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Cyclic peptides are an emerging therapeutic modality that can target protein-protein interaction sites with high affinity and selectivity. A common medicinal chemistry strategy for the optimization of peptide hits is conformational stabilization through macrocyclization. We present a method based on explicit solvent enhanced sampling molecular dynamics simulations for estimating the impact of varying linker lengths and chemistry on the conformational stability of a peptide. The method is demonstrated on three cyclic peptide series that bind to proteins PCSK9, trypsin, and MDM2 adopting loop, β-sheet, and helical secondary structures. In general, the simulations show greater solution stability of the receptor-bound conformation for the higher-affinity peptides, consistent with the idea that preorganizing a ligand for binding can enhance binding affinity. The impact of the force field and sampling is discussed for one series that does not follow this trend. We have successfully applied this method to internal discovery programs to design peptides with increased potency and chemical stability.

Background: Mechanisms triggering the pathogenesis of chronic spontaneous urticaria (CSU) have been identified as type I autoallergic (which is associated with IgE antibodies against autoantigens) and type IIb autoimmune (which is driven by autoantibodies to FceR1 and/or IgE)., Objective: Our aim was to define presumptive endotypes in patients with CSU by using tests amenable to use in routine clinical practice., Methods: A retrospective analysis of the medical records of 394 patients with CSU with or without chronic inducible urticaria or angioedema was performed. Patients were assigned to 1 of 4 groups as follows: (1) type I endotype of CSU, if they presented at least 1 of the following: allergic disease, total IgE level of at least 40UI/mL, and positive result of skin tests to inhalant allergen(s), (2) type IIb endotype of CSU, if they presented at least 1 of following: autoimmune disease, low total IgE level less than 40 IU/mL, positive autologous serum skin test result, positive for antinuclear antibodies in a titer of at least 1:160, and elevated level of anti-thyroid peroxidase, (3) overlap of type I/type IIb endotypes of CSU, if they presented with at least 1 marker of both type I and type IIb, and (4) non-type I/type IIb endotype of CSU, if they presented with none of the markers of type I or type IIb., Results: The mean age at onset of symptoms was 34 years; 82.2% of those with CSU were female, and angioedema and chronic inducible urticaria were found in 74.8% and 31.9% of patients, respectively. Of the patients with CSU, 38% presented with the type I endotype and 51% presented with type I/type IIb overlap, whereas 9% presented with the type IIb endotype and 2% presented with the non-type I/type IIb endotype. Eosinopenia was associated with type IIb and type I/type IIb overlap as opposed to the type I and non-type I/type IIb endotypes ( P  = .02)., Conclusions: Most patients with CSU presented with features of the type 1 (autoallergic) endotype, whether associated with type IIb (autoimmune) endotype or not., (© 2023 The Author(s).)

Peptides and peptidomimetics represent the middle space between small molecules and large proteins-they retain the relatively small size and synthetic accessibility of small molecules while providing high binding specificity for biomolecular partners typically observed with proteins. During the course of our efforts to target intracellular protein-protein interactions in cancer, we observed that the cellular uptake of peptides is critically determined by the cell line-specifically, we noted that peptides show better uptake in cancer cells with enhanced macropinocytic indices. Here, we describe the results of our analysis of cellular penetration by different classes of conformationally stabilized peptides. We tested the uptake of linear peptides, peptide macrocycles, stabilized helices, β-hairpin peptides, and cross-linked helix dimers in 11 different cell lines. Efficient uptake of these conformationally defined constructs directly correlated with the macropinocytic activity of each cell line: high uptake of compounds was observed in cells with mutations in certain signaling pathways. Significantly, the study shows that constrained peptides follow the same uptake mechanism as proteins in macropinocytic cells, but unlike proteins, peptide mimics can be readily designed to resist denaturation and proteolytic degradation. Our findings expand the current understanding of cellular uptake in cancer cells by designed peptidomimetics and suggest that cancer cells with certain mutations are suitable mediums for the study of biological pathways with peptide leads.

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2'-F-monomer metabolites was low and transient in rats and humans. In vitro, 2'-F-nucleoside 5'-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2'-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2'-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2'-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Single-stranded (ss) 2'-fluoro (2'-F)-modified oligonucleotides (ONs) with a full phosphorothioate (PS) backbone have been reported to be cytotoxic and cause DNA double-strand breaks (DSBs) when transfected into HeLa cells. However, the molecular determinants of these effects have not been fully explored. In this study, we investigated the impact of ON structure, chemistry, delivery method, and cell type on in vitro cytotoxicity and DSBs. We found that ss PS-ONs were more cytotoxic than double-stranded (ds) PS-ONs, irrespective of the 2'-ribose chemistry, inclusive of the 2'-F modification. Cytotoxicity of ss ONs was most affected by the total PS content, with an additional contribution of 2'-F substitutions in HeLa, but not HepG2, cells. The relatively mild cytotoxicity of ds ONs was most impacted by long contiguous PS stretches combined with 2'-F substitutions. None of the tested ds 2'-F-modified PS-ONs caused DSBs, while the previously reported DSBs caused by ss 2'-F-modified PS-ONs were PS dependent. HeLa cells were more sensitive to ON-mediated toxicity when transfected with Lipofectamine 2000 versus Lipofectamine RNAiMax. Importantly, asialoglycoprotein receptor-mediated uptake of N-acetylgalactosamine-conjugated ss or ds PS-ONs, even those with long PS stretches and high 2'-F content, was neither cytotoxic nor caused DSBs at transfection-equivalent exposures. These results suggest that in vitro cytotoxicity and DSBs associated with ONs are delivery method dependent and primarily determined by single-stranded nature and PS content of ONs.

Registration of pharmaceuticals requires an assessment of their genotoxic potential using in vitro and in vivo tests outlined in the International Conference on Harmonisation (ICH) guidance S2(R1). We have evaluated numerous siRNA-N-acetylgalactosamine (GalNAc) conjugates containing phosphorothioate linkages and various combinations of 2'-fluoro and 2'-O-methyl ribose modifications of multiple nucleotides in the ICH battery of assays, all of which have uniformly yielded negative results. To verify these negative genotoxicity results, in this study we confirm test article exposure using toolkit small interfering RNAs (siRNAs) representative of those in the clinic. In the Ames test, the highest uptake of the siRNA-GalNAc conjugates occurred at 1 h postdose in all bacterial strains independent of siRNA sequence or chemistry (up to ∼14,000 siRNA molecules per cell), followed by metabolic degradation of the parent siRNA at 6, 24, and 48 h postdose. siRNA-GalNAc conjugates were internalized by bacteria as assessed by protection from the addition of nucleases to the culture media following uptake and by the requirement of cell lysis for detection of the siRNA. In the in vitro chromosome aberration assay, uptake was observed in Chinese hamster ovary cells (up to ∼5,500 siRNA molecules per cell at 21 h postdose) and in CD3 + human peripheral blood lymphocytes (up to ∼500 siRNA molecules per cell at 21 h postdose). In the in vivo micronucleus assay in rat bone marrow, exposure to parent siRNA was 100-350 μg of antisense strand per gram of protein at 24 and 48 h postlimit dose of 2 g/kg. Loss of terminal nucleotides was detected in bone marrow by mass spectrometry, indicating exposure to monomer metabolites as well. Negative genotoxicity results were also confirmed in an in vitro double-strand DNA break assay in HeLa and HepG2 cells where exposure was maximized using transfection reagents. Thus negative genotoxicity assay results for siRNA-GalNAc conjugates were valid and not the result of poor or no intracellular exposure.

Regulation of the heat shock response (HSR) is essential in all living systems. In E. coli, the HSR is regulated by an alternative σ factor, σ(32) , which is encoded by the rpoH gene. The mRNA of rpoH adopts a complex secondary structure that is critical for the proper translation of the σ(32) protein. At low temperatures, the rpoH gene transcript forms a highly structured mRNA containing several three-way junctions, including a rare perfectly paired three-way junction (3WJ). This complex secondary structure serves as a primitive but highly effective strategy for the thermal control of gene expression. In this work, the first small-molecule modulators of the E. coli σ(32) mRNA temperature sensor are reported., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recently, the utility of triptycene as a scaffold for targeting nucleic acid three-way junctions was demonstrated. A rapid, efficient route for the synthesis of bridgehead-substituted triptycenes is reported, in addition to solid-phase diversification to a new class of triptycene peptides. The triptycene peptides were evaluated for binding to a d(CAG)·(CTG) repeat DNA junction exhibiting potent affinities. The bridgehead-substituted triptycenes provide new building blocks for rapid access to diverse triptycene ligands with novel architectures.

The Oligonucleotide Safety Working Group subcommittee on genotoxicity testing considers therapeutic oligonucleotides (ONs) unlikely to be genotoxic based on their properties and on the negative results for ONs tested to date. Nonetheless, the subcommittee believes that genotoxicity testing of new ONs is warranted because modified monomers could be liberated from a metabolized ON and incorporated into DNA and could hypothetically cause chain termination, miscoding, and/or faulty replication or repair. The standard test battery as described in Option 1 of International Conference on Harmonisation S2(R1) is generally adequate to assess such potential. However, for the in vitro assay for gene mutations, mammalian cells are considered more relevant than bacteria for most ONs due to their known responsiveness to nucleosides and their greater potential for ON uptake; on the other hand, bacterial assays may be more appropriate for ONs containing non-ON components. Testing is not recommended for ONs with only naturally occurring chemistries or for ONs with chemistries for which there is documented lack of genotoxicity in systems with demonstrated cellular uptake. Testing is recommended for ONs that contain non-natural chemical modifications and use of the complete drug product (including linkers, conjugates, and liposomes) is suggested to provide the most clinically relevant assessment. Documentation of uptake into cells comparable to those used for genotoxicity testing is proposed because intracellular exposure cannot be assumed for these large molecules. ONs could also hypothetically cause mutations through triple helix formation with genomic DNA and no tests are available for detection of such sequence-specific mutations across the entire genome. However, because the potential for triplex formation by therapeutic ONs is extremely low, this potential can be assessed adequately by sequence analysis.

Triptycenes have been shown to bind nucleic acid three-way junctions, but rapid and efficient methods to diversify the triptycene core are lacking. An efficient synthesis of a 9-substituted triptycene scaffold is reported that can be used as a building block for solid-phase peptide synthesis and rapid diversification. The triptycene building block was diversified to produce a new class of tripeptide-triptycenes, and their binding abilities toward d(CAG)·(CTG) repeat junctions were investigated., Competing Interests: The authors declare no competing financial interest.

Nucleic acid three-way junctions (3WJs) play key roles in biological processes such as nucleic acid replication in addition to being implicated as dynamic transient intermediates in trinucleotide repeat sequences. Structural modulation of specific nucleic acid junctions could allow for control of biological processes and disease states at the nucleic acid level. Trinucleotide repeat expansions are associated with several neurodegenerative diseases where dynamic slippage is thought to occur during replication, forming transient 3WJ intermediates with the complementary strand. Here, we report triptycene-based molecules that bind to a d(CAG)·(CTG) repeat using a gel shift assay, fluorescence-quenching and circular dichroism.

One of the most challenging goals of modern synthetic chemistry is to develop multi-step reactions for rapid and efficient access to complex molecules. We report a triple aryne-tetrazine reaction that enables rapid access to a new class of polyaromatic heterocycles. This new reaction, which couples diverse reactivity modes between simple aryne and tetrazine starting materials, proceeds in a single operation and takes less than 5 minutes in air with no metal catalyst.

The modulation of nucleic acids by small molecules is an essential process across the kingdoms of life. Targeting nucleic acids with small molecules represents a significant challenge at the forefront of chemical biology. Nucleic acid junctions are ubiquitous structural motifs in nature and in designed materials. Herein, we describe a new class of structure-specific nucleic acid junction stabilizers based on a triptycene scaffold. Triptycenes provide significant stabilization of DNA and RNA three-way junctions, providing a new scaffold for the development of nucleic acid junction binders with enhanced recognition properties. Additionally, we report cytotoxicity and cell uptake data in two human ovarian carcinoma cell lines., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Formulation of short interfering RNA (siRNA) into multicomponent lipid nanoparticles (LNP) is an effective strategy for hepatic delivery and therapeutic gene silencing. This study systematically evaluated the effect of polyethylene glycol (PEG) density on LNP physicochemical properties, innate immune response stimulation, and in vivo efficacy. Increased PEG density not only shielded LNP surface charge but also reduced hemolytic activity, suggesting the formation of a steric barrier. In addition, increasing the PEG density reduced LNP immunostimulatory potential as reflected in cytokine induction both in vivo and in vitro. Higher PEG density also hindered in vivo efficacy, presumably due to reduced association with apolipoprotein E (ApoE), a protein which serves as an endogenous targeting ligand to hepatocytes. This effect could be overcome by incorporating an exogenous targeting ligand into the highly shielded LNPs, thereby circumventing the requirement for ApoE association. Therefore, these studies provide useful information for the rational design of LNP-based siRNA delivery systems with an optimal safety and efficacy profile.

siRNA therapeutics have promise for the treatment of a wide range of genetic disorders. Motivated by lipoproteins, we report lipopeptide nanoparticles as potent and selective siRNA carriers with a wide therapeutic index. Lead material cKK-E12 showed potent silencing effects in mice (ED50 ∼ 0.002 mg/kg), rats (ED50 < 0.01 mg/kg), and nonhuman primates (over 95% silencing at 0.3 mg/kg). Apolipoprotein E plays a significant role in the potency of cKK-E12 both in vitro and in vivo. cKK-E12 was highly selective toward liver parenchymal cell in vivo, with orders of magnitude lower doses needed to silence in hepatocytes compared with endothelial cells and immune cells in different organs. Toxicity studies showed that cKK-E12 was well tolerated in rats at a dose of 1 mg/kg (over 100-fold higher than the ED50). To our knowledge, this is the most efficacious and selective nonviral siRNA delivery system for gene silencing in hepatocytes reported to date.

The emerging class of RNA interference (RNAi) therapeutics is a fundamentally novel approach to treating human disease by enabling the pursuit of molecular targets considered "undruggable" by small molecules and traditional protein therapeutics. A key challenge toward realizing the full potential of this technology is the safe and efficient delivery of siRNA to target tissues. The physical chemical properties of siRNAs preclude passive diffusion across most cell membranes. For systemic administration, novel delivery systems are required to confer "drug-like" pharmacokinetic and pharmacodynamic properties. Engineered nanomaterials and the emerging field of nanomedicine are important drivers of turning the promise of RNAi therapeutics into reality. The current clinical progress of systemically administered siRNA therapeutics is reviewed, with special attention to the toxicity profiles associated with RNAi nanomedicines. As a case study, the preclinical development of ALN-VSP, the first lipid nanoparticle (LNP)-formulated siRNA therapeutic to be tested in cancer patients, is reviewed to broadly highlight some of the preclinical safety challenges and areas of investigation for "next generation" LNP systems., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.

Organic anion transporters (OATs) play a pivotal role in the clearance of small organic anions by the kidney, yet little is known about how their activity is regulated. A yeast two-hybrid assay was used to identify putative OAT3-associated proteins in the kidney. Atypical protein kinase Czeta (PKCzeta) was shown to bind to OAT3. Binding was confirmed in immunoprecipitation assays. The OAT3/PKCzeta interaction was investigated in rodent renal cortical slices from fasted animals. Insulin, an upstream activator of PKCzeta, increased both OAT3-mediated uptake of estrone sulfate (ES) and PKCzeta activity. Both effects were abolished by a PKCzeta-specific pseudosubstrate inhibitor. Increased ES transport was not observed in renal slices from OAT3-null mice. Transport of the shared OAT1/OAT3 substrate, rho-aminohippurate, behaved similarly, except that stimulation was reduced, not abolished, in the OAT3-null mice. This suggested that OAT1 activity was also modified by PKCzeta, subsequently confirmed using an OAT1-specific substrate, adefovir. Inhibition of PKCzeta also blocked the increase in ES uptake seen in response to epidermal growth factor and to activation of protein kinase A. Thus, PKCzeta acted downstream of the epidermal growth factor to protein kinase A signaling pathway. Activation of transport was accompanied by an increase in V(max) and was blocked by microtubule disruption, indicating that activation may result from trafficking of OAT3 into the plasma membrane. These data demonstrate that PKCzeta activation up-regulates OAT1 and OAT3 function, and that protein-protein interactions play a central role controlling these two important renal drug transporters.

The failure of drug candidates during clinical trials due to toxicity, especially hepatotoxicity, is an important and continuing problem in the pharmaceutical industry. This chapter explores new predictive toxicogenomics approaches to better understand the hepatotoxic potential of human drug candidates and to assess their toxicity earlier in the drug development process. The underlying data consisted of two commercial knowledgebases that employed a hybrid experimental design in which human drug-toxicity information was extracted from the literature, dichotomized, and merged with rat-based gene expression measures (primary isolated hepatocytes and whole liver). Toxicity classification rules were built using a stochastic gradient boosting machine learner, with classification error estimated using a modified bootstrap estimate of true error. Several types of clustering methods were also applied, based on sets of compounds and genes. Robust classification rules were constructed for both in vitro (hepatocytes) and in vivo (liver) data, based on a high-dose, 24-h design. There appeared to be little overlap between the two classifiers, at least in terms of their gene lists. Robust classifiers could not be fitted when earlier time points and/or low-dose data were included, indicating that experimental design is important for these systems. Our results suggest development of a compound screening assay based on these toxicity classifiers appears feasible, with classifier operating characteristics used to tune a screen for a specific implementation within preclinical testing paradigms.

We report the isolation and structural characterization of the full-length gene and cDNA for a novel mouse ATP-binding cassette (ABC) transporter, Abca13. The mRNA, isolated from mouse kidney, is 6.7 kb in size and encodes a protein consisting of 2143 amino acids with a predicted molecular weight of 240 kDa. The Abca13 gene consists of 44 exons which span 360 kb of genomic sequence. Abca13 has been mapped to mouse chromosome 11.a2, revealing the human orthologue highly conserved on a syntenic region of human chromosome 7p12. The deduced mouse Abca13 protein shows highest amino acid sequence homology to human ABCA1 (50%), ABCA4 (50%), and ABCA12 (56%). Analysis of the putative Abca13 promoter region revealed potential transcription factor binding sites associated with myeloid- and lymphoid-derived cell types. mRNA transcript levels were highest in mouse submaxillary gland, epididymus, ovary, and thymus; with lower levels in a variety of other tissues. An alternative transcript was discovered in mouse kidney devoid of exon 11. The removal of exon 11 by post-transcriptional splicing causes a frameshift in the open reading frame and results in a premature termination codon. We hypothesize that the excision of exon 11 may serve as a regulatory mechanism in kidney, and perhaps other tissues as well. The molecular characterization of the mouse Abca13 gene will establish the foundation for future functional studies of the human ABCA13 transporter.