Infrared Reflection-Absorption Spectroscopy of α-Keto Acids at the Air-Water Interface: Effects of Chain Length and Headgroup on Environmentally Relevant Surfactant Films.

A variety of organic surfactants are found at air-water interfaces in natural environments, including on the surfaces of aqueous aerosols. The structure and morphology of these organic films can have profound impacts on material transfer between the gas and condensed phases, the optical properties of atmospheric aerosol, and chemical processing at air-water interfaces. Combined, these effects can have significant impacts on climate via radiative forcing, but our understanding of organic films at air-water interfaces is incomplete. Here, we examine the impact of the polar headgroup and alkyl tail length on the structure and morphology of organic monolayers at the air-water interfaces. First, we focus on the substituted carboxylic acids, α-keto acids, using Langmuir isotherms and infrared reflection absorption spectroscopy (IR-RAS) to elucidate key structures and phase behaviors of α-keto acids with a range of surface activities. We show that the structure of α-keto acids, both soluble and insoluble, at water surfaces is a compromise between van der Waals interactions of the hydrocarbon tail and hydrogen bonding interactions involving the polar headgroup. Then, we use this new data set regarding α-keto acid films at water surfaces to examine the role of the polar headgroup on organic films using a similar substituted carboxylic acid (α-hydroxystearic acid), an unsubstituted carboxylic acid (stearic acid), and an alcohol (stearyl alcohol). We show that the polar headgroup and its hydrogen bonding interactions can significantly affect the orientation of amphiphiles at air-water interfaces. Here, we provide side-by-side comparisons of Langmuir isotherms and IR-RA spectra for a set of environmentally relevant organic amphiphiles with a range of alkyl tail lengths and polar headgroup structures.

Fluorogenic monomer activation for protein-initiated atom transfer radical polymerization.

Fluorogenic atom transfer radical polymerization (ATRP) directly detects initiator-dependent polymer formation, as initially non-fluorescent polycyclic aromatic probe monomers reveal visible fluorescence upon polymerization in real time. Advancement of this initial proof-of-concept toward biodetection applications requires both a more detailed mechanistic understanding of probe fluorescence activation, and the ability to initiate fluorogenic polymerization directly from a biomolecule surface. Here, we show that simple monomer hydrogenation, independent of polymerization, reveals probe fluorescence, supporting the critical role of covalent enone attachment in fluorogenic probe quenching and subsequent fluorescence activation. We next demonstrate bioorthogonal, protein-initiated fluorogenic ATRP by the surface conjugation and characterization of protein-initiator conjugates of a model protein, bovine serum albumin (BSA). Fluorogenic ATRP from initiator-modified protein allows for real-time visualization of polymer formation with negligible background fluorescence from unmodified BSA controls. We further probe the bioorthogonality of this fluorogenic ATRP assay by assessing polymer formation in a complex biological environment, spiked with fetal bovine serum. Taken together, we demonstrate the potential of aqueous fluorogenic ATRP as a robust, bioorthogonal method for biomolecular-initiated polymerization by real-time fluorescence activation.

Pharmacologic IRE1/XBP1s activation confers targeted ER proteostasis reprogramming.

Activation of the IRE1/XBP1s signaling arm of the unfolded protein response (UPR) is a promising strategy to correct defects in endoplasmic reticulum (ER) proteostasis implicated in diverse diseases. However, no pharmacologic activators of this pathway identified to date are suitable for ER proteostasis remodeling through selective activation of IRE1/XBP1s signaling. Here, we use high-throughput screening to identify non-toxic compounds that induce ER proteostasis remodeling through IRE1/XBP1s activation. We employ transcriptional profiling to stringently confirm that our prioritized compounds selectively activate IRE1/XBP1s signaling without activating other cellular stress-responsive signaling pathways. Furthermore, we demonstrate that our compounds improve ER proteostasis of destabilized variants of amyloid precursor protein (APP) through an IRE1-dependent mechanism and reduce APP-associated mitochondrial toxicity in cellular models. These results establish highly selective IRE1/XBP1s activating compounds that can be widely employed to define the functional importance of IRE1/XBP1s activity for ER proteostasis regulation in the context of health and disease.

Reactive Oxygen Species (ROS)-Activatable Prodrug for Selective Activation of ATF6 after Ischemia/Reperfusion Injury.

We describe here the design, synthesis, and biological evaluation of a reactive oxygen species (ROS)-activatable prodrug for the selective delivery of 147, a small molecule ATF6 activator, for ischemia/reperfusion injury. ROS-activatable prodrug 1 and a negative control unable to release free drug were synthesized and examined for peroxide-mediated activation. Prodrug 1 blocks activity of 147 by its inability to undergo metabolic oxidation by ER-resident cytochrome P450 enzymes such as Cyp1A2, probed directly here for the first time. Biological evaluation of ROS-activatable prodrug 1 in primary cardiomyocytes demonstrates protection against peroxide-mediated toxicity and enhances viability following simulated I/R injury. The ability to selectively target ATF6 activation under diseased conditions establishes the potential for localized stress-responsive signaling pathway activation as a therapeutic approach for I/R injury and related protein misfolding maladies.

Fluorogenic atom transfer radical polymerization in aqueous media as a strategy for detection.

The development of novel approaches to signal amplification in aqueous media could enable new diagnostic platforms for the detection of water-soluble analytes, including biomolecules. This paper describes a fluorogenic polymerization approach to amplify initiator signal by the detection of visible fluorescence upon polymerization in real-time. Fluorogenic monomers were synthesized and co-polymerized by atom transfer radical polymerization (ATRP) in water to reveal increasing polymer fluorescence as a function of both reaction time and initiator concentration. Optimization of the fluorogenic ATRP reaction conditions allowed for the quantitative detection of a small-molecule initiator as a model analyte over a broad linear concentration range (pM to mM). Raising the reaction temperature from 30 °C to 60 °C facilitated sensitive initiator detection at sub-picomolar concentrations in as little as 1 h of polymerization. This method was then applied to the detection of streptavidin as a model biological analyte by fluorogenic polymerization from a designed biotinylated ATRP initiator. Taken together, these studies represent the first example of a fluorogenic ATRP reaction and establish fluorogenic polymerization as a promising approach for the direct detection of aqueous analytes and biomolecular recognition events.

Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation.

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases.

Unfolded protein response activation reduces secretion and extracellular aggregation of amyloidogenic immunoglobulin light chain.

Light-chain amyloidosis (AL) is a degenerative disease characterized by the extracellular aggregation of a destabilized amyloidogenic Ig light chain (LC) secreted from a clonally expanded plasma cell. Current treatments for AL revolve around ablating the cancer plasma cell population using chemotherapy regimens. Unfortunately, this approach is limited to the ∼ 70% of patients who do not exhibit significant organ proteotoxicity and can tolerate chemotherapy. Thus, identifying new therapeutic strategies to alleviate LC organ proteotoxicity should allow AL patients with significant cardiac and/or renal involvement to subsequently tolerate established chemotherapy treatments. Using a small-molecule screening approach, the unfolded protein response (UPR) was identified as a cellular signaling pathway whose activation selectively attenuates secretion of amyloidogenic LC, while not affecting secretion of a nonamyloidogenic LC. Activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the absence of stress recapitulates the selective decrease in amyloidogenic LC secretion by remodeling the endoplasmic reticulum proteostasis network. Stress-independent activation of XBP1s, or especially ATF6, also attenuates extracellular aggregation of amyloidogenic LC into soluble aggregates. Collectively, our results show that stress-independent activation of these adaptive UPR transcription factors offers a therapeutic strategy to reduce proteotoxicity associated with LC aggregation.

Broadly applicable methodology for the rapid and dosable small molecule-mediated regulation of transcription factors in human cells.

Direct and selective small molecule control of transcription factor activity is an appealing avenue for elucidating the cell biology mediated by transcriptional programs. However, pharmacologic tools to modulate transcription factor activity are scarce because transcription factors are not readily amenable to small molecule-mediated regulation. Moreover, existing genetic approaches to regulate transcription factors often lead to high nonphysiologic levels of transcriptional activation that significantly impair our ability to understand the functional implications of transcription factor activity. Herein, we demonstrate that small molecule-mediated conformational control of protein degradation is a generally applicable, chemical biological methodology to obtain small molecule-regulated transcription factors that modulate transcriptional responses at physiologic levels in human cells. Our establishment of this approach allows for the rapid development of genetically encoded, small molecule-regulated transcription factors to explore the biologic and therapeutic impact of physiologic levels of transcription factor activity in cells.

Designed guanidinium-rich amphipathic oligocarbonate molecular transporters complex, deliver and release siRNA in cells.

The polyanionic nature of oligonucleotides and their enzymatic degradation present challenges for the use of siRNA in research and therapy; among the most notable of these is clinically relevant delivery into cells. To address this problem, we designed and synthesized the first members of a new class of guanidinium-rich amphipathic oligocarbonates that noncovalently complex, deliver, and release siRNA in cells, resulting in robust knockdown of target protein synthesis in vitro as determined using a dual-reporter system. The organocatalytic oligomerization used to synthesize these co-oligomers is step-economical and broadly tunable, affording an exceptionally quick strategy to explore chemical space for optimal siRNA delivery in varied applications. The speed and versatility of this approach and the biodegradability of the designed agents make this an attractive strategy for biological tool development, imaging, diagnostics, and therapeutic applications.

Beyond Cell Penetrating Peptides: Designed Molecular Transporters.

Inspired originally by peptides that traverse biological barriers, research on molecular transporters has since identified the key structural requirements that govern cellular entry, leading to new, significantly more effective and more readily available agents. These new drug delivery systems enable or enhance cellular and tissue uptake, can be targeted, and provide numerous additional advantages of significance in imaging, diagnostics and therapy.

Oligocarbonate molecular transporters: oligomerization-based syntheses and cell-penetrating studies.

A new family of guanidinium-rich molecular transporters featuring a novel oligocarbonate backbone with 1,7-side chain spacing is described. Conjugates can be rapidly assembled irrespective of length in a one-step oligomerization strategy that can proceed with concomitant introduction of probes (or by analogy drugs). The new transporters exhibit excellent cellular entry as determined by flow cytometry and fluorescence microscopy, and the functionality of their drug delivery capabilities was confirmed by the delivery of the bioluminescent small molecule probe luciferin and turnover by its intracellular target enzyme.