Rajendra Joshi

Discovery of diacylphloroglucinols as a new class of GPR40 (FFAR1) agonists

In this letter, we report discovery of diacylphloroglucinol compounds as a new class of GPR40 (FFAR1) agonists. Several diacylphloroglucinols with varying length of acyl functionality and substitution on aromatic hydroxyls were synthesized and evaluated for GPR40 agonism using functional calcium-flux assay. Out of 17 compounds evaluated, 14, 17, 19 and 25 exhibited good GPR40 agonistic activity with EC(50) values ranging from 0.07 to 8 microM (pEC(50) 7.12-5.09), respectively, with maximal agonistic response of 84-102%.

Multifunctional silica nanoparticles for optical and magnetic resonance imaging.

Abstract The surface of spherical, nonporous silica nanoparticles (SiO2-NPs) was modified with gadolinium (Gd) complexes, fluorophores, and cell-penetrating peptides to achieve multifunctionality on a single particle. The Gd surface concentrations were 9–16 μmol/g resulting in nanomaterials with high local longitudinal and transversal relaxivities (~1×105 and ~5×105 /mm/s/NP, respectively). Rapid cellular uptake was observed in vitro; however, larger extracellular agglomerates were also formed. In vivo administration revealed a fast distribution throughout the body followed by a nearly complete disappearance of fluorescence in all organs except the lungs, liver, and spleen after 24 h. Such NPs have the potential to serve as efficient multimodal probes in molecular imaging.

MR contrast agent composed of cholesterol and peptide nucleic acids: Design, synthesis and cellular uptake

A new mRNA targeting contrast agent consisting of three main functional domains, (i) gadolinium based magnetic resonance reporter part, (ii) antisense peptide nucleic acids targeted to mRNA, and (iii) cholesterol as the delivery vector, was developed and synthesized. The new contrast agent showed efficient cellular uptake and significant contrast enhancement at very low labeling concentrations (0.5 microM). However, after uptake into cells the agent was located predominantly in endosomes like a similar cell penetrating peptide conjugated probe. Our results indicate that this newly developed contrast agent could be used for the labeling of cells for optical as well as magnetic resonance imaging.

Facile synthesis of peptide nucleic acids and peptide nucleic acid-peptide conjugates on an automated peptide synthesizer

Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence‐specific manner. Therefore, they are widely used in molecular diagnosis of antisense‐targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost‐effective semi‐automated synthesis of PNAs and PNA‐peptide constructs on an automated peptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries usable, e.g. for high‐throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure. Copyright

Synthesis and in Vitro Evaluation of a Biotinylated Dextran-Derived Probe for Molecular Imaging

Herein we report the design, synthesis, and in vitro evaluation of a gadolinium-containing biotinylated dextran-derived molecular imaging probe as a prospective neuroanatomical tracer by means of magnetic resonance imaging (MRI). The probe was effectively taken up by cultured differentiated murine neuroblastoma cells and significantly enhanced the contrast in T(1)- and T(2)-weighted MR images of labeled cells under physiological conditions. A significant longitudinal relaxation rate enhancement in the presence of avidin was observed allowing the verification of the results in the end of noninvasive longitudinal MRI connectivity studies by post-mortem histology. The in vitro results indicate that the probe has the potential to be used in vivo to identify the organization of global neuronal networks in the brain with MRI.

CPP or Cholesterol Conjugated Antisense PNA for Cellular Delivery

Gonadotropin Releasing Hormone (pGlu-His-Trp-Ser-Tyr-Gly-Leu- Arg-Pro-Gly-NH2, GnRH) plays a signifi cant role in the controlling of gonadotropins and steroids hormones. A large number of linear GnRH analogues has been synthesized and tested for several medical uses. Leuprolide acetate (pGlu-His-Trp-Ser-Tyr-(D)Leu-Leu-Arg-Pro-NHEt, LPA) is a potent GnRH agonist and is used to treat a wide range of sex hormone related disorders, including prostatic cancer, endometriosis and precocious puberty. Despite its widespread use, only limited information based on spectroscopic evidence regarding the solution conformation of Leuprolide are known. Moreover, non crystallographic data is available for the receptor of GnRH (G protein-coupled receptor). The aim of this study was to characterize the conformation of Leuprolide and its modifi ed linear analogue (pGlu-His-Trp-Ser-Tyr(OMe)-(D)Leu-Leu- Arg-Aze-NHEt) in DMSO solution (which simulates better the receptor environment) using Nuclear Magnetic Resonance (NMR) and Molecular Modeling techniques. By using both NMR and Molecular Modeling we have characterized the secondary structural preferences of these GnRH analogues.

Role of exofacial protein thiols in the cytosolic delivery of the cysteine-rich cell penetrating peptide CyLoP-1

The Phosphatidylinositide 3-kinase (PI3-K) pathway is deregulated in a range of cancers, and several inhibitors of this enzyme are entering the clinic. A current problem for such targeted therapeutics in drug discovery pipelines is the lack of biomarkers for use in clinical trials, to allow the assessment of early therapeutic response and thus better patient management. Here, we assessed uptake of the PET tracer [18F]-Fluorodeoxythymidine (FLT) in response to reduction of PI3-K signalling mediated by the novel PI-3K inhibitor GDC-0941 in both ectopic and orthotopic xenograft models. Methods Nude mice bearing U87 and HCT116 xenografts were imaged for 105 minutes dynamically with [18F]-FLT at baseline (when tumours were~200mm3) and after acute (18h) or chronic (186h) treatment with 50mg/kg GDC-0941 or vehicle twice daily by gavage. An orthotopic model consisting of HCT116 liver metastases obtained after primary inoculation of cells into the spleen was also imaged at baseline and after acute/chronic GDC-0941 therapy. Regions of Interest (ROIs) were drawn manually over tumours/metastases and maximum standard uptake value (SUV)s for tumour uptake were calculated, as was uptake normalised to mean activity in blood pool as estimated by an ROI drawn on the heart contents (NUV). Tumour tissue was analysed at sacrifice for expression of TK1, cleaved PARP and phospho-AKT. Results Growth of U87 xenografts was significantly inhibited by GDC-0941 treatment throughout the course of the study (average growth rate of treated = -1.0±9.3 mm3, control = 69.2±22.1mm3 per day, p<0.05). Growth of HCT116 xenografts was not significantly different between groups (average growth rate of treated = 37.3±8.4 mm3, control =46.2±11.9 mm3 per day); however growth of HCT116 liver metastases was significantly reduced (Liver:bodyweight ratio of 0.11±0.01 vs 0.06±0.01, p<0.01). Uptake of [18F]-FLT was reduced in GDC-0941 treated animals bearing U87 xenografts or HCT116 liver metastases at the acute timepoint compared to baseline (NUVmax of 2.17±0.38 vs 1.59±0.29, p<0.01; 2.42±0.19,1.8±0.12 p<0.05 respectively), but was unchanged in unresponsive HCT116 xenografts. Conclusion [18F]-FLT is a strong candidate for the non-invasive measurement of GDC-0941 action. In addition to FLT, we are assessing the performance of a range of other imaging agents, including proprietary tracers, that can potentially bring value in monitoring of PI3-kinase targeting therapies. Disclosure of author financial interest or relationships: C. Cawthorne, GE Healthcare, Grant/research support; I. Wilson, GE Healthcare Medical Diagnostics, Employment; N. Burrows, None; R. Gieling, None; J. Gregory, None; A.M. Smigova, None; M. Babur, None; K. Williams, GE, Grant/research support . S1286

Correction to Synthesis and in Vitro Evaluation of a Biotinylated Dextran-Derived Probe for Molecular Imaging

In the version of this paper published on the web on January 24, 2012, and in the April 2012 issue, ref 20 was omitted. The reference is corrected in the online version published on July 13, 2012. The correct (20) is provided here.