Peptide Nucleic Acids Containing Cationic/Amino-Alkyl Modified Bases Promote Enhanced Hybridization Kinetics and Thermodynamics with Single-Strand DNA.

Peptide nucleic acids (PNAs) are antisense molecules with excellent polynucleotide hybridization properties; they are resistant to nuclease degradation but often have poor cell permeability leading to moderate cellular activity and limited clinical results. The addition of cationic substitutions (positive charges) to PNA molecules greatly increases cell permeability. In this report, we describe the synthesis and polynucleotide hybridization properties of a novel cationic/amino-alkyl nucleotide base-modified PNA (OPNA). This study was designed to quantitate the effect the cationic/amino-alkyl nucleotide base modification had on the kinetic and thermodynamic properties of OPNA-DNA hybridization using surface plasmon resonance and UV thermal melt studies. Kinetic studies reveal a favorable 10-30 fold increase in affinity for a single cationic modification on the base of an adenine, cytosine, or guanidine OPNA sequence compared to the nonmodified PNA strand. The increase in affinity is correlated directly with a favorable decrease in the dissociation rate constant and increase in the association rate constant. Introducing additional amino-alkyl base modifications further favors a decrease in the dissociation rate (3-10-fold per amino-alkyl). The thermodynamics driving the OPNA hybridization is promoted by an additional favorable -80 kJ/mol enthalpy of binding for a single amino-alkyl modification compared to the PNA strand. This increase in enthalpy is consistent with an ion-ion interaction with the DNA strand. These kinetic and thermodynamic hybridization studies reveal for the first time that this type of cationic/amino-alkyl base-modified PNA has favorable hybridization properties suitable for development as an antisense oligomer.

Design and biological characterization of a series of dual mechanism ERK1/2 inhibitors with a Triazolopyridinone core.

Oncology clinical development programs have targeted the RAS/RAF/MEK/ERK signaling pathway with small molecule inhibitors for a variety of cancers during the past decades, and most therapies have shown limited or minimal success. Specific BRAF and MEK inhibitors have shown clinical efficacy in patients for the treatment of BRAF-mutant melanoma. However, most cancers have shown treatment resistance after several months of inhibitor usage, and reports indicate resistance is often associated with the reactivation of the MAPK signaling pathway. It is widely accepted that an effective MAPK therapy will have a significant impact on curtailing cancer growth and improving patient survival. However, despite more than three decades of intense research and pharmaceutical industry efforts, an FDA-approved, effective anti-cancer ERK inhibitor has yet to be developed. Here, we present the design, optimization, and biological characterization of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate the phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. Our series of dual mechanism ERK1/2 inhibitors, in which we incorporated a triazolopyridinone core, may present potential benefits for enhancing efficacy and addressing the emergence of treatment resistance.

Regulation of Lipoprotein Homeostasis by Self-Assembling Peptides.

High levels of serum low-density lipoprotein (LDL) cholesterol contribute to atherosclerosis, a key risk factor of cardiovascular diseases. PCSK9 is a circulatory enzyme that downregulates expression of hepatic LDL receptors, concomitantly increasing serum LDL-C. This work investigates a small, self-assembling peptide, EPep2-8, as a peptide inhibitor of PCSK9. EPep2-8 is a multidomain peptide comprising a self-assembling domain, E2, conjugated to a bioactive domain, Pep2-8, previously shown to inhibit PCSK9. The E2 domain facilitates self-assembly of EPep2-8 into long, nanofibrous polymers with an underlying supramolecular ß-sheet secondary structure. Intermolecular interactions between nanofibers drive EPep2-8 to form a thixotropic and cytocompatible hydrogel in aqueous and charge-neutral solutions. These properties enable EPep2-8 to be delivered as an in situ depot for regulation of lipoprotein homeostasis. In surface plasmon resonance studies, EPep2-8 bound specifically to PCSK9 with an apparent, noncovalent, and irreversible dissociation, significantly improving the binding affinity of Pep2-8 alone (KD = 667 ± 48 nM). Increased binding affinity of EPep2-8 is primarily due to the superstoichiometric interaction of the peptide with PCSK9. Promisingly, EPep2-8 retains bioactivity in vitro, engendering dose-dependent uptake of LDL-C in hepatocytes. This mechanism of self-assembly on a target site may be a simple method to improve the affinity of peptide inhibitors.

Recent advances in cancer drug discovery targeting RAS.

Mutated RAS is present in 30% of human tumors, appearing in 90% of pancreatic, 45% of colon and 35% of lung cancers. These high occurrences make RAS one of the most important drug targets in oncology. Three decades of effort to target RAS have been unsuccessful in generating drug therapies suggesting that it might represent an 'undruggable' target. However, recent reports highlighting new approaches for targeting RAS have uncovered more information on protein structure and identified new binding pockets. Efforts to target the KRAS G12C mutation specifically have shown promising results whereas other approaches have targeted various protein complexes. These advances could lead to development of new effective cancer drugs targeting RAS.

Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche.

We previously reported a new approach for culturing difficult-to-preserve primary patient-derived multiple myeloma cells (MMC) using an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment and a culture medium supplemented with patient plasma. In the current study, we used this biomimetic model to show, for the first time, that the long-term survival of OSB is the most critical factor in maintaining the ex vivo viability and proliferative capacity of MMC. We found that the adhesion and retention of MMC to the tissue scaffold was meditated by osteoblastic N-cadherin, as one of potential mechanisms that regulate MMC-OSB interactions. However, in the presence of MMC and patient plasma, the viability and osteogenic activity of OSB became gradually compromised, and consequently MMC could not remain viable over 3 weeks. We demonstrated that the long-term survival of both OSB and MMC could be enhanced by: (1) optimizing perfusion flow rate and patient-derived plasma composition in the culture medium and (2) replenishing OSB during culture as a practical means of prolonging MMC's viability beyond several weeks. These findings were obtained using a high-throughput well plate-based perfusion device from the perspective of optimizing the ex vivo preservation of patient-derived MM biospecimens for downstream use in biological studies and chemosensitivity analyses.

Use of surface-enhanced Raman scattering as a prognostic indicator of acute kidney transplant rejection.

We report an early, noninvasive and rapid prognostic method of predicting potential acute kidney dysfunction using surface-enhanced Raman scattering (SERS). Our analysis was performed on urine samples collected prospectively from 58 kidney transplant patients using a He-Ne laser (632.8 nm) as the excitation source. All abnormal kidney function episodes (three acute rejections and two acute kidney failures that were eventually diagnosed independently by clinical biopsy) consistently exhibited unique SERS spectral features in just one day following the transplant surgery. These results suggested that SERS analysis provides an early and more specific indication to kidney function than the clinically used biomarker, serum creatinine (sCr).

Patient-specific 3D microfluidic tissue model for multiple myeloma.

In vitro culturing of primary multiple myeloma cells (MMC) has been a major challenge as this plasma cell malignancy depends on the bone marrow environment for its survival. Using a microfluidic platform to emulate the dynamic physiology of the bone marrow microenvironment, we report here a new approach for culturing difficult to preserve primary human MMC. The system uses a three-dimensional ossified tissue to mimic the tumor niche and recapitulate interactions between bone marrow cells and osteoblasts (OSB). To this end, the human fetal OSB cell line hFOB 1.19 was cultured in an eight-chamber microfluidic culture device to facilitate the seeding of mononuclear cells from bone marrow aspirates from three multiple myeloma patients. Optical microscopy, used for real-time monitoring of mononuclear cell interactions with the ossified tissue, confirmed that these are drawn toward the OSB layer. After 3 weeks, cocultures were characterized by flow cytometry to evaluate the amount of expansion of primary MMC (with CD138(+) and CD38(+)CD56(+) phenotypes) in this system. For each of the three patients analyzed, bone marrow mononuclear cells underwent, on an average, 2 to 5 expansions; CD38(+)CD56(+) cells underwent 1 to 3 expansions and CD138(+) cells underwent 2.5 to 4.6 expansions. This approach is expected to provide a new avenue that can facilitate: (1) testing of personalized therapeutics for multiple myeloma patients; (2) evaluation of new drugs without the need for costly animal models; and (3) studying the biology of multiple myeloma, and in particular, the mechanisms responsible for drug resistance and relapse.

Antidepressant-induced suicidality: how translational epidemiology incorporating pharmacogenetics into controlled trials can improve clinical care.

Randomized controlled trials (RCTs) have been a staple of the drug development process for several decades. Here, we review the origins of RCTs and their adoption within drug development, highlighting shortcomings that tend to be ignored and possible solutions offered from personalized medicine. While RCTs play an important role in development of therapeutics, we underscore how if used indiscriminately, their adverse effects may outweigh the benefits. As an example, we focus on the development of antidepressants and how a severe adverse drug response - suicidal ideation - can be overlooked. We conclude with a discussion of how pharmacogenetics may address some of the deficiencies of RCTs, bringing the focus of drug response back to the individual patient rather than the population, using as an example the discovery of genetic markers associated with antidepressant-induced suicidal ideation.

Single-test parallel assessment of multiple genetic disorders.

We advocate a new paradigm for genetic diagnosis based on using customized array panels, each of which groups multiple genes and mutations associated with clinical profiles that are common to particular syndromic diseases. This parallel approach, based on a single-test multigene multiplexing strategy, compared with traditional sequential testing by gene-by-gene genetic analysis, drastically reduces the time and cost of diagnosis while maintaining accuracy and reliability. Faster diagnosis enables early decision-making to facilitate better patient management and outcomes at reduced costs to the healthcare system.

Noise filtering and nonparametric analysis of microarray data underscores discriminating markers of oral, prostate, lung, ovarian and breast cancer.

BACKGROUND: A major goal of cancer research is to identify discrete biomarkers that specifically characterize a given malignancy. These markers are useful in diagnosis, may identify potential targets for drug development, and can aid in evaluating treatment efficacy and predicting patient outcome. Microarray technology has enabled marker discovery from human cells by permitting measurement of steady-state mRNA levels derived from thousands of genes. However many challenging and unresolved issues regarding the acquisition and analysis of microarray data remain, such as accounting for both experimental and biological noise, transcripts whose expression profiles are not normally distributed, guidelines for statistical assessment of false positive/negative rates and comparing data derived from different research groups. This study addresses these issues using Affymetrix HG-U95A and HG-U133 GeneChip data derived from different research groups. RESULTS: We present here a simple non parametric approach coupled with noise filtering to identify sets of genes differentially expressed between the normal and cancer states in oral, breast, lung, prostate and ovarian tumors. An important feature of this study is the ability to integrate data from different laboratories, improving the analytical power of the individual results. One of the most interesting findings is the down regulation of genes involved in tissue differentiation. CONCLUSIONS: This study presents the development and application of a noise model that suppresses noise, limits false positives in the results, and allows integration of results from individual studies derived from different research groups.

Fructose-responsive genes in the small intestine of neonatal rats.

The intestinal brush border fructose transporter GLUT5 (SLC2A5) typically appears in rats after weaning is completed. However, precocious consumption of dietary fructose or in vivo perfusion for 4 h of the small intestine with high fructose (HF) specifically stimulates de novo synthesis of GLUT5 mRNA and protein before weaning is completed. Intermediary signals linking the substrate, fructose, to GLUT5 transcription are not known but should also respond to fructose perfusion. Hence, we used microarray hybridization and RT-PCR to identify genes whose expression levels change during HF relative to high-glucose (HG) perfusion. Expression of GLUT5 and NaPi2b, the intestinal Na+-dependent phosphate transporter, dramatically increased and decreased, respectively, with HF perfusion for 4 h. Expression of >20 genes, including two key gluconeogenic enzymes, glucose-6-phosphatase (G6P) and fructose-1,6-bisphosphatase, also increased markedly, along with fructose-2,6-bisphosphatase, an enzyme unique to fructose metabolism and regulating fructose-1,6-bisphosphatase activity. GLUT5 and G6P mRNA abundance, which increased dramatically with HF relative to HG, alpha-methylglucose, and normal Ringer perfusion, may be tightly and specifically linked to changes in intestinal luminal fructose but not glucose concentrations. G6P but not GLUT5 mRNA abundance increased after just 20 min of HF perfusion. This cluster of gluconeogenic enzymes and their common metabolic intermediate fructose-6-phosphate may regulate fructose metabolism and GLUT5 expression in the small intestine.

In vivo pharmacokinetics and regulation of gene expression profiles by isothiocyanate sulforaphane in the rat.

Sulforaphane (SUL) is one member of the isothiocyanate class of cancer chemopreventive compounds that has been shown to be effective in blocking initiation and progression of carcinogenesis. Previously, many studies have shown that SUL can potently induce phase II detoxifying enzymes, which contributes to its chemopreventive functions. In this study, we used 4967 oligonucleotides microarray to assess the genes that are modulated by SUL in in vivo rat livers, as well as time course of expression of these genes. The pharmacokinetics of SUL was assessed after oral dose of 50 micromol of SUL. The plasma concentration occurred at 1 h and peaked around 20 microM at 4 h after dosing and declined with a half-life of about 2.2 h. Analysis of the gene expression data found various clusters of genes that are important in cellular defense mechanisms and cell cycle regulation. The most robust cluster of genes is the metallothionein-like genes (MT-1/2 and MT-1a), which are increased up to 10-fold by 2 to 4 h after SUL dosing. The second cluster of genes is the glutathione S-transferase-A3-like genes, which include aflatoxin B1 aldehyde reductase and aldehyde oxidase. These genes are increased slightly by 4 h and peaked at 12 h. Real-time polymerase chain reaction was performed to authenticate the mRNA expression of some of these genes. In summary, this in vivo study of SUL provides the first clue as to the plasma concentrations of SUL, in vivo mitogen-activated protein kinase activations in rat livers, as well as what other genes are modulated in addition to phase II detoxifying genes. The results from this study may yield better insights for its chemopreventive functions.

Comprehensive genome-wide comparison of DNA and RNA level scan using microarray technology for identification of candidate cancer-related genes in the HL-60 cell line.

Genome-wide scans for DNA and RNA changes in the HL-60 cell line relative to normal leukocytes were conducted. Microarray-based comparative genome hybridization (CGH) studies were performed with the Spectral Genomics Human Bacterial Artificial Chromosome (BAC) 3MB system. Transcriptional measurements of approximately 12,500 human genes were monitored using Affymetrix U95A GeneChips. In HL-60, genomic DNA amplification of the 8q24 locus, trisomy 18, and deletions at loci 5q11.2 approximately q31, 6q12, 9p21.3 approximately p22, 10p12 approximately p15, 14q22 approximately q31, 17p12 approximately p13.3, and monosomy X were detected. After obtaining locus information about the RNA transcripts from the Affymetrix database, 4368 genes were stratified both according to status of RNA expression and the DNA copy number of their designated loci. The expression level of 2326 (53.25%) of 4368 transcripts is concordant with DNA copy number. Examples of specific, highly expressed, cancer-associated genes in amplified loci include SERPINB10, MYC, TYMS, HEC, and EPB41L3, while CD14, GZMK, TCF7, FOS, MLH3, CTNNA1, IRF1, VIM, CRK, MAP3K1, STAM, MAX, SFRG5, ENC1, PURA, MNT, RASA1, GLRX, UBE2B, NR3C1, PTENP1, BS69, COPEB, SKIP, PIM2, and MIC2 represent cancer-associated genes in deleted loci with decreased expression. The complementary usage of genome-wide DNA and RNA scans should enhance the identification of candidate genes in the neoplastic process.

Functional cloning of genes encoding dsRNA binding proteins.

Over a dozen human genes code for proteins that specifically bind to double-stranded RNA. These proteins have been implicated in several important cellular processes such as transcriptional activation, inhibition of translational initiation, RNA editing, mRNA localization, signal transduction, and posttranscriptional gene silencing (PTGS or RNAi). The recent discovery that PTGS or RNAi is a dsRNA-mediated pathway has further added to the study of dsRNA binding proteins. A method that enables the cloning of genes encoding dsRNA binding proteins would greatly facilitate the identification and study of these molecules. Here we describe a method for isolating such genes from an expression library using radiolabeled poly(I):poly(C) as a binding substrate.

C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression.

To verify the genome annotation and to create a resource to functionally characterize the proteome, we attempted to Gateway-clone all predicted protein-encoding open reading frames (ORFs), or the 'ORFeome,' of Caenorhabditis elegans. We successfully cloned approximately 12,000 ORFs (ORFeome 1.1), of which roughly 4,000 correspond to genes that are untouched by any cDNA or expressed-sequence tag (EST). More than 50% of predicted genes needed corrections in their intron-exon structures. Notably, approximately 11,000 C. elegans proteins can now be expressed under many conditions and characterized using various high-throughput strategies, including large-scale interactome mapping. We suggest that similar ORFeome projects will be valuable for other organisms, including humans.