All-in-one disulfide bridging enables the generation of antibody conjugates with modular cargo loading.

Antibody-drug conjugates (ADCs) are valuable therapeutic entities which leverage the specificity of antibodies to selectively deliver cytotoxins to antigen-expressing targets such as cancer cells. However, current methods for their construction still suffer from a number of shortcomings. For instance, using a single modification technology to modulate the drug-to-antibody ratio (DAR) in integer increments while maintaining homogeneity and stability remains exceptionally challenging. Herein, we report a novel method for the generation of antibody conjugates with modular cargo loading from native antibodies. Our approach relies on a new class of disulfide rebridging linkers, which can react with eight cysteine residues, thereby effecting all-in-one bridging of all four interchain disulfides in an IgG1 antibody with a single linker molecule. Modification of the antibody with the linker in a 1 : 1 ratio enabled the modulation of cargo loading in a quick and selective manner through derivatization of the linker with varying numbers of payload attachment handles to allow for attachment of either 1, 2, 3 or 4 payloads (fluorescent dyes or cytotoxins). Assessment of the biological activity of these conjugates demonstrated their exceptional stability in human plasma and utility for cell-selective cytotoxin delivery or imaging/diagnostic applications.

New approaches to moderate CRISPR-Cas9 activity: Addressing issues of cellular uptake and endosomal escape.

CRISPR-Cas9 is rapidly entering molecular biology and biomedicine as a promising gene-editing tool. A unique feature of CRISPR-Cas9 is a single-guide RNA directing a Cas9 nuclease toward its genomic target. Herein, we highlight new approaches for improving cellular uptake and endosomal escape of CRISPR-Cas9. As opposed to other recently published works, this review is focused on non-viral carriers as a means to facilitate the cellular uptake of CRISPR-Cas9 through endocytosis. The majority of non-viral carriers, such as gold nanoparticles, polymer nanoparticles, lipid nanoparticles, and nanoscale zeolitic imidazole frameworks, is developed with a focus toward optimizing the endosomal escape of CRISPR-Cas9 by taking advantage of the acidic environment in the late endosomes. Among the most broadly used methods for in vitro and ex vivo ribonucleotide protein transfection are electroporation and microinjection. Thus, other delivery formats are warranted for in vivo delivery of CRISPR-Cas9. Herein, we specifically revise the use of peptide and nanoparticle-based systems as platforms for CRISPR-Cas9 delivery in vivo. Finally, we highlight future perspectives of the CRISPR-Cas9 gene-editing tool and the prospects of using non-viral vectors to improve its bioavailability and therapeutic potential.

Enhanced target cell specificity and uptake of lipid nanoparticles using RNA aptamers and peptides.

Lipid nanoparticles (LNPs) constitute a facile and scalable approach for delivery of payloads to human cells. LNPs are relatively immunologically inert and can be produced in a cost effective and scalable manner. However, targeting and delivery of LNPs across the blood-brain barrier (BBB) has proven challenging. In an effort to target LNPs composed of an ionizable cationic lipid (DLin-MC3-DMA), cholesterol, the phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) to particular cell types, as well as to generate LNPs that can cross the BBB, we developed and assessed two approaches. The first was centered on the BBB-penetrating trans-activator of transcription (Tat) peptide or the peptide T7, and the other on RNA aptamers targeted to glycoprotein gp160 from human immunodeficiency virus (HIV) or C-C chemokine receptor type 5 (CCR5), a HIV-1 coreceptor. We report herein a CCR5-selective RNA aptamer that acts to facilitate entry through a simplified BBB model and that drives the uptake of LNPs into CCR5-expressing cells, while the gp160 aptamer did not. We further observed that the addition of cell-penetrating peptides, Tat and T7, did not increase BBB penetration above the aptamer-loaded LNPs alone. Moreover, we found that these targeted LNPs exhibit low immunogenic and low toxic profiles and that targeted LNPs can traverse the BBB to potentially deliver drugs into the target tissue. This approach highlights the usefulness of aptamer-loaded LNPs to increase target cell specificity and potentially deliverability of central-nervous-system-active RNAi therapeutics across the BBB.

Production of allergen-specific immunotherapeutic agents for the treatment of food allergy.

Allergen-specific immunotherapy (IT) is emerging as a viable avenue for the treatment of food allergies. Clinical trials currently investigate raw or slightly processed foods as therapeutic agents, as trials using food-grade agents can be performed without the strict regulations to which conventional drugs are subjected. However, this limits the ability of standardization and may affect clinical trial outcomes and reproducibility. Herein, we provide an overview of methods used in the production of immunotherapeutic agents for the treatment of food allergies, including processed foods, allergen extracts, recombinant allergens, and synthetic peptides, as well as the physical and chemical processes for the reduction of protein allergenicity. Commercial interests currently favor producing standardized drug-grade allergen extracts for therapeutic use, and clinical trials are ongoing. In the near future, recombinant production could replace purification strategies since it allows the manufacturing of pure, native allergens or sequence-modified allergens with reduced allergenicity. A recurring issue within this field is the inadequate reporting of production procedures, quality control, product physicochemical characteristics, allergenicity, and immunological properties. This information is of vital importance in assessing therapeutic standardization and clinical safety profile, which are central parameters for the development of future therapeutic agents.

Structure-Activity Relationship Studies of Tetrahydroquinolone Free Fatty Acid Receptor 3 Modulators.

Free fatty acid receptor 3 (FFA3, previously GPR41) is activated by short-chain fatty acids, mediates health effects of the gut microbiota, and is a therapeutic target for metabolic and inflammatory diseases. The shortage of well-characterized tool compounds has however impeded progress. Herein, we report structure-activity relationship of an allosteric modulator series and characterization of physicochemical and pharmacokinetic properties of selected compounds, including previous and new tools. Two representatives, 57 (TUG-1907) and 63 (TUG-2015), showed improved solubility and preserved potency. Of these, 57, with EC50 = 145 nM and a solubility of 33 µM, showed high clearance in vivo but is a preferred tool in vitro. In contrast, 63, with EC50 = 162 nM and a solubility of 9 µM, showed lower clearance and seems better suited for in vivo studies. Using 57, we demonstrate for the first time that FFA3 activation leads to calcium mobilization in murine dorsal root ganglia.

Lipidated Polyaza Crown Ethers as Membrane Anchors for DNA-Controlled Content Mixing between Liposomes.

The ability to manipulate and fuse nano-compartmentalized volumes addresses a demand for spatiotemporal control in the field of synthetic biology, for example in the bottom-up construction of (bio)chemical nanoreactors and for the interrogation of enzymatic reactions in confined space. Herein, we mix entrapped sub-attoliter volumes of liposomes (~135 nm diameter) via lipid bilayer fusion, facilitated by the hybridization of membrane-anchored lipidated oligonucleotides. We report on an improved synthesis of the membrane-anchor phosphoramidites that allows for a flexible choice of lipophilic moiety. Lipid-nucleic acid conjugates (LiNAs) with and without triethylene glycol spacers between anchor and the 17 nt binding sequence were synthesized and their fusogenic potential evaluated. A fluorescence-based content mixing assay was employed for kinetic monitoring of fusion of the bulk liposome populations at different temperatures. Data obtained at 50 °C indicated a quantitative conversion of the limiting liposome population into fused liposomes and an unprecedently high initial fusion rate was observed. For most conditions and designs only low leakage during fusion was observed. These results consolidate LiNA-mediated membrane fusion as a robust platform for programming compartmentalized chemical and enzymatic reactions.

Control of LDL Uptake in Human Cells by Targeting the LDLR Regulatory Long Non-coding RNA BM450697.

Hypercholesterolemia is a condition that is characterized by very high levels of cholesterol in the blood and is a major correlating factor with heart disease. Indeed, high levels of the low-density lipoprotein (LDL) have been causally linked to the development of atherosclerotic cardiovascular disease (ASCVD). A method to specifically reduce cholesterol in the blood in a long-term, stable manner could prove therapeutically relevant. Cholesterol is removed from the blood by the LDL receptor (LDLR) in the liver. Others and we have discovered that a long non-coding RNA (lncRNA; BM450697) functions as an endogenous epigenetic regulator of LDLR and that the repression of this lncRNA by the action of small interfering RNAs (siRNAs) results in the activation of LDLR. We found here, through the interrogation of two siRNAs that can target this lncRNA, both in a transcriptional and post-transcriptional manner, that BM450697 functions as a local scaffold for modulating LDLR transcription. Moreover, we found that conjugation of α-N-acetylgalactosamine (GalNAc) with two lncRNA-directed siRNAs allows for direct liver cell targeting of this lncRNA and functional enhanced uptake of cholesterol. Collectively, these data suggest that targeting the BM450697 lncRNA regulator of LDLR may result in a more specific, long-term, targeted approach to regulating cholesterol in the blood.

Discovery of a Potent Thiazolidine Free Fatty Acid Receptor 2 Agonist with Favorable Pharmacokinetic Properties.

Free fatty acid receptor 2 (FFA2/GPR43) is a receptor for short-chain fatty acids reported to be involved in regulation of metabolism, appetite, fat accumulation, and inflammatory responses and is a potential target for treatment of various inflammatory and metabolic diseases. By bioisosteric replacement of the central pyrrolidine core of a previously disclosed FFA2 agonist with a synthetically more tractable thiazolidine, we were able to rapidly synthesize and screen analogues modified at both the 2- and 3-positions on the thiazolidine core. Herein, we report SAR exploration of thiazolidine FFA2 agonists and the identification of 31 (TUG-1375), a compound with significantly increased potency (7-fold in a cAMP assay) and reduced lipophilicity (50-fold reduced clog P) relative to the pyrrolidine lead structure. The compound has high solubility, high chemical, microsomal, and hepatocyte stability, and favorable pharmacokinetic properties and was confirmed to induce human neutrophil mobilization and to inhibit lipolysis in murine adipocytes.

Studies of Impending Oligonucleotide Therapeutics in Simulated Biofluids.

Synthetic oligonucleotides, their complexes and conjugates with other biomolecules represent valuable research tools and therapeutic agents. In spite of growing applications in basic research and clinical science, only few studies have addressed the issue of such compounds' stability in biological media. Herein, we studied the stability of two therapeutically relevant oligonucleotide probes in simulated biofluids; the 21 nucleotide-long DNA/locked nucleic acid oligonucleotide ON targeted toward cancer-associated BRAF V600E mutation, and a longer DNA analog (TTC) originating from BRAF gene. We found that stability of peptide-oligonucleotide conjugates (POCs) in human serum (HS) was superior compared with the naked or complexed 21mer oligonucleotide, whereas stability of POCs in simulated gastric juice (GJ) was dependent on the peptide sequence. Addition of pepstatin A in general increased the stability of oligonucleotides after 24 h digestion in HS and simulated GJ. Similarly, complexation with optimal amounts of histone proteins was found to rescue oligonucleotide stability after 24 h digestion in hydrochloric acid.

A single extracellular amino acid in Free Fatty Acid Receptor 2 defines antagonist species selectivity and G protein selection bias.

Free Fatty Acid Receptor 2 is a GPCR activated by short chain fatty acids produced in high levels in the lower gut by microbial fermentation of non-digestible carbohydrates. A major challenge in studying this receptor is that the mouse ortholog does not have significant affinity for antagonists that are able to block the human receptor. Docking of exemplar antagonists from two chemical series to homology models of both human and mouse Free Fatty Acid Receptor 2 suggested that a single lysine - arginine variation at the extracellular face of the receptor might provide the basis for antagonist selectivity and mutational swap studies confirmed this hypothesis. Extending these studies to agonist function indicated that although the lysine - arginine variation between human and mouse orthologs had limited effect on G protein-mediated signal transduction, removal of positive charge from this residue produced a signalling-biased variant of Free Fatty Acid Receptor 2 in which Gi-mediated signalling by both short chain fatty acids and synthetic agonists was maintained whilst there was marked loss of agonist potency for signalling via Gq/11 and G12/13 G proteins. A single residue at the extracellular face of the receptor thus plays key roles in both agonist and antagonist function.

Development and Characterization of a Fluorescent Tracer for the Free Fatty Acid Receptor 2 (FFA2/GPR43).

The free fatty acid receptor 2 (FFA2/GPR43) is considered a potential target for treatment of metabolic and inflammatory diseases. Here we describe the development of the first fluorescent tracer for FFA2 intended as a tool for assessment of thermodynamic and kinetic binding parameters of unlabeled ligands. Starting with a known azetidine FFA2 antagonist, we used a carboxylic acid moiety known not to be critical for receptor interaction as attachment point for a nitrobenzoxadiazole (NBD) fluorophore. This led to the development of 4 (TUG-1609), a fluorescent tracer for FFA2 with favorable spectroscopic properties and high affinity, as determined by bioluminescence resonance energy transfer (BRET)-based saturation and kinetic binding experiments, as well as a high specific to nonspecific BRET binding signal. A BRET-based competition binding assay with 4 was also established and used to determine binding constants and kinetics of unlabeled ligands.

Optimization and modeling of the remote loading of luciferin into liposomes.

We carried out a mechanistic study to characterize and optimize the remote loading of luciferin into preformed liposomes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPC/DPPG) 7:3 mixtures. The influence of the loading agent (acetate, propionate, butyrate), the metal counterion (Na(+), K(+), Ca(+2), Mg(+2)), and the initial extra-liposomal amount of luciferin (nL(add)) on the luciferin Loading Efficiency (LE%) and luciferin-to-lipid weight ratio, i.e., Loading Capacity (LC), in the final formulation was determined. In addition, the effect of the loading process on the colloidal stability and phase behavior of the liposomes was monitored. Based on our experimental results, a theoretical model was developed to describe the course of luciferin remote loading. It was found that the highest luciferin loading was obtained with magnesium acetate. The use of longer aliphatic carboxylates or inorganic proton donors pronouncedly reduced luciferin loading, whereas the effect of the counterion was modest. The remote-loading process barely affected the colloidal stability and drug retention of the liposomes, albeit with moderate luciferin-induced membrane perturbations. The correlation between luciferin loading, expressed as LE% and LC, and nL(add) was established, and under our conditions the maximum LC was attained using an nL(add) of around 2.6µmol. Higher amounts of luciferin tend to pronouncedly perturb the liposome stability and luciferin retention. Our theoretical model furnishes a fair quantitative description of the correlation between nL(add) and luciferin loading, and a membrane permeability coefficient for uncharged luciferin of 1×10(-8)cm/s could be determined. We believe that our study will prove very useful to optimize the remote-loading strategies of moderately polar carboxylic acid drugs in general.

Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer.

The free fatty acid receptor 1 (FFA1/GPR40) is a potential target for treatment of type 2 diabetes. Although several potent agonists have been described, there remains a strong need for suitable tracers to interrogate ligand binding to this receptor. We address this by exploring fluorophore-tethering to known potent FFA1 agonists. This led to the development of 4, a high affinity FFA1 tracer with favorable and polarity-dependent fluorescent properties. A close to ideal overlap between the emission spectrum of the NanoLuciferase receptor tag and the excitation spectrum of 4 enabled the establishment of a homogeneous BRET-based binding assay suitable for both detailed kinetic studies and high throughput competition binding studies. Using 4 as a tracer demonstrated that the compound acts fully competitively with selected synthetic agonists but not with lauric acid and allowed for the characterization of binding affinities of a diverse selection of known FFA1 agonists, indicating that 4 will be a valuable tool for future studies at FFA1.

Non-equivalence of Key Positively Charged Residues of the Free Fatty Acid 2 Receptor in the Recognition and Function of Agonist Versus Antagonist Ligands.

Short chain fatty acids (SCFAs) are produced in the gut by bacterial fermentation of poorly digested carbohydrates. A key mediator of their actions is the G protein-coupled free fatty acid 2 (FFA2) receptor, and this has been suggested as a therapeutic target for the treatment of both metabolic and inflammatory diseases. However, a lack of understanding of the molecular determinants dictating how ligands bind to this receptor has hindered development. We have developed a novel radiolabeled FFA2 antagonist to probe ligand binding to FFA2, and in combination with mutagenesis and molecular modeling studies, we define how agonist and antagonist ligands interact with the receptor. Although both agonist and antagonist ligands contain negatively charged carboxylates that interact with two key positively charged arginine residues in transmembrane domains V and VII of FFA2, there are clear differences in how these interactions occur. Specifically, although agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two. Moreover, different chemical series of antagonist interact preferentially with different arginine residues. A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor.

Complex pharmacology of novel allosteric free fatty acid 3 receptor ligands.

Analysis of the roles of the short chain fatty acid receptor, free fatty acid 3 receptor (FFA3), has been severely limited by the low potency of its endogenous ligands, the crossover of function of these on the closely related free fatty acid 2 receptor, and a dearth of FFA3-selective synthetic ligands. From a series of hexahydroquinolone-3-carboxamides, we demonstrate that 4-(furan-2-yl)-2-methyl-5-oxo-N-(o-tolyl)-1,4,5,6,7,8-hexahydroquinoline-3-carboxamide is a selective and moderately potent positive allosteric modular (PAM)-agonist of the FFA3 receptor. Modest chemical variations within this series resulted in compounds completely lacking activity, acting as FFA3 PAMs, or appearing to act as FFA3-negative allosteric modulators. However, the pharmacology of this series was further complicated in that certain analogs displaying overall antagonism of FFA3 function actually appeared to generate their effects via a combined positive allosteric binding cooperativity and negative allosteric effect on orthosteric ligand maximal signaling response. These studies show that various PAM-agonist and allosteric modulators of FFA3 can be identified and characterized. However, within the current chemical series, considerable care must be taken to define the pharmacological characteristics of specific compounds before useful predictions of their activity and their use in defining specific roles of FFA3 in either in vitro and in vivo settings can be made.

Propofol modulates the lipid phase transition and localizes near the headgroup of membranes.

The compound 2,6-diisopropylphenol (Propofol, PRF) is widely used for inducing general anesthesia, but the mechanism of PRF action remains relatively poorly understood at the molecular level. This work examines the possibility that a potential mode of action of PRF is to modulate the lipid order in target membranes. The effect on monolayers and bilayers of dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) was probed using Langmuir monolayer isotherms, differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations. Increasing amounts of PRF in a DPPC monolayer causes a decrease in isothermal compressibility modulus at the phase transition. A partition constant for PRF in DPPC liposomes on the order of K≈1500 M(-1) was found, and the partitioning was found to be enthalpy-driven above the melting temperature (Tm). A decrease in Tm with PRF content was found whereas the bilayer melting enthalpy ΔHm remains almost constant. The last finding indicates that PRF incorporates into the membrane at a depth near the phosphatidylcholine headgroup, in agreement with our MD-simulations. The simulations also reveal that PRF partitions into the membrane on a timescale of 0.5 µs and has a cholesterol-like ordering effect on DPPC in the fluid phase. The vertical location of the PRF binding site in a bacterial ligand-gated ion channel coincides with the location found in our MD-simulations. Our results suggest that multiple physicochemical mechanisms may determine anesthetic potency of PRF, including effects on proteins that are mediated through the bilayer.