Quantitative Measurement of Cytosolic and Nuclear Penetration of Oligonucleotide Therapeutics.

A major obstacle in the development of effective oligonucleotide therapeutics is a lack of understanding about their cytosolic and nuclear penetration. To address this problem, we have applied the chloroalkane penetration assay (CAPA) to oligonucleotide therapeutics. CAPA was used to quantitate cytosolic delivery of antisense oligonucleotides (ASOs) and siRNAs and to explore the effects of a wide variety of commonly used chemical modifications and their patterning. We evaluated potential artifacts by exploring the effects of serum, comparing activity data and CAPA data, and assessing the impact of the chloroalkane tag and its linker chemistry. We also used viral transduction to expand CAPA to the nuclear compartment in epithelial and neuronal cell lines. Using this enhanced method, we measured a 48-h time course of nuclear penetration for a panel of chemically diverse modified RNAs. Moving forward, CAPA will be a useful tool for deconvoluting the complex processes of endosomal uptake, escape into the cytosol, and subcellular trafficking of oligonucleotide therapeutics in therapeutically relevant cell types.

Cytosolic delivery of peptidic STAT3 SH2 domain inhibitors.

The signal transducer and activator of transcription 3 (STAT3) protein is constitutively activated in several cancers. STAT3 activity can be blocked by inhibiting its Src Homology 2 (SH2) domain, but phosphotyrosine and its isosteres have poor bioavailability. In this work, we develop peptide-based inhibitors of STAT3-SH2 by combining chemical strategies that have proven effective for targeting other SH2 domains. These strategies include a STAT3-specific selectivity sequence, non-hydrolyzable phosphotyrosine isosteres, and a high-efficiency cell-penetrating peptide. Peptides that combined these three strategies had substantial biological stability and cytosolic delivery, as measured using highly quantitative cell-based assays. However, these peptides did not inhibit STAT3 activity in cells. By comparing in vitro binding affinity, cell penetration, and proteolytic stability, this work explores the delicate balance of factors that contribute to biological activity for peptidic inhibitors of STAT3.

Stapled Peptide Inhibitors of Autophagy Adapter LC3B.

A growing body of evidence suggests that autophagy inhibition enhances the effectiveness of chemotherapy, especially in difficult-to-treat cancers. Existing autophagy inhibitors are primarily lysosomotropic agents. More specific autophagy inhibitors are highly sought-after. The microtubule-associated protein 1A/1B light chain 3B protein, LC3B, is an adapter protein that mediates key protein-protein interactions at several points in autophagy pathways. In this work, we used a known peptide ligand as a starting point to develop improved LC3B inhibitors. We obtained structure-activity relationships that quantify the binding contributions of peptide termini, individual charged residues, and hydrophobic interactions. Based on these data, we used artificial amino acids and diversity-oriented stapling to improve affinity and resistance to biological degradation, while maintaining or improving LC3B affinity and selectivity. These peptides represent the highest-affinity LC3B-selective ligands reported to date, and they will be useful tools for further elucidation of LC3B's role in autophagy and in cancer.

Small-Molecule Inhibitors of Haemophilus influenzae IgA1 Protease.

Newly identified, nontypable Haemophilus influenzae (H. influenza) strains represent a serious threat to global health. Due to the increasing prevalence of antibiotic resistance, virulence factors have emerged as potential therapeutic targets that would be less likely to promote resistance. IgA1 proteases are secreted virulence factors of many Gram-negative human pathogens. These enzymes play important roles in tissue invasion as well as evasion of the immune response, yet there has been limited work on pharmacological inhibitors. Here, we report the discovery of the first small molecule, nonpeptidic inhibitors of H. influenzae IgA1 proteases. We screened over 47 000 compounds in a biochemical assay using recombinant protease and identified a hit compound with micromolar potency. Preliminary structure-activity relationships produced additional inhibitors, two of which showed improved inhibition and selectivity for IgA protease over other serine proteases. We further showed dose-dependent inhibition against four different IgA1 protease variants collected from clinical isolates. These data support further development of IgA protease inhibitors as potential therapeutics for antibiotic-resistant H. influenza strains. The newly discovered inhibitors also represent valuable probes for exploring the roles of these proteases in bacterial colonization, invasion, and infection of mucosal tissues.

Microfluidic Chemical Cytometry for Enzyme Assays of Single Cells.

Cellular heterogeneity occurs, and should be probed, at multiple levels of cellular structure and physiology from the genome to enzyme activity. In particular, single-cell measures of protein levels are complemented by single-cell measurements of the activity of these proteins. Microfluidic assays of enzyme activity at the single-cell level combine moderate to high throughput with low dead volumes and the potential for automation. Herein, we describe the steps required to fabricate and operate a microfluidic device for chemical cytometry of fluorescent or fluorogenic reporters of enzyme activity in individual cells.