BCR-ABL triggers a glucose-dependent survival program during leukemogenesis through the suppression of TXNIP.

Imatinib is highly effective in the treatment of chronic myelogenous leukemia (CML), but the primary and acquired imatinib resistance remains the big hurdle. Molecular mechanisms for CML resistance to tyrosine kinase inhibitors, beyond point mutations in BCR-ABL kinase domain, still need to be addressed. Here, we demonstrated that thioredoxin-interacting protein (TXNIP) is a novel BCR-ABL target gene. Suppression of TXNIP was responsible for BCR-ABL triggered glucose metabolic reprogramming and mitochondrial homeostasis. Mechanistically, Miz-1/P300 complex transactivates TXNIP through the recognition of TXNIP core promoter region, responding to the c-Myc suppression by either imatinib or BCR-ABL knockdown. TXNIP restoration sensitizes CML cells to imatinib treatment and compromises imatinib resistant CML cell survival, predominantly through the blockage of both glycolysis and glucose oxidation which results in the mitochondrial dysfunction and ATP production. In particular, TXNIP suppresses expressions of the key glycolytic enzyme, hexokinase 2 (HK2), and lactate dehydrogenase A (LDHA), potentially through Fbw7-dependent c-Myc degradation. In accordance, BCR-ABL suppression of TXNIP provided a novel survival pathway for the transformation of mouse bone marrow cells. Knockout of TXNIP accelerated BCR-ABL transformation, whereas TXNIP overexpression suppressed this transformation. Combination of drug inducing TXNIP expression with imatinib synergistically kills CML cells from patients and further extends the survival of CML mice. Thus, the activation of TXNIP represents an effective strategy for CML treatment to overcome resistance.

Integrative exploration of the mutual gene signatures and immune microenvironment between benign prostate hyperplasia and castration-resistant prostate cancer.

BACKGROUND: Benign prostate hyperplasia (BPH) and prostate cancer (CaP) are among the most frequently occurring prostatic diseases. When CaP progressed to castration-resistant CaP (CRPC), the prognosis is poor. Although CaP/CRPC and BPH frequently coexist in prostate, the inter-relational mechanism between them is largely unknown. METHODS: Single-cell RNA sequencing, bulk-RNA sequencing, and microarray data of BPH, CaP in the Gene Expression Omnibus database were obtained and comprehensively analyzed. Weighted Gene Co-Expression Network Analysis (WGCNA) and lasso regression analysis were performed to explore the potential biomarkers. RESULTS: With WGCNA, five modules in BPH, two in CaP, and three in CRPC were identified as significant modules. Pathway enrichment analysis found that the epigenetics and chromosomal-related signaling were dominantly clustered in the CaP group but not in BPH and CRPC. Lasso regression analysis was used to analyze further the mutual genes between the BPH module and the CRPC module. As a result, DDA1, ERG28, OGFOD1, and OXA1L were significantly correlated with the transcriptomic features in both BPH and CRPC. More importantly, the role of the four gene signatures was validated in two independent anti-PD-1 immunotherapy cohort. CONCLUSION: This study revealed the shared gene signatures and immune microenvironment between BPH and CRPC. The identified hub genes, including DDA1, ERG28, OGFOD1, and OXA1L, might be potential therapeutic targets for facilitating immunotherapy in prostate cancer.

AMP-activated protein kinase-farnesoid X receptor pathway contributes to oleanolic acid-induced liver injury.

Natural pentacyclic triterpenoid oleanolic acid (OA) is used as an over-the-counter drug for acute and chronic hepatitis. However, clinical use of OA-containing herbal medicines has been reported to cause cholestasis, and the specific mechanism is unknown. The purpose of this study was to explore how OA causes cholestatic liver injury via the AMP-activated protein kinase (AMPK)-farnesoid X receptor (FXR) pathway. In animal experiments, it was found that OA treatment activated AMPK and decreased FXR and bile acid efflux transport proteins expression. When intervened with the specific inhibitor Compound C (CC), it was observed that AMPK activation was inhibited, the reduction of FXR and bile acid efflux transport protein expression was effectively alleviated, serum biochemical indicators were significantly reduced, and liver pathological damage brought about by OA was effectively ameliorated. In addition, OA was found to downregulate the expression of FXR and bile acid efflux transport proteins by activating the ERK1/2-LKB1-AMPK pathway in cellular experiments. The ERK1/2 inhibitor U0126 was used to pretreat primary hepatocytes, and this drastically reduced the phosphorylation levels of LKB1 and AMPK. The inhibition effects of OA on FXR and bile acid efflux transport proteins were also effectively alleviated after pretreatment with CC. In addition, OA-induced downregulation of FXR gene and protein expression levels was significantly prevented after silencing AMPKα1 expression in AML12 cells. Our study demonstrated that OA inhibited FXR and bile acid efflux transporters through the activation of AMPK, thus leading to cholestatic liver injury.

A teenager with combined methylmalonic aciduria and homocystinuria (CblC type) presenting with neurological symptoms and congenital heart diseases: a case report.

Combined methylmalonic acidemia and homocystinuria, is a rare autosomal recessive disorder due to defective intracellular cobalamin metabolism. We report an 18-year-old Chinese male who presented with hypermyotonia, seizures, and congenital heart diseases. Mutation analysis revealed c.365A>T and c.482 G>A mutations in the MMACHC gene, diagnosed with methylmalonic aciduria and homocystinuria (CblC type). After treatment with vitamin B12, L-carnitine, betaine, and folate, which resulted in an improvement in his clinical symptoms and laboratory values. This case emphasizes that inborn errors of metabolism should be considered for a teenager presenting with challenging or neurologic symptoms, especially when combined with unexplained heart diseases.

Pilose antler polypeptides enhance chemotherapy effects in triple-negative breast cancer by activating the adaptive immune system.

Water-soluble polypeptides from pilose antler (PAWPs) are a traditional Chinese functional food and have been reported to inhibit triple-negative breast cancer (TNBC) in mice. Thus, in this study, we characterized PAWPs through peptidomics, and 405 total polypeptides were finally identified. Subsequently, our results indicate that PAWPs combined with neoadjuvant chemotherapy (NAC) result in a markedly lower spleen index compared with that in other groups. Next, 25 subpopulations of T cells were identified by multi-parametric flow cytometry in the lungs, spleen, and peripheral blood of different groups. These results indicated that PAWPs combined with NAC promote the proliferation of CD3+ T cells in the spleen and significantly affect the fate of the T-cell subpopulation. Moreover, PAWPs combined with NAC increased the infiltration of CD4+ interferon-γ+ T cells into tumor tissues. Our results showed that PAWPs have immunoregulatory functions and chemosensitizing effects, with good prospects for future clinical application.

MIR99AHG/miR-204-5p/TXNIP/Nrf2/ARE Signaling Pathway Decreases Glioblastoma Temozolomide Sensitivity.

Glioblastoma (GBM) is the most prevalent primary cerebral tumor in adults with high aggressiveness. Temozolomide (TMZ) is considered as the most widely used chemotherapy for GBM patients. Accumulating studies have proved that long non-coding RNAs (lncRNAs) participate in the pathogenesis of tumors. The aim of our study is to disclose the role of mir-99a-let-7c cluster host gene (MIR99AHG) in GBM. MIR99AHG expression was discovered to be elevated in GBM cells through quantitative real-time polymerase chain reaction (RT-qPCR) analysis. Loss-of-function experiments demonstrated that MIR99AHG silencing enhanced TMZ sensitivity of GBM both in vitro and in vivo. RNA pull down, RNA-binding protein immunoprecipitation (RIP), and luciferase reporter assays were implemented to unveil the underlying mechanism of MIR99AHG in GBM. The results of the mechanism assays implied that MIR99AHG interacted with microRNA-204-5p (miR-204-5p) and enhanced thioredoxin interacting protein (TXNIP) expression to inactivate the Nrf2/ARE signaling pathway. MIR99AHG/miR-204-5p/TXNIP regulatory axis was verified by rescue experiments in GBM. To summarize, MIR99AHG plays a promoting role in the TMZ resistance of GBM cells. The findings in this study might provide novel sight for the treatment for GBM.

The orexin story, sleep and sleep disturbances.

The orexins, also known as hypocretins, are two neuropeptides (orexin A and B or hypocretin 1 and 2) produced by a few thousand neurons located in the lateral hypothalamus that were independently discovered by two research groups in 1998. Those two peptides bind two receptors (orexin/hypocretin receptor 1 and receptor 2) that are widely distributed in the brain and involved in the central physiological regulation of sleep and wakefulness, orexin receptor 2 having the major role in the maintenance of arousal. They are also implicated in a multiplicity of other functions, such as reward seeking, energy balance, autonomic regulation and emotional behaviours. The destruction of orexin neurons is responsible for the sleep disorder narcolepsy with cataplexy (type 1) in humans, and a defect of orexin signalling also causes a narcoleptic phenotype in several animal species. Orexin discovery is unprecedented in the history of sleep research, and pharmacological manipulations of orexin may have multiple therapeutic applications. Several orexin receptor antagonists were recently developed as new drugs for insomnia, and orexin agonists may be the next-generation drugs for narcolepsy. Given the broad range of functions of the orexin system, these drugs might also be beneficial for treating various conditions other than sleep disorders in the near future.

Trifolirhizin regulates the balance of Th17/Treg cells and inflammation in the ulcerative colitis mice through inhibiting the TXNIP-mediated activation of NLRP3 inflammasome.

Ulcerative colitis (UC) is a chronic and recurrent autoimmune disease, characterized by recurrence and remission of mucosal inflammation. Although the understanding of the pathogenesis of UC has been improved, effective therapeutic drugs are required for treating patients with UC. In current work, the mouse model of colitis was established. Trifolirhizin was demonstrated to improve symptom in dextran sulfate sodium (DSS)-induced colitis mice. The body weight of mice was elevated, whereas the disease activity index (DAI) was reduced. Moreover, trifolirhizin was involved in inhibition of inflammation and regulation of the balance of T helper 17 (Th 17) cells and regulatory T (Treg) cells in DSS-induced colitis mice. Further, the activation NLRP3 inflammasome was suppressed by trifolirhizin in DSS-induced colitis mice. Trifolirhizin was also identified to regulate AMP-activated protein kinase (AMPK)-thioredoxin-interacting protein (TXNIP) pathway. The trifolirhizin-mediated anti-inflammatory effect was inhibited by suppressing AMPK in DSS-induced UC mice. In summary, the research suggested that administration of trifolirhizin significantly improved the symptoms and the pathological damage in DSS-induced UC mice. Trifolirhizin regulated the balance of Th17/Treg cells and inflammation in the UC mice through inhibiting the TXNIP-mediated activation of NLRP3 inflammasome.

Identification of a novel mechanism for reversal of doxorubicin-induced chemotherapy resistance by TXNIP in triple-negative breast cancer via promoting reactive oxygen-mediated DNA damage.

Given that triple-negative breast cancer (TNBC) lacks specific receptors (estrogen and progesterone receptors and human epidermal growth factor receptor 2) and cannot be treated with endocrine therapy, chemotherapy has remained the mainstay of treatment. Drug resistance is reportedly the main obstacle to the clinical use of doxorubicin (DOX) in this patient population. Accordingly, screening molecules related to chemoresistance and studying their specific mechanisms has clinical significance for improving the efficacy of chemotherapy in TNBC patients. Thioredoxin-interacting protein (TXNIP) is a metabolism-related protein that plays a tumor suppressor role in various malignant tumors; however, the specific role of TXNIP in tumor chemoresistance has not been reported. In the present study, we explored the potential molecular mechanism of TXNIP in the chemoresistance of TNBC for the first time. The results showed that TXNIP inhibited the proliferation of TNBC drug-resistant cells and promoted apoptosis in vitro and in vivo. Furthermore, TXNIP promoted the synthesis of reactive oxygen species (ROS) and the accumulation of DNA damage caused by DOX and increased γ-H2AX levels in a time and dose-dependent manner. Moreover, ROS scavenger pretreatment could block DNA damage induced by TXNIP and restore the resistance of TNBC resistant cells to DOX to a certain extent. In addition, we found that the small molecule c-Myc inhibitor 10058-F4 promoted TXNIP expression, increased ROS synthesis in cells, and could enhance the cytotoxicity of chemotherapy drugs in vitro and in vivo when combined with DOX. These results indicated that c-Myc inhibitor 10058-F4 could induce TXNIP upregulation in TNBC drug-resistant cells, and the upregulated TXNIP increased the accumulation of ROS-dependent DNA damage, thereby decreasing chemotherapy resistance of TNBC. Our findings reveal a new mechanism of mediating drug resistance and provide a new drug combination strategy to overcome DOX resistance in TNBC.

Amyloid seeding as a disease mechanism and treatment target in transthyretin cardiac amyloidosis.

Transthyretin (TTR) is a tetrameric transport protein mainly synthesized by the liver and choroid plexus. ATTR amyloidosis is characterized by the misfolding of TTR monomers and their accumulation within tissues as amyloid fibres. Current therapeutic options rely on the blockade of TTR production, TTR stabilization to maintain the native structure of TTR, amyloid degradation, or induction of amyloid removal from tissues. "Amyloid seeds" are defined as small fibril fragments that induce amyloid precursors to assume a structure rich in ß-sheets, thus promoting fibrillogenesis. Amyloid seeds are important to promote the amplification and spread of amyloid deposits. Further studies are needed to better understand the molecular structure of ATTR seeds (i.e. the characteristics of the most amyloidogenic species), and the conditions that promote the formation and multiplication of seeds in vivo. The pathological cascade may begin months to years before symptom onset, suggesting that seeds in tissues might potentially be used as biomarkers for the early disease stages. Inhibition of amyloid aggregation by anti-seeding peptides may represent a disease mechanism and treatment target in ATTR amyloidosis, with an additional benefit over current therapies.

Citrate transporter inhibitors: possible new anticancer agents.

The culmination of 80+ years of cancer research implicates the aberrant metabolism in tumor cells as a root cause of pathogenesis. Citrate is an essential molecule in intermediary metabolism, and its amplified availability to critical pathways in cancer cells via citrate transporters confers a high rate of cancer cell growth and proliferation. Inhibiting the plasma membrane and mitochondrial citrate transporters - whether individually, in combination, or partnered with complementary metabolic targets - in order to combat cancer may prove to be a consequential chemotherapeutic strategy. This review aims to summarize the use of different classes of citrate transporter inhibitors for anticancer activity, either individually or as part of a cocktail.

How Reciprocal Interactions Between the Tumor Microenvironment and Ion Transport Proteins Drive Cancer Progression.

Solid tumors comprise two major components: the cancer cells and the tumor stroma. The stroma is a mixture of cellular and acellular components including fibroblasts, mesenchymal and cancer stem cells, endothelial cells, immune cells, extracellular matrix, and tumor interstitial fluid. The insufficient tumor perfusion and the highly proliferative state and dysregulated metabolism of the cancer cells collectively create a physicochemical microenvironment characterized by altered nutrient concentrations and varying degrees of hypoxia and acidosis. Furthermore, both cancer and stromal cells secrete numerous growth factors, cytokines, and extracellular matrix proteins which further shape the tumor microenvironment (TME), favoring cancer progression.Transport proteins expressed by cancer and stromal cells localize at the interface between the cells and the TME and are in a reciprocal relationship with it, as both sensors and modulators of TME properties. It has been amply demonstrated how acid-base and nutrient transporters of cancer cells enable their growth, presumably by contributing both to the extracellular acidosis and the exchange of metabolic substrates and waste products between cells and TME. However, the TME also impacts other transport proteins important for cancer progression, such as multidrug resistance proteins. In this review, we summarize current knowledge of the cellular and acellular components of solid tumors and their interrelationship with key ion transport proteins. We focus in particular on acid-base transport proteins with known or proposed roles in cancer development, and we discuss their relevance for novel therapeutic strategies.

IRF7 and RNH1 are modifying factors of HIV-1 reservoirs: a genome-wide association analysis.

BACKGROUND: Combination antiretroviral treatment (cART) cannot eradicate HIV-1 from the body due to the establishment of persisting viral reservoirs which are not affected by therapy and reinitiate new rounds of HIV-1 replication after treatment interruption. These HIV-1 reservoirs mainly comprise long-lived resting memory CD4+ T cells and are established early after infection. There is a high variation in the size of these viral reservoirs among virally suppressed individuals. Identification of host factors that contribute to or can explain this observed variation could open avenues for new HIV-1 treatment strategies. METHODS: In this study, we conducted a genome-wide quantitative trait locus (QTL) analysis to probe functionally relevant genetic variants linked to levels of cell-associated (CA) HIV-1 DNA, CA HIV-1 RNA, and RNA:DNA ratio in CD4+ T cells isolated from blood from a cohort of 207 (Caucasian) people living with HIV-1 (PLHIV) on long-term suppressive antiretroviral treatment (median = 6.6 years). CA HIV-1 DNA and CA HIV-1 RNA levels were measured with corresponding droplet digital PCR (ddPCR) assays, and genotype information of 522,455 single-nucleotide variants was retrieved via the Infinium Global Screening array platform. RESULTS: The analysis resulted in one significant association with CA HIV-1 DNA (rs2613996, P < 5 × 10-8) and two suggestive associations with RNA:DNA ratio (rs7113204 and rs7817589, P < 5 × 10-7). Then, we prioritized PTDSS2, IRF7, RNH1, and DEAF1 as potential HIV-1 reservoir modifiers and validated that higher expressions of IRF7 and RNH1 were accompanied by rs7113204-G. Moreover, RNA:DNA ratio, indicating relative HIV-1 transcription activity, was lower in PLHIV carrying this variant. CONCLUSIONS: The presented data suggests that the amount of CA HIV-1 DNA and RNA:DNA ratio can be influenced through PTDSS2, RNH1, and IRF7 that were anchored by our genome-wide association analysis. Further, these observations reveal potential host genetic factors affecting the size and transcriptional activity of HIV-1 reservoirs and could indicate new targets for HIV-1 therapeutic strategies.

Efficacy and safety of maralixibat treatment in patients with Alagille syndrome and cholestatic pruritus (ICONIC): a randomised phase 2 study.

BACKGROUND: Alagille syndrome is a rare genetic disease that often presents with severe cholestasis and pruritus. There are no approved drugs for management. Maralixibat, an apical, sodium-dependent, bile acid transport inhibitor, prevents enterohepatic bile acid recirculation. We evaluated the safety and efficacy of maralixibat for children with cholestasis in Alagille syndrome. METHODS: ICONIC was a placebo-controlled, randomised withdrawal period (RWD), phase 2b study with open-label extension in children (aged 1-18 years) with Alagille syndrome (NCT02160782). Eligible participants had more than three times the normal serum bile acid (sBA) levels and intractable pruritus. After 18 weeks of maralixibat 380 µg/kg once per day, participants were randomly assigned (1:1) to continue maralixibat or receive placebo for 4 weeks. Subsequently, all participants received open-label maralixibat until week 48. During the long-term extension (204 weeks reported), doses were increased up to 380 µg/kg twice per day. The primary endpoint was the mean sBA change during the RWD in participants with at least 50% sBA reduction by week 18. Cholestastic pruritus was assessed using observer-rated, patient-rated, and clinician-rated 0-4 scales. The safety population was defined as all participants who had received at least one dose of maralixibat. This trial was registered with ClinicalTrials.gov, NCT02160782, and is closed to recruitment. FINDINGS: Between Oct 28, 2014, and Aug 14, 2015, 31 participants (mean age 5·4 years [SD 4·25]) were enrolled and 28 analysed at week 48. Of the 29 participants who entered the randomised drug withdrawal period, ten (34%) were female and 19 (66%) were male. In the RWD, participants switched to placebo had significant increases in sBA (94 µmol/L, 95% CI 23 to 164) and pruritus (1·7 points, 95% CI 1·2 to 2·2), whereas participants who continued maralixibat maintained treatment effect. This study met the primary endpoint (least square mean difference -117 µmol/L, 95% CI -232 to -2). From baseline to week 48, sBA (-96 µmol/L, -162 to -31) and pruritus (-1·6 pts, -2·1 to -1·1) improved. In participants who continued to week 204 (n=15) all improvements were maintained. Maralixibat was generally safe and well tolerated throughout. The most frequent adverse events were gastrointestinal related. Most adverse events were self-limiting in nature and mild-to-moderate in severity. INTERPRETATION: In children with Alagille syndrome, maralixibat is, to our knowledge, the first agent to show durable and clinically meaningful improvements in cholestasis. Maralixibat might represent a new treatment paradigm for chronic cholestasis in Alagille syndrome. FUNDING: Mirum Pharmaceuticals.

Role of Sciellin in gallbladder cancer proliferation and formation of neutrophil extracellular traps.

Apart from primary tumor development and metastasis, cancer-associated thrombosis is the second cause of cancer death in solid tumor malignancy. However, the mechanistic insight into the development of gallbladder cancer (GBC) and cancer-associated thrombosis remains unclear. This study aimed to investigate the mechanistic role of Sciellin (SCEL) in GBC cell proliferation and the development of venous thromboembolism. The expression level of SCEL was determined by immunohistochemical staining. Roles of SCEL in gallbladder cancer cell were determined by molecular and cell biology methods. SCEL was markedly upregulated in GBC and associated with advanced TNM stages and a poor prognosis. Furthermore, SCEL interacted with EGFR and stabilized EGFR expression that activates downstream PI3K and Akt pathway, leading to cell proliferation. In addition, SCEL induces tumor cell IL-8 production that stimulates the formation of neutrophil extracellular traps (NETs), accelerating thromboembolism. In xenografts, SCEL-expressing GBCs developed larger tumors and thrombosis compared with control cells. The present results indicate that SCEL promotes GBC cell proliferation and induces NET-associated thrombosis, thus serving as a potential therapeutic target.

Papel de la Nogina en obesidad: potencial biomarcador? / Noggin's role in obesity: Biomarker potential?

La obesidad es una enfermedad multifactorial resultado de la interacción entre factores genéticos, conductuales y ambientales que pueden influir en la respuesta individual a los hábitos alimenticios y de ejercicio físico. Su prevalencia ha aumentado dramáticamente durante la última década convirtiéndose en un problema de salud pública porque se asocia a patologías como diabetes tipo II, daño cardiovascular, hiperlipidemias y cáncer, que afectan a ambos sexos, todas las edades y todos los grupos étnicos. Actualmente, es la enfermedad metabólica más prevalente en los países desarrollados. Hay muchos loci y varios genes que se han asociado con la predisposición a la obesidad, a la delgadez, y al desarrollo de la obesidad y se clasifican según su expresión en diferentes etapas de esta condición, como inicio temprano, predisposición a la obesidad, inicio tardío, obesidad severa (mórbida). En este artículo se revisa el papel potencial del gen Nogina en la adipogénesis y los posibles mecanismos o vías de señalización en los que este gen interviene para conducir a la obesidad

Obesity is a multifactorial disease resulting from the interaction between genetic, behavioral and environmental factors that can influence the individual response to eating and exercise habits. Its prevalence has increased drastically in the last decade, becoming a public health problem because is associated with diseases such as type II diabetes, cardiovascular damage, hyperlipidemias and cancer, which affect both sexes, all ages and all ethnic groups. Currently, it is the most prevalent metabolic disease in developed countries. There are many loci and several genes that have been associated with the predisposition for obesity and thinness, obesity development and classified according to their expression in different stages of this condition, such as in early onset, predisposition to obesity, late onset, severe obesity (morbid). In this article I review the potential role of the Noggin gene in adipogenesis and the possible mechanisms or signaling pathways in which this gene intervenes to lead to obesity

Targeting the population for gene therapy with MYBPC3.

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited myocardial disease characterized by unexplained left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. Clinical heterogeneity is wide, ranging from asymptomatic individuals to heart failure, arrhythmias and sudden death. HCM is often caused by mutations in genes encoding components of the sarcomere. Among them, MYBPC3, encoding cardiac myosin-myosin binding protein C is the most frequently mutated gene. Three quarter of pathogenic or likely pathogenic mutations in MYBPC3 are truncating and the resulting protein was not detected in HCM myectomy samples. The overall prognosis of the patients is excellent if managed with contemporary therapy, but still remains a significant disease-related health burden, and carriers with double heterozygous, compound heterozygous and homozygous mutations often display a more severe clinical phenotype than single heterozygotes. We propose these individuals as a good target population for MYBPC3 gene therapy.

Click chemistry compared to thiol chemistry for the synthesis of site-selective glycoconjugate vaccines using CRM197 as carrier protein.

Conjugation chemistry is one of the main parameters affecting immunogenicity of glycoconjugate vaccines and a rational approach toward a deeper understanding of their mechanism of action will greatly benefit from highly-defined and well-characterized structures. Herein, different conjugation methods were investigated with the aim of controlling glycosylation site and glycosylation density on the carrier protein. S. Typhimurium lipopolysaccharide O-Antigen and CRM197 carrier protein were used as models. In particular, thiol and click chemistry were examined, both involving the linkage of the terminal reducing sugar unit of the O-Antigen chain to different amino acids on the carrier protein. Thiol chemistry allowed O-Antigen conjugation only when the carrier protein was activated on the lysines and with a relative high number of linkers, while click chemistry allowed conjugate generation even when just one position on the protein was activated and to both lysine and tyrosine sites. The study highlights click chemistry as a leading approach for the synthesis of well-defined glycoconjugates, useful to investigate the relationship between conjugate design and immune response.

Discovery and optimization of novel N-benzyl-3,6-dimethylbenzo[d]isoxazol-5-amine derivatives as potent and selective TRIM24 bromodomain inhibitors with potential anti-cancer activities.

Tripartite motif-containing protein 24 (TRIM24), recognized as an epigenetic reader for acetylated H3K23 (H3K23ac) via its bromodomain, has been closely involved in tumorigenesis or tumor progression of several cancers. Developing inhibitors of TRIM24 is significant for functional studies and drug discovery. Herein, we report the identification, optimization and evaluation of N-benzyl-3,6-dimethylbenzo[d]isoxazol-5-amines as TRIM24 bromodomain inhibitors starting from an in house library screening. Structure-based optimization leads to two potent and selective compounds 11d and 11h in an Alphascreen assay with IC50 values of 1.88 µM and 2.53 µM, respectively. The viability assay demonstrates the great potential of this series of compounds as inhibitors of proliferation of prostate cancer (PC) cells LNCaP, C4-2B. A colony formation assay further supports this inhibitory activity. Compounds 11d and 11h inhibit cell proliferation of other cancer types such as non-small cell lung cancer (NSCLC) cells A549 with IC50 values of 1.08 µM and 0.75 µM, respectively. These data suggests that compounds 11d and 11h are promising lead compounds for further research.

Antimicrobial and Antibiofilm Activities of Helical Antimicrobial Peptide Sequences Incorporating Metal-Binding Motifs.

Antimicrobial peptides (AMPs) represent alternative strategies to combat the global health problem of antibiotic resistance. However, naturally occurring AMPs are generally not sufficiently active for use as antibiotics. Optimized synthetic versions incorporating additional design principles are needed. Here, we engineered amino-terminal Cu(II) and Ni(II) (ATCUN) binding motifs, which can enhance biological function, into the native sequence of two AMPs, CM15 and citropin1.1. The incorporation of metal-binding motifs modulated the antimicrobial activity of synthetic peptides against a panel of carbapenem-resistant enterococci (CRE) bacteria, including carbapenem-resistant Klebsiella pneumoniae (KpC+) and Escherichia coli (KpC+). Activity modulation depended on the type of ATCUN variant utilized. Membrane permeability assays revealed that the in silico selected lead template, CM15, and its ATCUN analogs increased bacterial cell death. Mass spectrometry, circular dichroism, and molecular dynamics simulations indicated that coordinating ATCUN derivatives with Cu(II) ions did not increase the helical tendencies of the AMPs. CM15 ATCUN variants, when combined with Meropenem, streptomycin, or chloramphenicol, showed synergistic effects against E. coli (KpC+ 1812446) biofilms. Motif addition also reduced the hemolytic activity of the wild-type AMP and improved the survival rate of mice in a systemic infection model. The dependence of these bioactivities on the particular amino acids of the ATCUN motif highlights the possible use of size, charge, and hydrophobicity to fine-tune AMP biological function. Our data indicate that incorporating metal-binding motifs into peptide sequences leads to synthetic variants with modified biological properties. These principles may be applied to augment the activities of other peptide sequences.