Cross Cell Membranes Research Articles

Derivatization of Microcystins Can Increase Target Inhibition while Reducing Cellular Uptake.

Microcystins, a large family of nonribosomal cyclic heptapeptides known for their hepatotoxicity, are among the best-studied cyanobacterial toxins. Recently, they have been discussed as leads for the development of anticancer drug substances. Their main mode-of-action is inhibition of the eukaryotic serine/threonine protein phosphatases 1 and 2A. Unlike many cytotoxins that can cross cell membranes by passive diffusion, microcystins depend on active uptake via organic anion transporting polypeptides 1B1 or 1B3. Both phosphatase inhibition and transportability strongly depend on the structure of the individual microcystin. Here, we present how chemical modification of positions 2 and 4 of the microcystin core structure can alter these two properties. Aiming to reduce transportability and increase phosphatase inhibition, we used pharmacophore modeling to investigate the phosphatase inhibition potential of microcystins derivatized with small molecules containing a variety of functional groups. The respective derivatives were synthesized using click chemistry. We discovered that some derivatized microcystins can address a yet undescribed subpocket of the protein phosphatase 1. The derivatized microcystins were tested for phosphatase 1 inhibition and cytotoxicity on transporter-expressing cell lines, revealing that target inhibition and transportability of microcystins can independently be influenced by the physicochemical properties, especially of the residue located in position 2 of the microcystin. Derivatization with small acids or amino acids resulted in microcystins with a favorable ratio of inhibition to transportability, making these derivatives potentially suitable for drug development.

Engineering hyaluronic acid-based nanoassemblies for monoclonal antibody delivery – design, characterization, and biological insights

AbstractThe current spotlight of cancer therapeutics is shifting towards personalized medicine with the widespread use of monoclonal antibodies (mAbs). Despite their increasing potential, mAbs have an intrinsic limitation related to their inability to cross cell membranes and reach intracellular targets. Nanotechnology offers promising solutions to overcome this limitation, however, formulation challenges remain. These challenges are the limited loading capacity (often insufficient to achieve clinical dosing), the complex formulation methods, and the insufficient characterization of mAb-loaded nanocarriers. Here, we present a new nanocarrier consisting of hyaluronic acid-based nanoassemblies (HANAs) specifically designed to entrap mAbs with a high efficiency and an outstanding loading capacity (50%, w/w). HANAs composed by an mAb, modified HA and phosphatidylcholine (PC) resulted in sizes of ~ 100 nm and neutral surface charge. Computational modeling identified the principal factors governing the high affinity of mAbs with the amphiphilic HA and PC. HANAs composition and structural configuration were analyzed using the orthogonal techniques cryogenic transmission electron microscopy (cryo-TEM), asymmetrical flow field-flow fractionation (AF4), and small-angle X-ray scattering (SAXS). These techniques provided evidence of the formation of core-shell nanostructures comprising an aqueous core surrounded by a bilayer consisting of phospholipids and amphiphilic HA. In vitro experiments in cancer cell lines and macrophages confirmed HANAs’ low toxicity and ability to transport mAbs to the intracellular space. The reproducibility of this assembling process at industrial-scale batch sizes and the long-term stability was assessed. In conclusion, these results underscore the suitability of HANAs technology to load and deliver biologicals, which holds promise for future clinical translation.

Characterization and efficacy of C60 nano-photosensitive drugs in colorectal cancer treatment

BackgroundFullerenes C60 shows great potential for drug transport. C60 generates large amounts of singlet oxygen upon photoexcitation, which has a significant inhibitory effect on tumor cells, so the photosensitive properties of C60 were exploited for photodynamic therapy of tumors by laser irradiation. MethodsIn this study, C60-NH2 was functionalized by introducing amino acids on the surface of C60, coupled with 5-FU to obtain C60 amino acid-derived drugs (C60AF, C60GF, C60LF), and activated photosensitive drugs (C60AFL, C60GFL, C60LFL) were obtained by laser irradiation. The C60 nano-photosensitive drugs were characterized in various ways, and the efficacy and safety of C60 nano-photosensitive drugs were verified by cellular experiments and animal experiments. Bioinformatics methods and cellular experiments were used to confirm the photosensitive drug targets and verify the therapeutic targets with C60AF. ResultsPhotosensitised tumor-targeted drug delivery effectively crosses cell membranes, leads to more apoptotic cell death, and provides higher anti-tumor efficacy and safety in vitro and in vivo colorectal cancer pharmacodynamic assays compared to free 5-FU.C60 photosensitized drug promotes tumor killing by inhibiting the colorectal cancer FLOR1 tumor protein target, with no significant toxic effects on normal organs. ConclusionC60 photosensitized drug delivery systems are expected to improve efficacy and reduce side effects in the future treatment of colorectal cancer. Further and better development and design of drugs and vectors for colorectal cancer therapy.

Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation.

Macrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol-containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol-containing elements and used them for the combinatorial synthesis of a 2,688-member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X-ray structure analysis of macrocycle-target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation

AbstractMacrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol‐containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol‐containing elements and used them for the combinatorial synthesis of a 2,688‐member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X‐ray structure analysis of macrocycle‐target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

Peptide-based PROTACs: Current Challenges and Future Perspectives.

Proteolysis-targeting chimeras (PROTACs) are an attractive means to target previously undruggable or drug-resistant mutant proteins. While small molecule-based PROTACs are stable and can cross cell membranes, there is limited availability of suitable small molecule warheads capable of recruiting proteins to an E3 ubiquitin ligase for degradation. With advances in structural biology and in silico protein structure prediction, it is now becoming easier to define highly selective peptides suitable for PROTAC design. As a result, peptide-based PROTACs are becoming a feasible proposition for targeting previously "undruggable" proteins not amenable to small molecule inhibition. In this review, we summarize recent progress in the design and application of peptide-based PROTACs as well as several practical approaches for obtaining candidate peptides for PROTACs. We also discuss the major hurdles preventing the translation of peptide-based PROTACs from bench to bedside, such as their delivery and bioavailability, with the aim of stimulating discussion about how best to accelerate the clinical development of peptide- based PROTACs in the near future.

Irg1/itaconate activates Nrf2/HO-1 pathway and mitigates septic liver injury in mice

Objectives: Immune-responsive gene 1 ( Irg1)-catalyzed production of itaconate is a bioactive metabolite with antibacterial, anti-inflammatory and antioxidant properties. Liver injury is closely related to poor outcomes in septic patients, while prevention of liver injury is essential for the pharmacological control of sepsis. This study investigated the pathological and pharmacological significance of Irg1/itaconate in septic liver injury. Methods: Septic liver injury was induced in mice by injecting lipopolysaccharide (LPS; 15 mg/kg) intraperitoneally. The hepatic mRNA and protein contents of Irg1 were detected. To investigate the pathological meaning of Irg1, septic liver injury was induced in Irg1 knockout mice and their wild-type (WT) littermates, and the degree of liver injury, hepatic inflammation, oxidative stress and the activation of nuclear erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) were subsequently determined. Finally, to explore the gene’s pharmacological potential, 4-octyl itaconate (4-OI; 50 mg/kg), a derivative of itaconate that could cross cell membranes, was administered intraperitoneally. The mobilization of Nrf2/HO-1 and liver injury were then evaluated. Results: The level of Irg1 expression was dramatically enhanced in mice with septic liver injury. Additionally, Irg1 deletion leaded to higher ALT and AST levels, elevated inflammatory and oxidative-stress indicators and compromised stimulation of the Nrf2/HO-1 pathway. However, administering 4-OI restored the Nrf2/HO-1 pathway which mitigated liver injury and inhibited hepatic inflammation and oxidative stress. Conclusions: The results figured that elevation of Irg1 might be a protective event that control the progress of septic liver injury, while 4-OI might have latent significance for the pharmaceutical intervention of septic liver injury.

Angiotensin II-dependent aldosterone production in the adrenal cortex.

The adrenal cortex is responsible for production of adrenal steroid hormones and is anatomically divided into three distinct zones: zona glomerulosa secreting mineralocorticoids (mainly aldosterone), zona fasciculata secreting glucocorticoids (cortisol), and zona reticularis producing androgens. Importantly, due to their high lipophilicity, no adrenal steroid hormone (including aldosterone) is stored in vesicles but rather gets synthesized and secreted instantly upon cell stimulation with specific stimuli. Aldosterone is the most potent mineralocorticoid hormone produced from the adrenal cortex in response to either angiotensin II (AngII) or elevated K+ levels in the blood (hyperkalemia). AngII, being a peptide, cannot cross cell membranes and thus, uses two distinct G protein-coupled receptor (GPCR) types, AngII type 1 receptor (AT1R) and AT2R to exert its effects inside cells. In zona glomerulosa cells, AT1R activation by AngII results in aldosterone synthesis and secretion via two main pathways: (a) Gq/11 proteins that activate phospholipase C ultimately raising intracellular free calcium concentration; and (b) βarrestin1 and -2 (also known as Arrestin-2 and -3, respectively) that elicit sustained extracellular signal-regulated kinase (ERK) activation. Both pathways induce upregulation and acute activation of StAR (steroidogenic acute regulatory) protein, the enzyme that catalyzes the rate-limiting step in aldosterone biosynthesis. This chapter describes these two salient pathways underlying AT1R-induced aldosterone production in zona glomerulosa cells. We also highlight some pharmacologically important notions pertaining to the efficacy of the currently available AT1R antagonists, also known as angiotensin receptor blockers (ARBs) or sartans at suppressing both pathways, i.e., their inverse agonism efficacy at G proteins and βarrestins.

Understanding How Charged Particles Cross Living Cell Membranes

Understanding How Charged Particles Cross Living Cell Membranes

Redox-Responsive Polymeric Nanoparticle for Nucleic Acid Delivery and Cancer Therapy: Progress, Opportunities, and Challenges.

Cancer development and progression of cancer are closely associated with the activation of oncogenes and loss of tumor suppressor genes. Nucleic acid drugs (e.g., siRNA, mRNA, and DNA) are widely used for cancer therapy due to their specific ability to regulate the expression of any cancer-associated genes. However, nucleic acid drugs are negatively charged biomacromolecules that are susceptible to serum nucleases and cannot cross cell membrane. Therefore, specific delivery tools are required to facilitate the intracellular delivery of nucleic acid drugs. In the past few decades, a variety of nanoparticles (NPs) are designed and developed for nucleic acid delivery and cancer therapy. In particular, the polymeric NPs in response to the abnormal redox status in cancer cells have garnered much more attention as their potential in redox-triggered nanostructure dissociation and rapid intracellular release of nucleic acid drugs. In this review, the important genes or signaling pathways regulating the abnormal redox status in cancer cells are briefly introduced and the recent development of redox-responsive NPs for nucleic acid delivery and cancer therapy is systemically summarized. The future development of NPs-mediated nucleic acid delivery and their challenges in clinical translation are also discussed.

A novel self-assembled nucleobase-nanofiber platform of CDN to activate the STING pathway for synergistic cancer immunotherapy

2′, 3′-cGAMP (CDN) as cGAS-STING pathway agonist is extensively used in tumor treatment. However, due to its negatively charged nature (containing two phosphate groups) and high hydrophilicity, CDN faces challenges in crossing cell membranes, resulting in reduced efficiency of its use. Additionally, CDN is susceptible to inactivation through phosphodiesterase hydrolysis. Therefore, the development of a new drug delivery system for CDN is necessary to prevent hydrolysis and enhance targeted accumulation in tumors, as well as improve cellular uptake for STING activation. In this study, we have developed peptide-polymer nanofibers (PEG-Q11) that incorporate thymine (T) and arginine (R) residues to facilitate complexation with CDN through the principles of Watson-Crick base pairing with thymine and favorable electrostatic interactions and bidentate hydrogen bonding with arginine side chains. The entrapment efficiency (EE) of PEG-Q11T3R4@CDN was found to be 51% higher than that of PEG-Q11@CDN. Due to its favorable biocompatibility, PEG-Q11T3R4@CDN was employed for immunotherapy in mouse CT26 tumors. In local tumor treatment, the administration of PEG-Q11T3R4@CDN at a low dose and through a single injection exhibited inhibitory effects. Furthermore, the local injection of PEG-Q11T3R4@CDN resulted in systemic therapeutic responses, effectively suppressing tumor metastasis by activating CD8 + T cells to target distant tumors. This research not only underscores the potential of PEG-Q11T3R4@CDN as an efficient therapeutic agent but also highlights its ability to achieve long-lasting systemic therapeutic outcomes following local treatment. Consequently, PEG-Q11T3R4@CDN represents a promising strategy for immunization.

Extracellular Vesicles and Endocannabinoid Signaling in Patients with COVID-19.

Introduction: Endocannabinoids in COVID-19 have immunomodulatory and anti-inflammatory properties but the functional role and the regulation of endocannabinoid signaling in this pandemic disorder is controversial. To exercise their biologic function, endocannabinoids need to travel across the intercellular space and within the blood stream to reach their target cells. How the lipophilic endocannabinoids are transported in the vascular system and how these hydrophobic compounds cross cell membranes is still unclear. Extracellular vesicles (EVs) are released and incorporated by many cell types including immune cells. EVs are small lipid-membrane covered particles and contain RNA, lipids and proteins. They play an important role in intercellular communication by transporting these signaling molecules from their cells of origin to specific target cells. EVs may represent ideal transport vehicles for lipophilic signaling molecules like endocannabinoids and this effect could also be evident in COVID-19. Materials and Methods: We measured the endocannabinoids anandamide, 2-AG, SEA, PEA and OEA in patients with COVID-19 in EVs and plasma. RNA sequencing of microRNAs (miRNAs) derived from EVs (EV-miRNAs) and mRNA transcripts from blood cells was used for the construction of signaling networks reflecting endocannabinoid and miRNA communication by EVs to target immune cells. Results: With the exception of anandamide, endocannabinoid concentrations were significantly enriched in EVs in comparison to plasma and increased with disease severity. No enrichment in EVs was seen for the more hydrophilic steroid hormones cortisol and testosterone. High EV-endocannabinoid concentrations were associated with downregulation of CNR2 (CB2) by upregulated EV-miRNA miR-146a-5p and upregulation of MGLL by downregulated EV-miR-199a-5p and EV-miR-370-5p suggesting counterregulatory effects. In contrast, low EV-levels of anandamide were associated with upregulation of CNR1 by downregulation of EV-miR-30c-5p and miR-26a-5p along with inhibition of FAAH. Immunologically active molecules in immune cells regulated by endocannabinoid signaling included VEGFA, GNAI2, IGF1, BDNF, IGF1R and CREB1 and CCND1 among others. Discussion and Conclusions: EVs carry immunologically functional endocannabinoids in COVID-19 along with miRNAs which may regulate the expression of mRNA transcripts involved in the regulation of endocannabinoid signaling and metabolism. This mechanism could fine-tune and adapt endocannabinoid effects in recipient cells in relationship to the present biological context.

Cell Penetrating Peptides: Classification, Mechanisms, Methods of Study, and Applications.

Cell-penetrating peptides (CPPs) encompass a class of peptides that possess the remarkable ability to cross cell membranes and deliver various types of cargoes, including drugs, nucleic acids, and proteins, into cells. For this reason, CPPs are largely investigated in drug delivery applications in the context of many diseases, such as cancer, diabetes, and genetic disorders. While sharing this functionality and some common structural features, such as a high content of positively charged amino acids, CPPs represent an extremely diverse group of elements, which can differentiate under many aspects. In this review, we summarize the most common characteristics of CPPs, introduce their main distinctive features, mechanistic aspects that drive their function, and outline the most widely used techniques for their structural and functional studies. We highlight current gaps and future perspectives in this field, which have the potential to significantly impact the future field of drug delivery and therapeutics.

SiRNA a promising tool for diabetes complications

RNA interference (RNAi) is a naturally occurring process of gene regulation that has been harnessed to silence specific genes in various cell types, including those involved in diabetes complications. Small interfering RNA (siRNA) is an RNA molecule that activates RNAi and targets specific genes for degradation. Recent research has demonstrated that siRNA holds promise as a tool for treating diabetes complications, including diabetic neuropathy, retinopathy, and nephropathy. In preclinical studies, siRNA has been shown to effectively target genes involved in these complications, resulting in improved clinical outcomes. One potential advantage of siRNA therapy is its ability to selectively target specific genes without disrupting endogenous mRNA pathways, which reduces the risk of off-target effects. Additionally, siRNA has the potential to provide long-lasting effects with a single dose, which could result in reduced treatment frequency and improved patient compliance. While promising preclinical results have been, several challenges still need to be addressed before siRNA can be used in clinical practice. These include delivery issues, as siRNA molecules rapidly degrade in the bloodstream and cannot cross cell membranes without assistance. Despite these challenges, the potential of siRNA as a tool for treating diabetes complications is exciting, and further research is needed to determine its safety and efficacy in clinical trials. With continued investigation and refinement, siRNA has the potential to become an important therapeutic tool for the treatment of diabetes complications, improving patient outcomes and quality of life.

Inflammatory Bowel Disease-Associated Colorectal Cancer: Translational and Transformational Risks Posed by Exogenous Free Hemoglobin Alpha Chain, A By-Product of Extravasated Erythrocyte Macrophage Erythrophagocytosis.

Colonic inflammatory bowel disease (IBD) encompasses ulcerative colitis (UC) and Crohn's colitis (CC). Patients with IBD are at increased risk for colitis-associated colorectal cancer (CACRC) compared to the general population. CACRC is preceded by IBD, characterized by highly heterogenous, pharmacologically incurable, pertinacious, worsening, and immune-mediated inflammatory pathologies of the colon and rectum. The molecular and immunological basis of CACRC is highly correlated with the duration and severity of inflammation, which is influenced by the exogenous free hemoglobin alpha chain (HbαC), a byproduct of infiltrating immune cells; extravasated erythrocytes; and macrophage erythrophagocytosis. The exogenous free HbαC prompts oxygen free radical-arbitrated DNA damage (DNAD) through increased cellular reactive oxygen species (ROS), which is exacerbated by decreased tissue antioxidant defenses. Mitigation of the Fenton Reaction via pharmaceutical therapy would attenuate ROS, promote apoptosis and DNAD repair, and subsequently prevent the incidence of CACRC. Three pharmaceutical options that attenuate hemoglobin toxicity include haptoglobin, deferoxamine, and flavonoids (vitamins C/E). Haptoglobin's clearance rate from plasma is inversely correlated with its size; the smaller the size, the faster the clearance. Thus, the administration of Hp1-1 may prove to be beneficial. Further, deferoxamine's hydrophilic structure limits its ability to cross cell membranes. Finally, the effectiveness of flavonoids, natural herb antioxidants, is associated with the high reactivity of hydroxyl substituents. Multiple analyses are currently underway to assess the clinical context of CACRC and outline the molecular basis of HbαC-induced ROS pathogenesis by exposing colonocytes and/or colonoids to HbαC. The molecular immunopathogenesis pathways of CACRC herein reviewed are broadly still not well understood. Therefore, this timely review outlines the molecular and immunological basis of disease pathogenesis and pharmaceutical intervention as a protective measure for CACRC.

Purine containing carbonucleoside phosphonate analogues as novel chemotype for Plasmodium falciparum Inhibition

The nucleotidase ISN1 is a potential therapeutic target of the purine salvage pathway of the malaria parasite Plasmodium falciparum. We identified PfISN1 ligands by in silico screening of a small library of nucleos(t)ide analogues and by thermal shift assays. Starting from a racemic cyclopentyl carbocyclic phosphonate scaffold, we explored the diversity on the nucleobase moiety and also proposed a convenient synthetic pathway to access the pure enantiomers of our initial hit (compound (±)-2). 2,6-Disubstituted purine containing derivatives such as compounds 1, (±)-7e and β-L-(+)-2 showed the most potent inhibition of the parasite in vitro, with low micromolar IC50 values. These results are remarkable considering the anionic nature of nucleotide analogues, which are known to lack activity in cell culture experiments due to their scarce capacity to cross cell membranes. For the first time, we report the antimalarial activity of a carbocyclic methylphosphonate nucleoside with an L-like configuration.

In Vitro and In Vivo Models for Drug Transport Across the Blood-Testis Barrier.

The blood-testis barrier (BTB) is a selectively permeable membrane barrier formed by adjacent Sertoli cells (SCs) in the seminiferous tubules of the testes that develops intercellular junctional complexes to protect developing germ cells from external pressures. However, due to this inherent defense mechanism, the seminiferous tubule lumen can act as a pharmacological sanctuary site for latent viruses (e.g., Ebola, Zika) and cancers (e.g., leukemia). Therefore, it is critical to identify and evaluate BTB carrier-mediated drug delivery pathways to successfully treat these viruses and cancers. Many drugs are unable to effectively cross cell membranes without assistance from carrier proteins like transporters because they are large, polar, and often carry a charge at physiologic pH. SCs express transporters that selectively permit endogenous compounds, such as carnitine or nucleosides, across the BTB to support normal physiologic activity, although reproductive toxicants can also use these pathways, thereby circumventing the BTB. Certain xenobiotics, including select cancer therapeutics, antivirals, contraceptives, and environmental toxicants, are known to accumulate within the male genital tract and cause testicular toxicity; however, the transport pathways by which these compounds circumvent the BTB are largely unknown. Consequently, there is a need to identify the clinically relevant BTB transport pathways in in vitro and in vivo BTB models that recapitulate human pharmacokinetics and pharmacodynamics for these xenobiotics. This review summarizes the various in vitro and in vivo models of the BTB reported in the literature and highlights the strengths and weaknesses of certain models for drug disposition studies. SIGNIFICANCE STATEMENT: Drug disposition to the testes is influenced by the physical, physiological, and immunological components of the blood-testis barrier (BTB). But many compounds are known to cross the BTB by transporters, resulting in pharmacological and/or toxicological effects in the testes. Therefore, models that assess drug transport across the human BTB must adequately account for these confounding factors. This review identifies and discusses the benefits and limitations of various in vitro and in vivo BTB models for preclinical drug disposition studies.

RRx-001: a chimeric triple action NLRP3 inhibitor, Nrf2 inducer, and nitric oxide superagonist.

RRx-001 is a shape shifting small molecule with Fast Track designation for the prevention/amelioration of chemoradiation-induced severe oral mucositis (SOM) in newly diagnosed Head and Neck cancer. It has been intentionally developed or "engineered" as a chimeric single molecular entity that targets multiple redox-based mechanisms. Like an antibody drug conjugate (ADC), RRx-001 contains, at one end a "targeting" moiety, which binds to the NLRP3 inflammasome and inhibits it as well as Kelch-like ECH-associated protein 1 (KEAP1), the negative regulator of Nrf2, and, at the other end, a conformationally constrained, dinitro containing 4 membered ring, which fragments under conditions of hypoxia and reduction to release therapeutically active metabolites i.e., the payload. This "payload", which is delivered specifically to hypoperfused and inflamed areas, includes nitric oxide, nitric oxide related species and carbon-centered radicals. As observed with ADCs, RRx-001 contains a backbone amide "linker" attached to a binding site, which correlates with the Fab region of an antibody, and to the dinitroazetidine payload, which is microenvironmentally activated. However, unlike ADCs, whose large size impacts their pharmacokinetic properties, RRx-001 is a nonpolar small molecule that easily crosses cell membranes and the blood brain barrier (BBB) and distributes systemically. This short review is organized around the de novo design and in vivo pro-oxidant/pro-inflammatory and antioxidant/anti-inflammatory activity of RRx-001, which, in turn, depends on the reduced to oxidized glutathione ratio and the oxygenation status of tissues.

Cryo-EM analysis of scorpion toxin binding to Ryanodine Receptors reveals subconductance that is abolished by PKA phosphorylation.

Calcins are peptides from scorpion venom with the unique ability to cross cell membranes, gaining access to intracellular targets. Ryanodine Receptors (RyR) are intracellular ion channels that control release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. Calcins target RyRs and induce long-lived subconductance states, whereby single-channel currents are decreased. We used cryo-electron microscopy to reveal the binding and structural effects of imperacalcin, showing that it opens the channel pore and causes large asymmetry throughout the cytosolic assembly of the tetrameric RyR. This also creates multiple extended ion conduction pathways beyond the transmembrane region, resulting in subconductance. Phosphorylation of imperacalcin by protein kinase A prevents its binding to RyR through direct steric hindrance, showing how posttranslational modifications made by the host organism can determine the fate of a natural toxin. The structure provides a direct template for developing calcin analogs that result in full channel block, with potential to treat RyR-related disorders.

Insights into the synthesis strategies of plant-derived cyclotides.

Cyclotides are plant peptides characterized with a head-to-tail cyclized backbone and three interlocking disulfide bonds, known as a cyclic cysteine knot. Despite the variations in cyclotides peptide sequences, this core structure is conserved, underlying their most useful feature: stability against thermal and chemical breakdown. Cyclotides are the only natural peptides known to date that are orally bioavailable and able to cross cell membranes. Cyclotides also display bioactivities that have been exploited and expanded to develop as potential therapeutic reagents for a wide range of conditions (e.g., HIV, inflammatory conditions, multiple sclerosis, etc.). As such, in vitro production of cyclotides is of the utmost importance since it could assist further research on this peptide class, specifically the structure-activity relationship and its mechanism of action. The information obtained could be utilized to assist drug development and optimization. Here, we discuss several strategies for the synthesis of cyclotides using both chemical and biological routes.