David M. Tellers

Effective siRNA delivery and target mRNA degradation using an amphipathic peptide to facilitate pH-dependent endosomal escape

Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of α-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.

Synthesis of Vaniprevir (MK-7009): Lactamization To Prepare a 22-Membered Macrocycle

Development of a practical synthesis of MK-7009, a 20-membered [corrected] macrocycle, is described. A variety of ring-closing strategies were evaluated, including ring-closing metathesis, intermolecular palladium-catalyzed cross-couplings, and macrolactamization. Ring closure via macrolactamization was found to give the highest yields under relatively high reaction concentrations. Optimization of the ring formation step and the synthesis of key intermediates en route to MK-7009 are reported.

Computational Design and Experimental Characterization of Peptides Intended for pH-Dependent Membrane Insertion and Pore Formation

There are many opportunities to use macromolecules, such as peptides and oligonucleotides, for intracellular applications. Despite this, general methods for delivering these molecules to the cytosol in a safe and efficient manner are not available. Efforts to develop a variety of intracellular drug delivery systems such as viral vectors, lipoplexes, nanoparticles, and amphiphilic peptides have been made, but various challenges such as delivery efficiency, toxicity, and controllability remain. A central challenge is the ability to selectively perturb, not destroy, the membrane to facilitate cargo introduction. Herein, we describe our efforts to design and characterize peptides that form pores inside membranes at acidic pH, so-called pH-switchable pore formation (PSPF) peptides, as a potential means for facilitating cargo translocation through membranes. Consistent with pore formation, these peptides exhibit low-pH-triggered selective release of ATP and miRNA, but not hemoglobin, from red blood cells. Consistent with these observations, biophysical studies (tryptophan fluorescence, circular dichroism, size-exclusion chromatography, analytical ultracentrifugation, and attenuated total reflectance Fourier transformed infrared spectroscopy) show that decreased pH destabilizes the PSPF peptides in aqueous systems while promoting their membrane insertion. Together, these results suggest that reduced pH drives insertion of PSPF peptides into membranes, leading to target-specific escape through a proposed pore formation mechanism.

Molecular Umbrella Conjugate for the Ocular Delivery of siRNA

The synthesis, computer modeling, and biological activity of an octawalled molecular umbrella short interfacing RNA (siRNA) conjugate is described. This molecular umbrella–siRNA conjugate exhibited mRNA knockdown activity in vitro in the absence of a transfection reagent. Evaluation of this molecular umbrella conjugate in vivo, using the rat eye via intravitreal injection, resulted in sequence specific mRNA knockdown in the retina with no obvious signs of toxicity, as judged by ophthalmic examination.

Rapid HATU-mediated solution phase siRNA conjugation.

Conditions for facile solution-phase amide conjugation of amine-modified siRNA with a diverse set of carboxylic acid partners using the coupling reagent HATU are described. These conditions eliminate the need for isolated activated esters and allow for rapid access to conjugates with a wide range of lipophilicity and functionality in good yield.

CHEMGENIE: integration of chemogenomics data for applications in chemical biology

Increasing amounts of biological data are accumulating in the pharmaceutical industry and academic institutions. However, data does not equal actionable information, and guidelines for appropriate data capture, harmonization, integration, mining, and visualization need to be established to fully harness its potential. Here, we describe ongoing efforts at Merck & Co. to structure data in the area of chemogenomics. We are integrating complementary data from both internal and external data sources into one chemogenomics database (Chemical Genetic Interaction Enterprise; CHEMGENIE). Here, we demonstrate how this well-curated database facilitates compound set design, tool compound selection, target deconvolution in phenotypic screening, and predictive model building.

Identification of proximal biomarkers of PKC agonism and evaluation of their role in HIV reactivation.

Design: The HIV latent CD4+ T cell reservoir is broadly recognized as a barrier to HIV cure. Induction of HIV expression using protein kinase C (PKC) agonists is one approach under investigation for reactivation of latently infected CD4+ T cells (Beans et al., 2013; Abreu et al., 2014; Jiang et al., 2014; Jiang and Dandekar, 2015). We proposed that an increased understanding of the molecular mechanisms of action of PKC agonists was necessary to inform on biological signaling and pharmacodynamic biomarkers. RNA sequencing (RNA Seq) was applied to identify genes and pathways modulated by PKC agonists. Methods: Human CD4+ T cells were treated ex vivo with Phorbol 12‐myristate 13‐acetate, prostatin or ingenol‐3‐angelate. At 3 h and 24 h post‐treatment, cells were harvested and RNA‐Seq was performed on RNA isolated from cell lysates. The genes differentially expressed across the PKC agonists were validated by quantitative RT‐PCR (qPCR). A subset of genes was evaluated for their role in HIV reactivation using siRNA and CRISPR approaches in the Jurkat latency cell model. Results: Treatment of primary human CD4+ T cells with PKC agonists resulted in alterations in gene expression. qPCR of RNA Seq data confirmed upregulation of 24 genes, including CD69, Egr1, Egr2, Egr3, CSF2, DUSP5, and NR4A1. Gene knockdown of Egr1 and Egr3 resulted in reduced expression and decreased HIV reactivation in response to PKC agonist treatment, indicating a potential role for Egr family members in latency reversal. Conclusion: Overall, our results offer new insights into the mechanism of action of PKC agonists, biomarkers of pathway engagement, and the potential role of EGR family in HIV reactivation. HighlightsRNASeq of PKC agonists in human CD4 T cells reveals altered signalling pathways and time‐dependent changes in gene expression.Several biomarkers of PKC agonisms were identified and validated in human CD4+ T cells and in in vivo rat studies.Egr family members have a role in PKC mediated latent HIV‐1 reactivation.

High-throughput chemical modification of oligonucleotides for systematic structure-activity relationship evaluation.

Chemical modification of siRNA is achieved in a high-throughput manner (96-well plate format) by copper catalyzed azide-alkyne cycloadditions. This transformation can be performed in one synthetic operation at up to four positions with complete specificity, good yield, and acceptable purity. As demonstrated here, this approach extends the current synthetic options for oligonucleotide modifications and simultaneously facilitates the systematic, rapid biological evaluation of modified siRNA.