Esophageal adenocarcinoma is rising rapidly in incidence, and usually develops from Barrett’s esophagus, a precursor condition commonly found in patients with chronic acid reflux. Pre-malignant lesions are challenging to detect on conventional screening endoscopy because of their flat appearance. Molecular changes can be used to improve detection of early neoplasia. We have developed a peptide that binds specifically to high-grade dysplasia and adenocarcinoma. We first applied the peptide ex vivo to esophageal specimens from 17 patients to validate specific binding. Next, we performed confocal endomicroscopy in vivo in 25 human subjects after topical peptide administration and found 3.8-fold greater fluorescence intensity for esophageal neoplasia compared with Barrett’s esophagus and squamous epithelium with 75% sensitivity and 97% specificity. No toxicity was attributed to the peptide in either animal or patient studies. Therefore, our first-in-humans results show that this targeted imaging agent is safe, and may be useful for guiding tissue biopsy and for early detection of esophageal neoplasia and potentially other cancers of epithelial origin, such as bladder, colon, lung, pancreas, and stomach.

Ricin is a family member of the lethal ribosome-inactivating proteins (RIP) found in plants. Ricin toxin A-chain (RTA) from castor beans catalyzes the hydrolytic depurination of a single base from a GAGA tetraloop of eukaryotic ribosomal RNA to release a single adenine from the sarcin-ricin loop (SRL). Protein synthesis is inhibited by loss of elongation factor binding resulting in cell death. We report a sensitive coupled assay for the measurement of adenine released from ribosomes or small stem-loop RNAs by RTA catalysis. Adenine phosphoribosyl transferase (APRTase) and pyruvate orthophosphate dikinase (PPDK) convert adenine to ATP for quantitation by firefly luciferase. The resulting AMP is cycled to ATP to give sustained luminescence proportional to adenine concentration. Sub-picomole adenine quantitation permits the action of RTA on eukaryotic ribosomes to be followed in continuous, high-throughput assays. Facile analysis of RIP catalytic activity will have applications in plant toxin detection, inhibitor screens, mechanistic analysis of depurinating agents on oligonucleotides and intact ribosomes, and in cancer immunochemotherapy. Kinetic analysis of the catalytic action of RTA on rabbit reticulocyte 80S ribosomes establishes a catalytic efficiency of 2.6 × 10 8 M −1 s −1 , a diffusion limited reaction indicating catalytic perfection even with large reactants.

Ricin A-chain (RTA) and saporin-L1 (SAP) catalyze adenosine depurination of 28S rRNA to inhibit protein synthesis and cause cell death. We present the crystal structures of RTA and SAP in complex with transition state analogue inhibitors. These tight-binding inhibitors mimic the sarcin-ricin recognition loop of 28S rRNA and the dissociative ribocation transition state established for RTA catalysis. RTA and SAP share unique purine-binding geometry with quadruple -stacking interactions between adjacent adenine and guanine bases and 2 conserved tyrosines. An arginine at one end of the -stack provides cationic polarization and enhanced leaving group ability to the susceptible adenine. Common features of these ribosome-inactivating proteins include adenine leaving group activation, a remarkable lack of ribocation stabilization, and conserved glutamates as general bases for activation of the H2O nucleophile. Catalytic forces originate primarily from leaving group activation evident in both RTA and SAP in complex with transition state analogues.is the catalytic subunit of ricin, a Centers for Disease Control and Prevention category B bioterrorism agent derived from Ricinus communis seeds. RTA catalyzes the depurination of an invariant adenosine residue, A 4234, within the GA 4234 GA tetraloop motif of the highly conserved sarcin-ricin loop of eukaryotic 28S rRNA (1). Clinical trials have exploited the toxicity of RTA in RTA-antibody constructs to kill leukemia and lymphoma cells (e.g., RTA conjugated to anti-CD22) (2-5). Side effects limit the utility of RTA immunotoxins (6, 7). Targeted inhibitors against ribosome-inactivating proteins (RIPs) could improve immunotoxin cancer therapies by rescuing normal cells following toxin treatment.Saporin-L1 (SAP), a homologue of RTA from Saponaria officinalis (soapwort) leaves, exhibits N-glycohydrolase activities on 80S ribosomes, poly(A) RNA, and other cellular . SAP releases multiple adenines from ribosomes, whereas RTA shows exquisite specificity.Truncated oligonucleotide constructs of the ribosomal sarcinricin loop are RTA and SAP substrates (13-16). In addition to hairpin stem-loop structures, RTA hydrolyzes adenine from cyclic GAGA loops that possess a 5Ј-to 3Ј-covalently closed synthetic linker (17, 18) (Fig. 1).Transition state analysis of RTA-mediated depurinations established that hydrolysis of adenine involves a ribocation intermediate, followed by attack of an activated water. Adenine activation is a major driving force for RTA catalysis (19,20). Efficient catalysis by RTA requires the invariant Glu-177 and Arg-180 residues (21-25). RTA and other RIPs have evolved to become near-perfect catalysts for mammalian ribosomes (21,(26)(27)(28). Here, we use transition state analogues to establish the catalytic site features contributing to this remarkable catalytic activity.RTA transition state structures have guided the design and synthesis of potent RIP inhibitors (17, 29). Inhibitors for RTA and SAP include 1Ј-aza-sugars with a nonhydrolyzable 9-deazaadenine to mimic the...

INTRODUCTION: Duodenal epithelial barrier impairment and immune activation may play a role in the pathogenesis of functional dyspepsia (FD). This study was aimed to evaluate the duodenal epithelium of patients with FD and healthy individuals for detectable microscopic structural abnormalities. METHODS: This is a prospective study using esophagogastroduodenoscopy enhanced with duodenal confocal laser endomicroscopy (CLE) and mucosal biopsies in patients with FD (n = 16) and healthy controls (n = 18). Blinded CLE images analysis evaluated the density of epithelial gaps (cell extrusion zones), a validated endoscopic measure of the intestinal barrier status. Analyses of the biopsied duodenal mucosa included standard histology, quantification of mucosal immune cells/cytokines, and immunohistochemistry for inflammatory epithelial cell death called pyroptosis. Transepithelial electrical resistance (TEER) was measured using Ussing chambers. Epithelial cell-to-cell adhesion proteins expression was assessed by real-time polymerase chain reaction. RESULTS: Patients with FD had significantly higher epithelial gap density on CLE in the distal duodenum than that of controls (P = 0.002). These mucosal abnormalities corresponded to significant changes in the duodenal biopsy samples of patients with FD, compared with controls, including impaired mucosal integrity by TEER (P = 0.009) and increased number of epithelial cells undergoing pyroptosis (P = 0.04). Reduced TEER inversely correlated with the severity of certain dyspeptic symptoms. Furthermore, patients with FD demonstrated altered duodenal expression of claudin-1 and interleukin-6. No differences in standard histology were found between the groups. DISCUSSION: This is the first report of duodenal CLE abnormalities in patients with FD, corroborated by biopsy findings of epithelial barrier impairment and increased cell death, implicating that duodenal barrier disruption is a pathogenesis factor in FD and introducing CLE a potential diagnostic biomarker in FD.

Ribosome-inactivating proteins (RIPs) catalyze the hydrolytic depurination of one or more adenosine residues from eukaryotic ribosomes. Depurination of the ribosomal sarcin-ricin tetraloop (GAGA) causes inhibition of protein synthesis and cellular death. We characterized the catalytic properties of saporin-L1 from Saponaria officinalis (soapwort) leaves and demonstrate robust activity against defined nucleic acid substrates and mammalian ribosomes. Transition state analogue mimics of small oligonucleotide substrates of saporin-L1 are powerful, slow-onset inhibitors when adenosine is replaced with the transition state mimic 9-deazaadenine-9-methylene-N-hydroxypyrrolidine (DADMeA). Linear, cyclic and stem-loop oligonucleotide inhibitors containing DADMeA and based on the GAGA sarcin-ricin tetraloop gave slow-onset tight-binding inhibition constants (Ki*) of 2.3 to 8.7 nM at physiological conditions and bind up to 40,000-fold tighter than RNA substrates. Saporin-L1 inhibition of rabbit reticulocyte translation was protected by these inhibitors. Transition state analogues of saporin-L1 have potential in cancer therapy that employs saporin-L1 linked immunotoxins.

Ricin Toxin A-chain (RTA) catalyzes the hydrolytic depurination of A4324, the first adenosine of the GAGA tetra-loop portion of 28S eukaryotic ribosomal RNA. Truncated stem-loop versions of the 28S rRNA are RTA substrates. Here, we investigate circular DNA and DNA/RNA hybrid GAGA sequence oligonucleotides as minimal substrates and inhibitor scaffolds for RTA catalysis. Closing the 5'- and 3'-ends of a d(GAGA) tetraloop creates a substrate with 92-fold more activity with RTA (kcat/Km) than that for the d(GAGA) linear form. Circular substrates have catalytic rates (kcat) comparable to and exceeding those of RNA and DNA stem-loop substrates, respectively. RTA inhibition into the nanomolar range has been achieved by introducing an N-benzyl-hydroxypyrrolidine (N-Bn) transition state analogue at the RTA depurination site in a circular GAGA motif. The RNA/DNA hybrid oligonucleotide cyclic GdAGA provides a new scaffold for RTA inhibitor design, and cyclic G(N-Bn)GA is the smallest tight-binding RTA inhibitor (Ki = 70 nM). The design of such molecules that lack the base-paired stem-loop architecture opens new chemical synthetic approaches to RTA inhibition.

The rapid rise in incidence of oesophageal adenocarcinoma has motivated the need for improved methods for surveillance of Barrett’s oesophagus. Early neoplasia is flat in morphology and patchy in distribution and is difficult to detect with conventional white light endoscopy (WLE). Light offers numerous advantages for rapidly visualising the oesophagus, and advanced optical methods are being developed for wide-field and cross-sectional imaging to guide tissue biopsy and stage early neoplasia, respectively. We review key features of these promising methods and address their potential to improve detection of Barrett’s neoplasia. The clinical performance of key advanced imaging technologies is reviewed, including (1) wide-field methods, such as high-definition WLE, chromoendoscopy, narrow-band imaging, autofluorescence and trimodal imaging and (2) cross-sectional techniques, such as optical coherence tomography, optical frequency domain imaging and confocal laser endomicroscopy. Some of these instruments are being adapted for molecular imaging to detect specific biological targets that are overexpressed in Barrett’s neoplasia. Gene expression profiles are being used to identify early targets that appear before morphological changes can be visualised with white light. These targets are detected in vivo using exogenous probes, such as lectins, peptides, antibodies, affibodies and activatable enzymes that are labelled with fluorescence dyes to produce high contrast images. This emerging approach has potential to provide a ‘red flag’ to identify regions of premalignant mucosa, outline disease margins and guide therapy based on the underlying molecular mechanisms of cancer progression.

A series of novel taxane-based multidrug resistance (MDR) reversal agents (TRAs) has been designed and synthesized. Structure-activity relationship (SAR) study clearly indicates that modification of the C-7 position with hydrophobic arenecarbonylcinnamoyl groups brings about high potency against drug efflux mediated by P-glycoprotein (P-gp). Six TRAs exhibit ability to modulate a wide range of ATP-binding cassette (ABC) transporters, such as P-gp, multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP), which may serve as novel broad-spectrum modulators of ABC transporters.