Chemogenomic profiling predicts antifungal synergies

Chemotherapies, HIV infections, and treatments to block organ transplant rejection are creating a population of immunocompromised individuals at serious risk of systemic fungal infections. Since single‐agent therapies are susceptible to failure due to either inherent or acquired resistance, alternative therapeutic approaches such as multi‐agent therapies are needed. We have developed a bioinformatics‐driven approach that efficiently predicts compound synergy for such combinatorial therapies. The approach uses chemogenomic profiles in order to identify compound profiles that have a statistically significant degree of similarity to a fluconazole profile. The compounds identified were then experimentally verified to be synergistic with fluconazole and with each other, in both Saccharomyces cerevisiae and the fungal pathogen Candida albicans. Our method is therefore capable of accurately predicting compound synergy to aid the development of combinatorial antifungal therapies.     

Proteomic characterization of the cytotoxic mechanism of gold (III) porphyrin 1a, a potential anticancer drug

There has been increasing interest in the potential applications of gold (III) complexes as anticancer drugs with higher cytotoxicity and fewer side effects than existing metal anticancer drugs. Our previous findings demonstrated that gold (III) porphyrin 1a preferentially induced apoptosis in a cancer cell line (SUNE1). In this study, we identified differentially expressed proteins related to the drug's cytotoxic action by comparing the protein alterations induced by gold (III) porphyrin 1a and cisplatin treatments. Several clusters of altered proteins were identified, including cellular structure and stress-related chaperone proteins, proteins involved in reactive oxygen species and enzyme proteins, translation factors, proteins that mediate cell proliferation or differentiation, and proteins participating in the internal degradation systems. Our results indicated that multiple factors leading to apoptosis were involved in drug cytotoxicity in SUNE1 cells. The balance between pro-apoptotic and anti-apoptotic signals determined the final fate of cancer cells.     

Proteomic profiling of fibroblasts reveals a modulating effect of extracellular calumenin on the organization of the actin cytoskeleton

CREC proteins constitute a family of EF-hand calcium binding proteins localized to the secretory pathway. Calumenin is the only member known to be secreted. Recently, it was shown that thrombin-activated thrombocytes liberate calumenin, which also is found in atherosclerotic lesions but not in normal vasculature. To study the possible effects of calumenin extracellularly, we used proteomic profiling of fibroblasts cultured in absence and in presence of calumenin. Using 2-DE and MS/MS, we show that normal fibroblasts contain several 28-29-kDa N-terminal and a 16-kDa C-terminal fragment of beta- or gamma-actin. Extracellularly added calumenin decreases the levels of both the N-terminal and C-terminal actin fragments, and, in addition, decreases the expression level of septin 2, which interacts with the actin cytoskeleton and is involved in cytokinesis. Labeling of S-phase fibroblasts with bromo-2'deoxy-uridine indicates that calumenin added to the medium also modulates the cell cycle. Our study thus indicates that calumenin may have an autocrine or a paracrine effect on the cells in its vicinity, and, therefore, may be involved in the pathophysiology of thrombosis or in wound healing.     

Proteomic profiling reveals oxidative phosphorylation pathway is suppressed in longissimus dorsi muscle of weaned piglets fed low-protein diet supplemented with limiting amino acids

Feeding high-protein diets in animals can lead to a decrease of nitrogen utilization efficiency, and then promote the environmental pollution. Recently, more reports have demonstrated that lowering protein level in diets supplemented with specific amino acids can address these problems. However, the whole proteome alteration in the skeletal muscle of weaned piglets fed low-protein diets is poorly understood. Here, we applied the isobaric tags for relative and absolute quantification approach to investigate this alteration. We fed weaned piglets with normal protein diet (20% crude protein) and low-protein diet supplemented with lysine, methionine, threonine, and tryptophan (17% crude protein) for 25 days. Then proteomic profiling of skeletal muscles was performed. In total, 1354 proteins were quantified in the porcine skeletal muscle proteome. 132 proteins were identified as differentially expressed proteins between the two groups. Differentially expressed proteins were significantly enriched in various nutrient metabolism including lipid, carbohydrate, and amino acid metabolism. Interestingly, a total of 20 differentially expressed proteins, which are involved in the oxidative phosphorylation pathway, were all down-regulated by the low-protein diet feeding. Further immunoblotting confirmed the down-regulations of MT-ATP8, COX2, NDUFA6, and SDHB, four selected proteins among these 20 proteins. Meanwhile, the ATP level in the low-protein diet group was also reduced. These findings for the first time reveal that oxidative phosphorylation pathway is suppressed in longissimus dorsi muscle of weaned piglets fed low-protein diet supplemented with limiting amino acids, which may provide new insights into further formula design and the choice of limiting amino acids in diets.     

Engineered Fn3 protein has targeted therapeutic effect on mesothelin-expressing cancer cells and increases tumor cell sensitivity to chemotherapy

Mesothelin is a protein expressed at high levels on the cell surface in a variety of cancers, with limited expression in healthy tissues. The presence of mesothelin on tumor tissue correlates with increased invasion and metastasis, and resistance to traditional chemotherapies, through mechanisms that remain poorly understood. Molecules that specifically recognize mesothelin and interrupt its contribution to tumor progression have significant potential for targeted therapy and targeted drug delivery applications. A number of mesothelin-targeting therapies are in preclinical and clinical development, although none are currently approved for routine clinical use. In this work, we report the development of a mesothelin-targeting protein based on the fibronectin type-III non-antibody protein scaffold, which offers opportunities for applications where antibodies have limitations. We engineered protein variants that bind mesothelin with high affinity and selectively initiate apoptosis in tumor cells expressing mesothelin. Interestingly, apoptosis does not occur through a caspase-mediated pathway and does not require downregulation of cell-surface mesothelin, suggesting a currently unknown pathway through which mesothelin contributes to cancer progression. Importantly, simultaneous treatment with mesothelin-binding protein and chemotherapeutic mitomycin C had a greater cytotoxic effect on mesothelin-positive cells compared to either molecule alone, underscoring the potential for combination therapy including biologics targeting mesothelin.     

Bioluminescent kinase strips: A novel approach to targeted and flexible kinase inhibitor profiling

In addition to target efficacy, drug safety is a major requirement during the drug discovery process and is influenced by target specificity. Therefore, it is imperative that every new drug candidate be profiled against various liability panels that include protein kinases. Here, an effective methodology to streamline kinase inhibitor profiling is described. An accessible standardized profiling system for 112 protein kinases covering all branches of the kinome was developed. This approach consists of creating different sets of kinases and their corresponding substrates in multi-tube strips. The kinase stocks are pre-standardized for optimal kinase activity and used for inhibitor profiling using a bioluminescent ADP detection assay. We show that these strips can routinely generate inhibitor selectivity profiles for small or broad kinase family panels. Lipid kinases were also assembled in strip format and profiled together with protein kinases. We identified two specific PI3K inhibitors that have off-target effects on CK2 that were not reported before and would have been missed if compounds were not profiled against lipid and protein kinases simultaneously. To validate the accuracy of the data generated by this method, we confirmed that the inhibition potencies observed are consistent with published values produced by more complex technologies such as radioactivity assays.     

D-甘露糖修饰聚合物胶束的制备及其在靶向药物输送中的应用

聚合物胶束作为药物载体具有良好的稳定性和生物相容性,提高疏水性药物溶解性等优势,是一类很有应用潜力的药物传输系统。本研究以合成的共价键连D-甘露糖的双亲性聚合物分子(PGMA-Mannose)为药物载体,包载抗癌药物阿霉素(DOX)制备具有甘露糖受体靶向性和pH敏感药物释放特性的新型载药聚合物胶束。利用激光共聚焦显微镜和MTT细胞毒性评价方法对载药胶束的细胞内吞摄取和毒性进行评价。实验结果表明,载药胶束能特异性识别人乳腺癌细胞MDA-MB-231表面过度表达的甘露糖受体,被癌细胞大量摄取并在细胞溶酶体酸性环境内释放药物,而载药胶束在表面甘露糖受体低表达的HEK293细胞中只有少量摄取。与原药DOX相比,该载药胶束对癌细胞的毒性显著提高,而对正常细胞的毒性较低。因此,该PGMA-Mannose聚合物胶束有望成为一种新型的靶向药物输送系统应用于癌症的治疗。     

A protein chimera self-assembling unit for drug delivery

In the modern view of selective drug delivery of bioactive molecules, the attention is moving onto the setup of the perfect carrier more than in the optimization of the active compound. In this respect, virus-like particles constitute bioinspired nanodevices with the intrinsic ability to transport a large class of molecules, ranging from smart drugs to small interfering RNAs. In this work, we demonstrate the efficacy of a novel construct obtained by fusing a self-assembling protein from the human Rotavirus A, VP6, with the Small Ubiquitin Modifier domain, which maintains the ability to form nanoparticles and nanotubes and is able to be used as a drug carrier, even without specific targeting epitopes. The high expression and purification yield, combined with low toxicity of the empty particles, clearly indicate a good candidate for future studies of selective drug delivery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2769, 2019.     

PREDICT: a method for inferring novel drug indications with application to personalized medicine

Inferring potential drug indications, for either novel or approved drugs, is a key step in drug development. Previous computational methods in this domain have focused on either drug repositioning or matching drug and disease gene expression profiles. Here, we present a novel method for the large‐scale prediction of drug indications (PREDICT) that can handle both approved drugs and novel molecules. Our method is based on the observation that similar drugs are indicated for similar diseases, and utilizes multiple drug–drug and disease–disease similarity measures for the prediction task. On cross‐validation, it obtains high specificity and sensitivity (AUC=0.9) in predicting drug indications, surpassing existing methods. We validate our predictions by their overlap with drug indications that are currently under clinical trials, and by their agreement with tissue‐specific expression information on the drug targets. We further show that disease‐specific genetic signatures can be used to accurately predict drug indications for new diseases (AUC=0.92). This lays the computational foundation for future personalized drug treatments, where gene expression signatures from individual patients would replace the disease‐specific signatures.     

Phenotyping drug polypharmacology via eicosanoid profiling of blood

It is widely accepted that small-molecule drugs, despite their selectivity at primary targets, exert pharmacological effects (and safety liabilities) through a multiplicity of pathways. As such, it has proved extremely difficult to experimentally assess polypharmacology in an agnostic fashion. Profiling of metabolites produced as part of physiological responses to pharmacological stimuli provides a unique opportunity to explore drug pharmacology. A total of 122 eicosanoid lipids in human whole blood were monitored from 10 different donors upon stimulation with several inducers of immunological responses and treatment with modulators of prostaglandin (PG) and leukotriene biosynthesis, including clinical and investigational molecules. Such analysis revealed differentiation between drugs nominally targeting different eicosanoid biosynthetic enzymes, or even those designed to target the same enzyme. Profiled agents, some of them marketed products, affect eicosanoid biosynthesis in ways that cannot be predicted from information on their intended targets. As an example, we used this platform to discriminate drugs based on their ability to silence PG biosynthesis in response to bacterial lipopolysaccharide, resulting in differential pharmacological activity in an in vivo model of endotoxemia. Some of the observed effects are subject to variability among individuals, indicating a potential application of this methodology to the patient stratification, based on their responses to benchmark drugs and experimental compounds read on the eicosanome via a simple blood test.     

Gene expression profiling of the pH response in Shigella flexneri 2a

The pH response of Shigella flexneri 2a 301 was identified by gene expression profiling. Gene expression profiles of cells grown in pH 4.5 or 8.6 were compared with the profiles of cells grown at pH 7.0. Differential expression was observed for 307 genes: 97 were acid up-regulated, 102 were acid down-regulated, 91 were base up-regulated, and 86 were base down-regulated. Twenty-seven genes were found to be both acid and base up-regulated, and 29 genes were both acid and base down-regulated. This study showed that (1) the most pH-dependent genes regulate energy metabolism; (2) the RpoS-dependent acid-resistance system is induced, while the glutamate-dependent acid resistance system is not; (3) high pH up-regulates some virulence genes, while low pH down-regulates them, consistent with Shigella infection of the low gut; and (4) several cross-stress response genes are induced by pH changes. These results also illustrate that many unknown genes are significantly regulated under acid or basic conditions, providing researchers with important information to characterize their function.     

Synthesis and in vitro characterization of oxytocin receptor targeted PEGylated immunoliposomes for drug delivery to the uterus

Abstract

Targeted delivery of therapeutics to the uterus is an important goal in the treatment of obstetric complications, such as preterm labour, postpartum hemorrhage, and dysfunctional labour. Current treatment for these obstetric complications is challenging, as there are limited effective and safe therapeutic options available. We have developed a targeted drug delivery system for the uterus by conjugating anti-oxytocin receptor (OTR) antibodies to the surface of PEGylated liposomes (OTR-PEG-ILs). The functionality of the OTR-PEG-ILs has previously been evaluated on human and murine myometrial tissues as well as in vivo in a murine model of preterm labour. The aim of this study was to report the pharmaceutical synthesis and characterization of the OTR-PEG-ILs and investigate their specific cellular interaction with OTR-expressing myometrial cells in vitro. Immunoliposomes composed of 1,2-distearoyl-sn-glycero-2-phosphocholine (DSPC) and cholesterol were prepared using an optimized method for the coupling of low concentrations of antibody to liposomes. The liposomes were characterized for particle size, antibody conjugation, drug encapsulation, liposome stability, specificity of binding, cellular internalization, mechanistic pathway of cellular uptake, and cellular toxicity. Cellular association studies demonstrated specific binding of OTR-PEG-ILs to OTRs and significant cellular uptake following binding. Evaluation of the mechanistic pathway of cellular uptake indicated that they undergo internalization through both clathrin- and caveolin-mediated mechanisms. Furthermore, cellular toxicity studies have shown no significant effect of OTR-PEG-ILs or the endocytotic inhibitors on cell viability. This study further supports oxytocin receptors as a novel pharmaceutical target for drug delivery to the uterus.     

Potentiating effects of GHRH analogs on the response to chemotherapy

Growth hormone releasing hormone (GHRH) from hypothalamus nominatively stimulates growth hormone release from adenohypophysis. GHRH is also produced by cancers, acting as an autocrine/paracrine growth factor. This growth factor function is seen in lymphoma, melanoma, colorectal, liver, lung, breast, prostate, kidney, bladder cancers. Pituitary type GHRH receptors and their splice variants are also expressed in these malignancies. Synthetic antagonists of the GHRH receptor inhibit proliferation of cancers. Besides direct inhibitory effects on tumors, GHRH antagonists also enhance cytotoxic chemotherapy. GHRH antagonists potentiate docetaxel effects on growth of H460 non-small cell lung cancer (NSCLC) and MX-1 breast cancer plus suppressive action of doxorubicin on MX-1 and HCC1806 breast cancer. We investigated mechanisms of antagonists on tumor growth, inflammatory signaling, doxorubicin response, expression of drug resistance genes, and efflux pump function. Triple negative breast cancer cell xenografted into nude mice were treated with GHRH antagonist, doxorubicin, or their combination. The combination reduced tumor growth, inflammatory gene expression, drug-resistance gene expression, cancer stem-cell marker expression, and efflux-pump function. Thus, antagonists increased the efficacy of doxorubicin in HCC1806 and MX-1 tumors. Growth inhibition of H460 NSCLC by GHRH antagonists induced marked downregulation in expression of prosurvival proteins K-Ras, COX-2, and pAKT. In HT-29, HCT-116 and HCT-15 colorectal cancer lines, GHRH antagonist treatment caused cellular arrest in S-phase of cell cycle, potentiated inhibition of in vitro proliferation and in vivo growth produced by S-phase specific cytotoxic agents, 5-FU, irinotecan and cisplatin. This enhancement of cytotoxic therapy by GHRH antagonists should have clinical applications.     

Reinforcing targeted therapeutics with phenotypic stability factors

Deregulated cell cycle progression can often be traced to intrinsic defects in specific regulatory proteins in cancer cells. Knowledge of these primary defects has led to targeted approaches that exploit the defects and spare normal cells. However, the success of such targeted approaches is still hit-or-miss. Genetic and epigenetic variability inherent in most tumors often results in phenotypic heterogeneity that, in turn, results in de novo or acquired resistance to therapeutic agents. The ability of cells to compensate and adapt to the inhibition of a specific cell cycle mediator is not remarkable. What is novel and of great potential importance is that the ability of cells to exhibit such adaptability varies markedly. “Phenotypic stability factors” that restrict the ability of cells to undergo epithelial-mesenchymal transitions (EMT) may dictate the success or failure of targeted therapies by interfering with compensatory changes such as deregulation of CDK2 activity. Identification of existing and new agents that induce and maintain phenotypic stability factors will inform and enable synergistic approaches to the eradication of even the most aggressive tumors.     

Systematic profiling of staralog response to acquired drug resistant kinase gatekeeper mutations in targeted cancer therapy

Amino Acids - Kinase-targeted therapy has been widely used as a lifesaving strategy for cancer patients. However, many patients treated with targeted cancer drugs are clinically observed to rapidly...     

Distinct genomic traits of acral and mucosal melanomas revealed by targeted mutational profiling

The incidence of melanoma is rising globally including China. Comparing to Caucasians, the incidence of non‐cutaneous melanomas is significantly higher in Chinese. Herein, we performed genomic profiling of 89 Chinese surgically resected primary melanomas, including acral (n = 54), cutaneous (n = 22), and mucosal (n = 13), by hybrid capture‐based next‐generation sequencing. We show that mucosal melanomas tended to harbor more pathogenic mutations than other types of melanoma, though the biological significance of this finding remains uncertain. Chromosomal arm‐level alterations including 6q, 9p, and 10p/q loss were highly recurrent in all subtypes, but mucosal melanoma was significantly associated with increased genomic instability. Importantly, 7p gain significantly correlated with unfavorable clinical outcomes in non‐cutaneous melanomas, representing an intriguing prognostic biomarker of those subtypes. Furthermore, focal amplification of 4q12 (KIT, KDR, and PDGFRα) and RAD51 deletion were more abundant in mucosal melanoma, while NOTCH2 amplification was enriched in acral melanoma. Additionally, cutaneous melanomas had higher mutation load than acral melanomas, while mucosal melanomas did not differ from other subtypes in mutation burden. Together, our data revealed important features of acral and mucosal melanomas in Chinese including distinctive driver mutation pattern and increased genomic instability. These findings highlight the possibilities of combination therapies in the clinical management of melanoma.