Opportunities for pharmacoproteomics in biomarker discovery.

Proteomic data are a uniquely valuable resource for drug response prediction and biomarker discovery because most drugs interact directly with proteins in target cells rather than with DNA or RNA. Recent advances in mass spectrometry and associated processing methods have enabled the generation of large-scale proteomic datasets. Here we review the significant opportunities that currently exist to combine large-scale proteomic data with drug-related research, a field termed pharmacoproteomics. We describe successful applications of drug response prediction using molecular data, with an emphasis on oncology. We focus on technical advances in data-independent acquisition mass spectrometry (DIA-MS) that can facilitate the discovery of protein biomarkers for drug responses, alongside the increased availability of big biomedical data. We spotlight new opportunities for machine learning in pharmacoproteomics, driven by the combination of these large datasets and improved high-performance computing. Finally, we explore the value of pre-clinical models for pharmacoproteomic studies and the accompanying challenges of clinical validation. We propose that pharmacoproteomics offers the potential for novel discovery and innovation within the cancer landscape.

L-type Amino Acid Transporter 1 Utilizing Ferulic Acid Derivatives Show Increased Drug Delivery in the Mouse Pancreas Along with Decreased Lipid Peroxidation and Prostaglandin Production.

Oxidative stress and pathological changes of Alzheimer's disease (AD) overlap with metabolic diseases, such as diabetes mellitus (DM). Therefore, tackling oxidative stress with antioxidants is a compelling drug target against multiple chronic diseases simultaneously. Ferulic acid (FA), a natural antioxidant, has previously been studied as a therapeutic agent against both AD and DM. However, FA suffers from poor bioavailability and delivery. As a solution, we have previously reported about L-type amino acid transporter 1 (LAT1)-utilizing derivatives with increased brain delivery and efficacy. In the present study, we evaluated the pharmacokinetics and antioxidative efficacy of the two derivatives in peripheral mouse tissues. Furthermore, we quantified the LAT1 expression in studied tissues with a targeted proteomics method to verify the transporter expression in mouse tissues. Additionally, the safety of the derivatives was assessed by exploring their effects on hemostasis in human plasma, erythrocytes, and endothelial cells. We found that both derivatives accumulated substantially in the pancreas, with over a 100-times higher area under curve compared to the FA. Supporting the pharmacokinetics, the LAT1 was highly expressed in the mouse pancreas. Treating mice with the LAT1-utilizing derivative of FA lowered malondialdehyde and prostaglandin E2 production in the pancreas, highlighting its antioxidative efficacy. Additionally, the LAT1-utilizing derivatives were found to be hemocompatible in human plasma and endothelial cells. Since antioxidative derivative 1 was substantially delivered into the pancreas along the previously studied brain, the derivative can be considered as a safe dual-targeting drug candidate in both the pancreas and the brain.

Pharmacoproteomics of Brain Barrier Transporters and Substrate Design for the Brain Targeted Drug Delivery.

One of the major reasons why central nervous system (CNS)-drug development has been challenging in the past, is the barriers that prevent substances entering from the blood circulation into the brain. These barriers include the blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), blood-cerebrospinal fluid barrier (BCSFB), and blood-arachnoid barrier (BAB), and they differ from each other in their transporter protein expression and function as well as among the species. The quantitative expression profiles of the transporters in the CNS-barriers have been recently revealed, and in this review, it is described how they affect the pharmacokinetics of compounds and how these expression differences can be taken into account in the prediction of brain drug disposition in humans, an approach called pharmacoproteomics. In recent years, also structural biology and computational resources have progressed remarkably, enabling a detailed understanding of the dynamic processes of transporters. Molecular dynamics simulations (MDS) are currently used commonly to reveal the conformational changes of the transporters and to find the interactions between the substrates and the protein during the binding, translocation in the transporter cavity, and release of the substrate on the other side of the membrane. The computational advancements have also aided in the rational design of transporter-utilizing compounds, including prodrugs that can be actively transported without losing potency towards the pharmacological target. In this review, the state-of-art of these approaches will be also discussed to give insights into the transporter-mediated drug delivery to the CNS.

Pharmacoproteomic analysis of topical dapsone and prednisolone interventions in the aqueous humor of anterior uveitis.

Uveitis is the inflammation of uveal tract comprising of iris, ciliary body and choroid. Blood ocular barriers maintaining the homeostasis of eye breach during uveitis, leads to high risk for sight-threatening complications. The purpose of this study was to compare the anti-inflammatory activity enabled by two diverse pharmacological agents (prednisolone and dapsone) using their effect on aqueous humor proteome. Wistar rats of either sex (150-200g) were used and randomly divided into various groups. Normal group was injected with 0.1ml normal saline (NS), endotoxin (LPS) (200 µg/0.1ml NS) was injected into endotoxin induced inflammatory groups followed by 0.1% dapsone and 1% prednisolone treatment in endotoxin induced uveitis (EIU) groups, respectively. Aqueocentesis was performed post 24 hour inflammation and samples were subjected for clinical parameter evaluation, cytokine analysis as well as global proteomic analysis using High-resolution mass spectrometer. Following which spectrum analysis, production spectra of peptides were matched against R. Norvegicus Protein Database (Uniport) using Proteome Discoverer (v2.2). Upon clinical evaluation, the anterior segment images post dapsone and prednisolone treatment have shown marked decrease in hyperaemia, miosis and iridial vessels vasodilation in rat eyes as compared to inflammation group. The result of cytokine analysis revealed 0.1% dapsone and prednisolone both significantly decreased the TNF-α levels. HRMS studies analysis expressed 140, 160, 158 and 141 proteins unique to normal, EIU, Dapsone and prednisolone group respectively. To conclude aqueous humor pharmacoproteomic revealed the anti-inflammatory activity of the dapsone comparable to the prednisolone treatment in endotoxin induced uveitis. The topical dapsone may be used as an alternative therapeutic option in treating uveitis without elevating intraocular pressure.

Quantitative Proteomics-Based Blood-Brain Barrier Study.

From the viewpoint of drug discovery, it is an important issue to elucidate the drug permeability at the human central nervous system (CNS) barriers and the molecular mechanisms in the cells forming CNS barriers especially during CNS diseases. I introduced quantitative proteomics techniques into the blood-brain barrier (BBB) study, then quantitatively investigated the transport system at the human BBB and clarified the quantitative differences in protein expression levels and functions of transporters and receptors between animals and humans, or in vitro and in vivo. Based on the difference in the absolute expression level of transporters between in vitro and in vivo, I demonstrated that the drug efflux activity of P-glycoprotein (P-gp) at in vivo BBB can be accurately reconstructed from the in vitro system, not only in mouse models but also monkeys similar to humans and pathological conditions. Furthermore, I discovered Claudin-11 as another tight junction molecule expressed at the CNS barriers, and clarified that it contributes to the disruption of the CNS barriers in multiple sclerosis. Furthermore, it was also elucidated that the P-gp dysfunction causes excessive brain entry of glucocorticoid which causes a nerve damage in cerebral infarct, and it can be suppressed by targeting Abl/Src kinases. These suggest that targeting the tight junctions and transporters, which are important molecules at the CNS barriers, would potentially lead to the treatment of CNS diseases. In this review, I would like to introduce a new CNS barrier study opened by quantitative proteomics research.

Neurogranin Regulates Alcohol Sensitivity through AKT Pathway in the Nucleus Accumbens.

Dysfunction of glutamate neurotransmission in the nucleus accumbens (NAc) has been implicated in the pathophysiology of alcohol use disorders (AUD). Neurogranin (Ng) is exclusively expressed in the brain and mediates N-methyl-d-aspartate receptor (NMDAR) hypo-function by regulating the intracellular calcium-calmodulin (Ca2+ -CaM) pathway. Ng null mice (Ng-/- mice) demonstrate increased alcohol drinking compared to wild-type mice, while also showing less tolerance to the effect of alcohol. To identify the molecular mechanism related to alcohol seeking, both in vivo microdialysis and label-free quantification proteomics comparing Ng genotype and effects of alcohol treatment on the NAc are utilized. There is significant difference in glutamate and gamma-aminobutyric acid (GABA) neurotransmission between genotypes; however, alcohol administration normalizes both glutamate and GABA levels in the NAc. Using label-free proteomics, 427 protein expression changes are identified against alcohol treatment in the NAc among 4347 total proteins detected. Bioinformatics analyses reveal significant molecular differences in Ng null mice in response to acute alcohol treatment. Ingenuity pathway analysis found that the AKT network is altered significantly between genotypes, which may increase the sensitivity of alcohol in Ng null mice. The pharmacoproteomics results presented here illustrate a possible molecular basis of the alcohol sensitivity through Ng signaling in the NAc.

Organic Anion-Transporting Polypeptide 1a4 (Oatp1a4/Slco1a4) at the Blood-Arachnoid Barrier is the Major Pathway of Sulforhodamine-101 Clearance from Cerebrospinal Fluid of Rats.

The blood-arachnoid barrier (BAB), which is formed by arachnoid epithelial cells linked by tight junctions, has generally been considered impermeable to water-soluble substances. However, we recently demonstrated that organic anion transporters 1 and 3 (Oat1 and Oat3) play roles in drug clearance at the BAB. Here, we examined whether an organic anion-transporting polypeptide (Oatp) also plays a role, using the fluorescent organic anion sulforhodamine-101 (SR-101) as a model substrate. SR-101 was injected into the cisterna magna of rats in order to minimize the contribution of choroid plexus transport. The in vivo cerebrospinal fluid (CSF) elimination clearance of SR-101 after intracisternal administration was ninefold greater than that of fluorescein-labeled inulin, a bulk flow marker. In the case of pre-administration of taurocholate, a broad-spectrum inhibitor of Oatps, or digoxin, a strong substrate/inhibitor for Oatp1a4 but not for Oatp1a1, Oat1, and Oat3, the CSF elimination of SR-101 was significantly reduced, becoming similar to that of inulin, and thus indicating complete inhibition of SR-101 clearance from the CSF. The distribution of SR-101 fluorescence was restricted to the arachnoid mater in the absence of inhibitor, whereas the fluorescence was increased in the parenchyma of the spinal cord after co-injection of taurocholate or digoxin. Immunostaining confirmed the localization of Oatp1a4 in the arachnoid mater. These results indicate that Oatp1a4 at the BAB acts as an avid clearance pathway of SR-101 in the CSF to the blood. Thus, Oatp1a4 appears to play a major role in CSF detoxification by limiting the distribution of organic anions to the brain and spinal cord.

Pharmacoproteomics Profile in Response to Acamprosate Treatment of an Alcoholism Animal Model.

Acamprosate is an FDA-approved medication for the treatment of alcoholism that is unfortunately only effective in certain patients. Although acamprosate is known to stabilize the hyper-glutamatergic state in alcoholism, pharmacological mechanisms of action in brain tissue remains unknown. To investigate the mechanism of acamprosate efficacy, the authors employ a pharmacoproteomics approach using an animal model of alcoholism, type 1 equilibrative nucleoside transporter (ENT1) null mice. The results demonstrate that acamprosate treatment significantly decreased both ethanol drinking and preference in ENT1 null mice compared to that of wild-type mice. Then, to elucidate acamprosate efficacy mechanism in ENT1 null mice, the authors utilize label-free quantification proteomics comparing both genotype and acamprosate treatment effects in the nucleus accumbens (NAc). A total of 1040 protein expression changes are identified in the NAc among 3634 total proteins detected. The proteomics and Western blot result demonstrate that acamprosate treatment decreased EAAT expression implicating stabilization of the hyper-glutamatergic condition in ENT1 null mice. Pathway analysis suggests that acamprosate treatment in ENT1 null mice seems to rescue glutamate toxicity through restoring of RTN4 and NF-κB medicated neuroimmune signaling compared to wild-type mice. Overall, pharmacoproteomics approaches suggest that neuroimmune restoration is a potential efficacy mechanism in the acamprosate treatment of certain sub-populations of alcohol dependent subjects.

Inner Blood-Retinal Barrier Dominantly Expresses Breast Cancer Resistance Protein: Comparative Quantitative Targeted Absolute Proteomics Study of CNS Barriers in Pig.

The purpose of this study was to determine absolute protein expression levels of transporters at the porcine inner blood-retinal barrier (BRB) and to compare the transporter protein expression quantitatively among the inner BRB, outer BRB, blood-brain barrier (BBB), and blood-cerebrospinal fluid barrier (BCSFB). Crude membrane fractions of isolated retinal capillaries (inner BRB) and isolated retinal pigment epithelium (RPE, outer BRB) were prepared from porcine eyeballs, while plasma membrane fractions were prepared from isolated porcine brain capillaries (BBB) and isolated choroid plexus (BCSFB). Protein expression levels of 32 molecules, including 16 ATP-binding-cassette (ABC) transporters and 13 solute-carrier (SLC) transporters, were measured using a quantitative targeted absolute proteomic technique. At the inner BRB, five molecules were detected: breast cancer resistance protein (BCRP, ABCG2; 22.8 fmol/µg protein), multidrug resistance protein 1 (MDR1, ABCB1; 8.70 fmol/µg protein), monocarboxylate transporter 1 (MCT1, SLC16A1; 4.83 fmol/µg protein), glucose transporter 1 (GLUT1, SLC2A1; 168 fmol/µg protein), and sodium-potassium adenosine triphosphatase (Na+/K+-ATPase; 53.7 fmol/µg protein). Other proteins were under the limits of quantification. Expression of MCT1 was at least 17.6-, 11.0-, and 19.2-fold greater than those of MCT2, 3, and 4, respectively. The transporter protein expression at the inner BRB was most highly correlated with that at the BBB (R2 = 0.8906), followed by outer BRB (R2 = 0.7988) and BCSFB (R2 = 0.4730). Sodium-dependent multivitamin transporter (SMVT, SLC5A6) and multidrug resistance-associated protein 1 (MRP1, ABCC1) were expressed at the outer BRB (0.378 and 1.03 fmol/µg protein, respectively) but were under the limit of quantification at the inner BRB. These findings may be helpful for understanding differential barrier function.

Quantitative Targeted Absolute Proteomics of Transporters and Pharmacoproteomics-Based Reconstruction of P-Glycoprotein Function in Mouse Small Intestine.

The purpose of this study was to investigate whether a pharmacokinetic model integrating in vitro mdr1a efflux activity (which we previously reported) with in vitro/in vivo differences in protein expression level can reconstruct intestinal mdr1a function. In situ intestinal permeability-surface area product ratio between wild-type and mdr1a/1b (-/-) mice is one of the parameters used to describe intestinal mdr1a function. The reconstructed ratios of six mdr1a substrates (dexamethasone, digoxin, loperamide, quinidine, verapamil, vinblastine) and one nonsubstrate (diazepam) were consistent with the observed values reported by Adachi et al. within 2.1-fold difference. Thus, intestinal mdr1a function can be reconstructed by our pharmacoproteomic modeling approach. Furthermore, we evaluated regional differences in protein expression levels of mouse intestinal transporters. Sixteen (mdr1a, mrp4, bcrp, abcg5, abcg8, glut1, 4f2hc, sglt1, lat2, pept1, mct1, slc22a18, ostß, villin1, Na(+)/K(+)-ATPase, γ-gtp) out of 46 target molecules were detected by employing our established quantitative targeted absolute proteomics technique. The protein expression amounts of mdr1a and bcrp increased progressively from duodenum to ileum. Sglt1, lat2, and 4f2hc were highly expressed in jejunum and ileum. Mct1 and ostß were highly expressed in ileum. The quantitative expression profiles established here should be helpful to understand and predict intestinal transporter functions.

Recent advances in defining the ubiquitylome.

Ubiquitin is a small 8.5 kDa protein that is conjugated to a target protein in a concerted three step enzymatic process. Ubiquitin addition can drastically affect function or target the modified protein for degradation. Ubiquitin modifications have important regulatory roles in disease progression, such as in cancer and neurodegenerative diseases to name a few. As a consequence, it is imperative to identify important ubiquitin targets to elucidate the role of the modification. Proteomic studies have sought to understand this role by identifying proteome-wide ubiquitylated proteins. Two central ideas have developed to characterize the ubiquitylome: affinity purification of ubiquitylated proteins and optimization of GG-peptide enrichment. In this review, we will discuss recent advances in both approaches and discuss how these studies are essential to pharmacoproteomics.

Quantitative targeted proteomics for understanding the blood-brain barrier: towards pharmacoproteomics.

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells linked together via complex tight junctions, and serves to prevent entry of drugs into the brain. Multiple transporters are expressed at the BBB, where they control exchange of materials between the circulating blood and brain interstitial fluid, thereby supporting and protecting the CNS. An understanding of the BBB is necessary for efficient development of CNS-acting drugs and to identify potential drug targets for treatment of CNS diseases. Quantitative targeted proteomics can provide detailed information on protein expression levels at the BBB. The present review highlights the latest applications of quantitative targeted proteomics in BBB research, specifically to evaluate species and in vivo-in vitro differences, and to reconstruct in vivo transport activity. Such a BBB quantitative proteomics approach can be considered as pharmacoproteomics.

Literature Analysis Regarding the Combination of Substances: Glucosamine + Chondroitin in the Treatment of Osteoarthritis.

An essential component of joint quality is cartilage. Therefore, the protection of this is a prerequisite for maintaining the condition of each joint. The assessment of the presence of articular cartilage is shown by X-ray of both joints in the standing position. Cartilage protection is possible for 1, 2 and 3 degree of cartilage damage according to the Kellgren and Lawrence scale.The challenge for the physician is to identify the cause of OA in accordance with the principles of Evidence Based Orthopedics/Traumatology, and not merely treat symptomatically, which is usually ineffective.In order to objectively present treatment methods, indications and the period of their implementation, it is biologically reasonable to refer to the needs of cartilage tissue resulting from the analysis of the causes of its damage and indications for justified methods of its protection.Biomechanical and biological elements are important in the process of implementing articular cartilage protection.The biomechanical elements are: limb axis disorders, differences in length, distortions at the level of the support quadrilateral, pelvic triangle and shoulder triangle, as well as balance disorders resulting from disturbances in the segmental proportion of the Fi number according to Leonardo da Vinci.There are many biological elements of the discussed disorder and they concern: the state of articular cartilage structure, matrix structure, matrix biophysical elements, molecular sponge mechanism, chondrocytes, cartilage nutrition and the severity of osteoarthritis (OA).The improvement of the conditions of the biological elements of damaged articular cartilage is considered fundamental and concerns the positive impact on numerous cartilage matrix proteins by chondroprotection. This element of treatment consists in the use of chondroitin sulphate and glucosamine as a drug, administered together in the appropriate dose and for a long time depending on the degree of degradation of the articular cartilage, usually from several to several months. The combination of chondroitin sulfate with glucosamine causes the activation of a much larger number of matrix proteins than each of the preparations separately.The pharmacokinetics of chondroitin sulfate and glucosamine are positive and favor their chondroprotective effect.The pharmacoproteomics of chondroitin sulfate and glucosamine administered together result from the activation of as many joint cartilage matrix proteins as possible. The development of proteomic techniques creates completely new therapeutic possibilities and is used to study the action of individual molecules.A clinically significant fact is that both chondroitin and glucosamine are natural, endogenous components of bone tissue and articular cartilage, so the use of both drugs is biologically compatible and results in numerous elements of cartilage protection.

E-cadherin is a robust prognostic biomarker in colorectal cancer and low expression is associated with sensitivity to inhibitors of topoisomerase, aurora, and HSP90 in preclinical models.

Cell-cell and cell-matrix adhesion proteins that have been implicated in colorectal epithelial integrity and epithelial-to-mesenchymal transition could be robust prognostic and potential predictive biomarkers for standard and novel therapies. We analyzed in situ protein expression of E-cadherin (ECAD), integrin ß4 (ITGB4), zonula occludens 1 (ZO-1), and cytokeratins in a single-hospital series of Norwegian patients with colorectal cancer (CRC) stages I-IV (n = 922) using multiplex fluorescence-based immunohistochemistry (mfIHC) on tissue microarrays. Pharmacoproteomic associations were explored in 35 CRC cell lines annotated with drug sensitivity data on > 400 approved and investigational drugs. ECAD, ITGB4, and ZO-1 were positively associated with survival, while cytokeratins were negatively associated with survival. Only ECAD showed independent prognostic value in multivariable Cox models. Clinical and molecular associations for ECAD were technically validated on a different mfIHC platform, and the prognostic value was validated in another Norwegian series (n = 798). In preclinical models, low and high ECAD expression differentially associated with sensitivity to topoisomerase, aurora, and HSP90 inhibitors, and EGFR inhibitors. E-cadherin protein expression is a robust prognostic biomarker with potential clinical utility in CRC.

[Development of Novel Methodology and Its Application for Clarifying the Transport Function of the Blood-brain Barrier].

The blood-brain barrier (BBB) consists of brain capillary endothelial cells linked by tight junctions and serves to regulate the transfer of endogenous compounds and xenobiotics between the circulating blood and brain interstitial fluid. We have developed a methodology to characterize brain-to-blood efflux transport in vivo, using the Brain Efflux Index and an in vitro culture model of the BBB, i.e., a conditionally immortalized cell line of the neurovascular unit. Employing these methods, we showed that the BBB plays an important role in protecting the brain by transporting neurotransmitters, neuromodulators, metabolites, uremic toxins, and xenobiotics together with atrial natriuretic peptide from the brain interstitial fluid to the circulating blood. We also developed a highly selective, sensitive LC-MS/MS method for simultaneous protein quantification. We found significant species differences in the expression amounts of various BBB transporter proteins among mice, rats, marmosets, cynomolgus monkeys, and humans. Among transporter proteins at the BBB, multidrug resistance protein 1 (Mdr1/Abcb1) is known to generate a concentration gradient of unbound substrate drugs between the blood and brain. Based on measurements of the intrinsic efflux transport rate of Mdr1 and the protein expression amounts of Mdr1 in mouse brain capillaries and Mdr1-expressing cell lines, we predicted the unbound drug concentration gradients of 7 drugs in the mouse brain in vivo. This was the first successful prediction of in vivo drug transport activity from in vitro experimental data and transporter protein concentration in tissues. This methodology and findings should greatly advance central nervous system barrier research.

Distinct Transport Properties of Human Pannexin 1 and Connexin 32 Hemichannels.

Pannexin (Px) and connexin (Cx) hemichannels mediate bidirectional membrane transport in response to various stimuli and are involved in drug efficacy and toxicity. The purpose of the present study was to clarify in detail the transport characteristics of Px1 and Cx32 hemichannels by establishing transport assay systems using human Px1- and P2RX7 receptor-overexpressing HEK293 cells (Px1/P2RX7/HEK293) and Cx32-overexpressing HEK293 cells (Cx32/HEK293), in which P2RX7 and an extracellular Ca2+-depleted condition serve as the opening trigger, respectively. Uptake of the cationic fluorescent dye propidium iodide (PI) was significantly increased in Px1/P2RX7/HEK293 cells compared to that in mock cells, whereas there was no significant uptake of the anionic fluorescent dye sulforhodamine 101 (SR101). Uptake of [3H]cholesterol by Px1/P2RX7/HEK293 cells was significantly decreased, whereas that of [3H]taurine was not, compared to mock cells. On the other hand, uptakes of PI and SR-101 by Cx32/HEK293 cells were both significantly increased compared to mock cells. The PI uptake by Cx32/HEK293 cells was significantly inhibited by thioacetamide, acetaminophen, and N-acetyl-p-benzoquinoneimine. Cellular uptake of [3H]cholesterol was significantly increased in Cx32/HEK293 cells and that of [3H]taurine was significantly decreased. These results support the idea that Px1 and Cx32 hemichannels have distinct substrate recognition specificities and transport directions.

Targeted Proteomics-Based Quantitative Protein Atlas of Pannexin and Connexin Subtypes in Mouse and Human Tissues and Cancer Cell Lines.

The pannexin (Px) and connexin (Cx) families form multimeric hemichannels that mediate cellular transport of a wide variety of signaling and other molecules and exhibit pathophysiological and pharmacological functions. Twenty-four Px and Cx subtypes have been identified in humans and 23 in mice. The purpose of this study is to establish a quantitative protein atlas of Px and Cx subtypes in mouse and human tissues and cancer cell lines by means of quantitative targeted absolute proteomics, using an internal standard protein in which stable-isotope-labeled target peptides selected according to in silico criteria are concatenated together with internal reference peptides for the determination of the protein amount. This quantification system enabled us to cover 20 of 24 subtypes (83%) in humans, and 21 of 23 subtypes (91%) in mice. In mice, Px1, Cx32, and Cx43 were most abundantly expressed in the small intestine, liver and pancreas, and brain capillary, brain, and heart, respectively. Human blood-brain barrier endothelial cells (human cerebral microvessel endothelial cells) highly expressed Px1 and Cx43. Among human cancer cells, Panc-1 selectively expressed Px1, and Caco-2 cells abundantly expressed Cx32, while MCF-7 and AsPC-1 did not express any subtypes of hemichannels tested. These results suggest that Px1, Cx32, and Cx43 appear to play predominant roles in normal tissues and some cancer cells.

Pharmacoproteomics Identifies Kinase Pathways that Drive the Epithelial-Mesenchymal Transition and Drug Resistance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a complex and deadly disease lacking druggable genetic mutations. The limited efficacy of systemic treatments for advanced HCC implies that predictive biomarkers and drug targets are urgently needed. Most HCC drugs target protein kinases, indicating that kinase-dependent signaling networks drive HCC progression. To identify HCC signaling networks that determine responses to kinase inhibitors (KIs), we apply a pharmacoproteomics approach integrating kinome activity in 17 HCC cell lines with their responses to 299 KIs, resulting in a comprehensive dataset of pathway-based drug response signatures. By profiling patient HCC samples, we identify signatures of clinical HCC drug responses in individual tumors. Our analyses reveal kinase networks promoting the epithelial-mesenchymal transition (EMT) and drug resistance, including a FZD2-AXL-NUAK1/2 signaling module, whose inhibition reverses the EMT and sensitizes HCC cells to drugs. Our approach identifies cancer drug targets and molecular signatures of drug response for personalized oncology.

Discovery of the Consistently Well-Performed Analysis Chain for SWATH-MS Based Pharmacoproteomic Quantification.

Sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) has emerged as one of the most popular techniques for label-free proteome quantification in current pharmacoproteomic research. It provides more comprehensive detection and more accurate quantitation of proteins comparing with the traditional techniques. The performance of SWATH-MS is highly susceptible to the selection of processing method. Till now, ≥27 methods (transformation, normalization, and missing-value imputation) are sequentially applied to construct numerous analysis chains for SWATH-MS, but it is still not clear which analysis chain gives the optimal quantification performance. Herein, the performances of 560 analysis chains for quantifying pharmacoproteomic data were comprehensively assessed. Firstly, the most complete set of the publicly available SWATH-MS based pharmacoproteomic data were collected by comprehensive literature review. Secondly, substantial variations among the performances of various analysis chains were observed, and the consistently well-performed analysis chains (CWPACs) across various datasets were for the first time generalized. Finally, the log and power transformations sequentially followed by the total ion current normalization were discovered as one of the best performed analysis chains for the quantification of SWATH-MS based pharmacoproteomic data. In sum, the CWPACs identified here provided important guidance to the quantification of proteomic data and could therefore facilitate the cutting-edge research in any pharmacoproteomic studies requiring SWATH-MS technique.

Systematic Analysis of Primary Sequence Domain Segments for the Discrimination Between Class C GPCR Subtypes.

G-protein-coupled receptors (GPCRs) are a large and diverse super-family of eukaryotic cell membrane proteins that play an important physiological role as transmitters of extracellular signal. In this paper, we investigate Class C, a member of this super-family that has attracted much attention in pharmacology. The limited knowledge about the complete 3D crystal structure of Class C receptors makes necessary the use of their primary amino acid sequences for analytical purposes. Here, we provide a systematic analysis of distinct receptor sequence segments with regard to their ability to differentiate between seven class C GPCR subtypes according to their topological location in the extracellular, transmembrane, or intracellular domains. We build on the results from the previous research that provided preliminary evidence of the potential use of separated domains of complete class C GPCR sequences as the basis for subtype classification. The use of the extracellular N-terminus domain alone was shown to result in a minor decrease in subtype discrimination in comparison with the complete sequence, despite discarding much of the sequence information. In this paper, we describe the use of Support Vector Machine-based classification models to evaluate the subtype-discriminating capacity of the specific topological sequence segments.