Cost-utility analysis of add-on vericiguat for the treatment of chronic heart failure with reduced ejection fraction in China.

OBJECTIVE: This study aimed to evaluate the cost-utility of the addition of vericiguat for treating chronic heart failure (CHF) in China from the healthcare payer's perspective. METHODS: A Markov model was built to estimate the cost and utility of treating CHF using vericiguat plus standard treatment (vericiguat group) vs. standard treatment alone (standard treatment group). The clinical parameters (mortality of cardiovascular and hospitalization rate of HF) were calculated according to the VICTORIA clinical trial. The HF cost and utility data were obtained from the literature published in China. One-way sensitivity analysis and probability sensitivity analysis were performed. RESULTS: According to the 13-year model, vericiguat was more expensive (155599.07 CNY vs. 259396.83 CNY) and more effective (4.41 QALYs vs. 4.54 QALYs). The incremental cost-utility ratio (ICUR) was 802389.27 CNY per QALY. One-way sensitivity analysis revealed that cardiovascular mortality in the two groups was the parameter that had the greatest impact on the results. The GDP per capita in 2022 in China was 85,700 CNY. The probability sensitivity analysis (PSA) showed that the probability of vericiguat being cost-effective was only 41.7% at the willingness-to-pay (WTP) threshold of 3 times GDP per capita (257,100 CNY). CONCLUSIONS: In China, the treatment of CHF with vericiguat is not cost-effective. The drug price could decrease to 145.8 CNY, which could be considered cost-effective.

JAK/STAT Inhibition Normalizes Lipid Composition in 3D Human Epidermal Equivalents Challenged with Th2 Cytokines.

Derangement of the epidermal barrier lipids and dysregulated immune responses are key pathogenic features of atopic dermatitis (AD). The Th2-type cytokines interleukin IL-4 and IL-13 play a prominent role in AD by activating the Janus Kinase/Signal Transduction and Activator of Transcription (JAK/STAT) intracellular signaling axis. This study aimed to investigate the role of JAK/STAT in the lipid perturbations induced by Th2 signaling in 3D epidermal equivalents. Tofacitinib, a low-molecular-mass JAK inhibitor, was used to screen for JAK/STAT-mediated deregulation of lipid metabolism. Th2 cytokines decreased the expression of elongases 1, 3, and 4 and serine-palmitoyl-transferase and increased that of sphingolipid delta(4)-desaturase and carbonic anhydrase 2. Th2 cytokines inhibited the synthesis of palmitoleic acid and caused depletion of triglycerides, in association with altered phosphatidylcholine profiles and fatty acid (FA) metabolism. Overall, the ceramide profiles were minimally affected. Except for most sphingolipids and very-long-chain FAs, the effects of Th2 on lipid pathways were reversed by co-treatment with tofacitinib. An increase in the mRNA levels of CPT1A and ACAT1, reduced by tofacitinib, suggests that Th2 cytokines promote FA beta-oxidation. In conclusion, pharmacological inhibition of JAK/STAT activation prevents the lipid disruption caused by the halted homeostasis of FA metabolism.

FGFR inhibition blocks NF-ĸB-dependent glucose metabolism and confers metabolic vulnerabilities in cholangiocarcinoma.

Genomic alterations that activate Fibroblast Growth Factor Receptor 2 (FGFR2) are common in intrahepatic cholangiocarcinoma (ICC) and confer sensitivity to FGFR inhibition. However, the depth and duration of response is often limited. Here, we conduct integrative transcriptomics, metabolomics, and phosphoproteomics analysis of patient-derived models to define pathways downstream of oncogenic FGFR2 signaling that fuel ICC growth and to uncover compensatory mechanisms associated with pathway inhibition. We find that FGFR2-mediated activation of Nuclear factor-κB (NF-κB) maintains a highly glycolytic phenotype. Conversely, FGFR inhibition blocks glucose uptake and glycolysis while inciting adaptive changes, including switching fuel source utilization favoring fatty acid oxidation and increasing mitochondrial fusion and autophagy. Accordingly, FGFR inhibitor efficacy is potentiated by combined mitochondrial targeting, an effect enhanced in xenograft models by intermittent fasting. Thus, we show that oncogenic FGFR2 signaling drives NF-κB-dependent glycolysis in ICC and that metabolic reprogramming in response to FGFR inhibition confers new targetable vulnerabilities.