Light-controlled inhibition of BRAFV600E kinase

Metastatic melanoma is amongst the most difficult types of cancer to treat, with current therapies mainly relying on the inhibition of the BRAFV600E mutant kinase. However, systemic inhibition of BRAF by small molecule drugs in cancer patients results - paradoxically - in increased wild-type BRAF activity in healthy tissue, causing side-effects and even the formation of new tumors. Here we show the development of BRAFV600E kinase inhibitors of which the activity can be switched on and off reversibly with light, offering the possibility to overcome problems of systemic drug activity by selectively activating the drug at the desired site of action. Based on a known inhibitor, eight photoswitchable effectors containing an azobenzene photoswitch were designed, synthesized and evaluated. The most promising inhibitor showed an approximately 10-fold increase in activity upon light-activation. This research offers inspiration for the development of therapies for metastatic melanoma in which tumor tissue is treated with an active BRAFV600E inhibitor with high spatial and temporal resolution, thus limiting the damage to other tissues

HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity

AbstractThe increasing number of patients suffering from chronic obstructive pulmonary disease (COPD) represents a major and increasing health problem. Therefore, novel therapeutic approaches are needed. Class I HDACs 1, 2 and 3 play key roles in the regulation of inflammatory gene expression with a particular pro-inflammatory role for HDAC 3. HDAC 3 has been reported to be an important player in inflammation by deacetylating NF-κB p65, which has been implicated in the pathology of COPD. Here, we applied the pharmacological HDAC 3-selective inhibitor RGFP966, which attenuated pro-inflammatory gene expression in models for inflammatory lung diseases. Consistent with this, a robust decrease of the transcriptional activity of NF-κB p65 was observed. HDAC 3 inhibition affected neither the acetylation status of NF-κB p65 nor histone H3 or histone H4. This indicates that HDAC 3 inhibition does not inhibit NF-κB p65 transcriptional activity by affecting its deacetylation but rather by inhibiting enzymatic activity of HDAC 3. Taken together, our findings indicate that pharmacological HDAC 3-selective inhibition by inhibitors such as RGFP966 may provide a novel and effective approach toward development of therapeutics for inflammatory lung diseases

Catalytic Modification of Dehydroalanine in Peptides and Proteins by Palladium-Mediated Cross-Coupling

Dehydroalanine (Dha) is a remarkably versatile non-canonical amino acid often found in antimicrobial peptides. Here we present the catalytic modification of Dha via a palladium mediated cross coupling reaction. Using Pd(EDTA)(OAc)2 as water soluble catalyst, a variety of arylboronic acids was coupled to the dehydrated residues in proteins and peptides such as nisin. The cross coupling reaction yields both the Heck product, in which the sp2-hybridisation of the α-carbon is retained, as well as the conjugated addition product. The reaction can be performed under mild aqueous conditions, which makes this method an attractive addition to the palette of bio-orthogonal catalytic methods

HDAC1-3 inhibitor MS-275 enhances IL10 expression in RAW264.7 macrophages and reduces cigarette smoke-induced airway inflammation in mice

Chronic obstructive pulmonary disease (COPD) constitutes a major health burden. Studying underlying molecular mechanisms could lead to new therapeutic targets. Macrophages are orchestrators of COPD, by releasing pro-inflammatory cytokines. This process relies on transcription factors such as NF-κB, among others. NF-κB is regulated by lysine acetylation; a post-translational modification installed by histone acetyltransferases and removed by histone deacetylases (HDACs). We hypothesized that small molecule HDAC inhibitors (HDACi) targeting class I HDACs members that can regulate NF-κB could attenuate inflammatory responses in COPD via modulation of the NF-κB signaling output. MS-275 is an isoform-selective inhibitor of HDAC1-3. In precision-cut lung slices and RAW264.7 macrophages, MS-275 upregulated the expression of both pro- and anti-inflammatory genes, implying mixed effects. Interestingly, anti-inflammatory IL10 expression was upregulated in these model systems. In the macrophages, this was associated with increased NF-κB activity, acetylation, nuclear translocation, and binding to the IL10 promoter. Importantly, in an in vivo model of cigarette smoke-exposed C57Bl/6 mice, MS-275 robustly attenuated inflammatory expression of KC and neutrophil influx in the lungs. This study highlights for the first time the potential of isoform-selective HDACi for the treatment of inflammatory lung diseases like COPD

The role of Fyn kinase in CRH regulation of energy homeostasis.

Corticotropin-releasing hormone (CRH) is the master regulator of stress responses mediated by the hypothalamic-pituitary-adrenal (HPA) axis in mammals. CRH and CRH-related ligands, termed urocortins (UCNs), along with their receptors, CRHR1 and CRHR2, are expressed both centrally and peripherally. Evidence has implicated the CRH family of peptides in mechanisms regulating energy balance in the animal. Interestingly, Fyn kinase, a member of the Src kinase family, is also involved in homeostasis regulation. Fyn knockout animals display reduced adiposity partly due to increased energy expenditure and lipid utilization in white adipose tissues. Fyn kinase expression is down-regulated by glucocorticoids, suggesting that HPA axis might regulate Fyn, thereby implying a possible interaction with CRH. I sought to determine a possible crosstalk between CRH and Fyn kinase. Using a mouse model with total Crh deficiency (Crh KO mouse) and applying various metabolic challenges, such as cold exposure and induction of lipolysis processes, Fyn kinase expression was studied in tissues harvested from age-matched WT and Crh KO mice. I uncovered a novel positive correlation between CRH and Fyn kinase that was unmasked upon normalization of the Crh KO blood glucocorticoid. Activation of lipolysis pathways did not affect Fyn kinase expression, but unraveled differences between the two genotypes, both in vivo and in vitro. CRH plays an important role in activating thermogenesis of brown adipose tissue and therefore I sought to determine whether Fyn kinase had similar actions. A novel role for Fyn kinase activity in controlling brown adipogenesis was reported with the use of the brown preadipocyte line, T37i. Additionally, treatment of brown adipocytes with the Fyn kinase activity inhibitor, SU6656, provided evidence of an interaction between Fyn and Akt, whereas ERK1/2 remained unaffected

Chemistry-based enzyme detection and inhibition in epigenetics

Increasing evidence supports the significant role of epigenetic modifications in regulation of gene expression. Aberrant activity of the responsible chromatin-remodeling enzymes has been linked to serious pathologies such as inflammation and cancer. In this dissertation we focus on the development of novel tools to detect protein modifications as well as small-molecule inhibitors with potential therapeutic properties. More specifically, we introduce the oxidative Heck reaction as an efficient chemoselective bioorthogonal reaction for coupling of arylboronic acids to protein-bound alkenes and its successful application in in vitro monitoring of protein acylation. Additionally, photoswitchable histone deacetylase (HDAC) inhibitors were developed as potential antitumor agents and evaluated for their potency against HDAC isoforms and viability of cancer cells, with one compound exhibiting very promising results in its photoactivated cis form. Azobenzene analogues of the clinically-used HDAC1-3 inhibitor Entinostat were also designed and tested for their effect on macrophage inflammatory responses in an attempt to elucidate the role of HDAC1-3 in such pathways. Notifying, one analogue, that inhibited all three isoforms, displayed an improved anti-inflammatory profile in comparison to the parental agent, underlying that maintenance of inhibition through HDAC1-3 is required for the desirable pharmacological effects. Finally, we describe our efforts on developing activity-based functionalized probes for detection of human recombinant and endogenous lysine-specific demethylase-1 (LSD1) activity. In all cases dose-dependent labeling was achieved while control experiments supported an activity-based profile. However, the effects on labeling upon treatment with LSD1 inhibitors were inconclusive and require further investigation of the mechanism of action of such probes

Bioorthogonal metabolic labelling with acyl-CoA reporters: targeting protein acylation

Protein acylation is an abundant post-translational modification with a pivotal role in a plethora of biological processes. To date, metabolic labelling with functionalized precursors of acyl-CoA and subsequent bio-orthogonal ligation to a complementary detection tag has offered an attractive approach for monitoring endogenous protein acylation with excellent selectivity. This review focuses on the applications of alkyne- and alkene-based bioorthogonal chemistries in the study of enzyme activity in vitro and summarizes the carboxylate-type chemical reporters that have enabled the visualization and identification of cellular acylated proteins. However, despite their importance, serious limitations question the use of this two-step labelling method in the quantification of the protein acylome

Olive oil intake and cancer risk: A systematic review and meta-analysis

Background Research evidence has established the beneficial effects of diet in cancer prevention; various epidemiological studies have suggested that olive oil component could play a role in decreasing cancer risk. This systematic review and meta-analysis aims to investigate the association between olive oil consumption, cancer risk and prognosis. Methods A systematic search was conducted in PubMed, EMBASE and Google Scholar databases (end-of-search: May 10, 2020). Pooled relative risk (RR) and 95% confidence intervals (95% CIs) were estimated with random-effects (DerSimonian-Laird) models. Subgroup analyses, sensitivity analyses and meta-regression analysis were also performed. Results 45 studies were included in the meta-analysis; 37 were case-control (17,369 cases and 28,294 controls) and 8 were cohort studies (12,461 incident cases in a total cohort of 929,771 subjects). Highest olive oil consumption was associated with 31% lower likelihood of any cancer (pooled RR = 0.69, 95%CI: 0.62–0.77), breast (RR = 0.67, 95%CI: 0.52–0.86), gastrointestinal (RR = 0.77, 95%CI: 0.66–0.89), upper aerodigestive (RR = 0.74, 95% CI: 0.60–0.91) and urinary tract cancer (RR = 0.46, 95%CI: 0.29–0.72). Significant overall effects spanned both Mediterranean and non-Mediterranean participants, studies presenting a multivariate and a univariate analysis and all subgroups by study quality. Conclusions Olive oil consumption seems to exert beneficial actions in terms of cancer prevention. Additional prospective cohort studies on various cancer types and survivors, as well as large randomized trials, seem desirable. © 2022 Markellos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited