Automated Fast-Flow Synthesis of Chromosome 9 Open Reading Frame 72 Dipeptide Repeat Proteins.

An expansion of the hexanucleotide (GGGGCC) repeat sequence in chromosome 9 open frame 72 (c9orf72) is the most common genetic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The mutation leads to the production of toxic dipeptide repeat proteins (DPRs) that induce neurodegeneration. However, the fundamental physicochemical properties of DPRs remain largely unknown due to their limited availability. Here, we synthesized the c9orf72 DPRs poly-glycine-arginine (poly-GR), poly-proline-arginine (poly-PR), poly-glycine-proline (poly-GP), poly-proline-alanine (poly-PA), and poly-glycine-alanine (poly-GA) using automated fast-flow peptide synthesis (AFPS) and achieved single-domain chemical synthesis of proteins with up to 200 amino acids. Circular dichroism spectroscopy of the synthetic DPRs revealed that proline-containing poly-PR, poly-GP, and poly-PA could adopt polyproline II-like helical secondary structures. In addition, structural analysis by size-exclusion chromatography indicated that longer poly-GP and poly-PA might aggregate. Furthermore, cell viability assays showed that human neuroblastoma cells cultured with poly-GR and poly-PR with longer repeat lengths resulted in reduced cell viability, while poly-GP and poly-PA did not, thereby reproducing the cytotoxic property of endogenous DPRs. This research demonstrates the potential of AFPS to synthesize low-complexity peptides and proteins necessary for studying their pathogenic mechanisms and constructing disease models.

In-Cell Penetration Selection-Mass Spectrometry Produces Noncanonical Peptides for Antisense Delivery.

Peptide-mediated delivery of macromolecules in cells has significant potential therapeutic benefits, but no therapy employing cell-penetrating peptides (CPPs) has reached the market after 30 years of investigation due to challenges in the discovery of new, more efficient sequences. Here, we demonstrate a method for in-cell penetration selection-mass spectrometry (in-cell PS-MS) to discover peptides from a synthetic library capable of delivering macromolecule cargo to the cytosol. This method was inspired by recent in vivo selection approaches for cell-surface screening, with an added spatial dimension resulting from subcellular fractionation. A representative peptide discovered in the cytosolic extract, Cyto1a, is nearly 100-fold more active toward antisense phosphorodiamidate morpholino oligomer (PMO) delivery compared to a sequence identified from a whole cell extract, which includes endosomes. Cyto1a is composed of d-residues and two non-α-amino acids, is more stable than its all-l isoform, and is less toxic than known CPPs with comparable activity. Pulse-chase and microscopy experiments revealed that while the PMO-Cyto1a conjugate is likely taken up by endosomes, it can escape to localize to the nucleus without nonspecifically releasing other endosomal components. In-cell PS-MS introduces a means to empirically discover unnatural synthetic peptides for subcellular delivery of therapeutically relevant cargo.

A Tumor-Homing Peptide Platform Enhances Drug Solubility, Improves Blood-Brain Barrier Permeability and Targets Glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common and deadliest malignant primary brain tumor, contributing significant morbidity and mortality among patients. As current standard-of-care demonstrates limited success, the development of new efficacious GBM therapeutics is urgently needed. Major challenges in advancing GBM chemotherapy include poor bioavailability, lack of tumor selectivity leading to undesired side effects, poor permeability across the blood-brain barrier (BBB), and extensive intratumoral heterogeneity. METHODS: We have previously identified a small, soluble peptide (BTP-7) that is able to cross the BBB and target the human GBM extracellular matrix (ECM). Here, we covalently attached BTP-7 to an insoluble anti-cancer drug, camptothecin (CPT). RESULTS: We demonstrate that conjugation of BTP-7 to CPT improves drug solubility in aqueous solution, retains drug efficacy against patient-derived GBM stem cells (GSC), enhances BBB permeability, and enables therapeutic targeting to intracranial GBM, leading to higher toxicity in GBM cells compared to normal brain tissues, and ultimately prolongs survival in mice bearing intracranial patient-derived GBM xenograft. CONCLUSION: BTP-7 is a new modality that opens the door to possibilities for GBM-targeted therapeutic approaches.

Palladium-Mediated Incorporation of Carboranes into Small Molecules, Peptides, and Proteins.

Carboranes represent a class of compounds with increasing therapeutic potential. However, few general approaches to readily embed carboranes into small molecules, peptides, and proteins are available. We report a strategy based on palladium-mediated C-X (X = C, S, and N) bond formation for the installation of carborane-containing moieties onto small molecules and peptides. We demonstrate the ability of Pd-based reagents with appropriate ligands to overcome the high hydrophobicity of the carborane group and enable chemoselective conjugation of cysteine residues at room temperature in aqueous buffer. Accordingly, carboranes can be efficiently installed on proteins by employing a combination of a bis-sulfonated biarylphosphine-ligated Pd reagent in an aqueous histidine buffer. This method is successfully employed on nanobodies, a fully synthetic affibody, and the antibody therapeutics trastuzumab and cetuximab. The conjugates of the affibody ZHER2 and the trastuzumab antibody retained binding to their target antigens. Conjugated proteins maintain their activity in cell-based functional assays in HER2-positive BT-474 cell lines. This approach enables the rapid incorporation of carborane moieties into small molecules, peptides, and proteins for further exploration in boron neutron capture therapy, which requires the targeted delivery of boron-dense groups.

Automated Flow Synthesis of Peptide-PNA Conjugates.

Antisense peptide nucleic acids (PNAs) have yet to translate to the clinic because of poor cellular uptake, limited solubility, and rapid elimination. Cell-penetrating peptides (CPPs) covalently attached to PNAs may facilitate clinical development by improving uptake into cells. We report an efficient technology that utilizes a fully automated fast-flow instrument to manufacture CPP-conjugated PNAs (PPNAs) in a single shot. The machine is rapid, with each amide bond being formed in 10 s. Anti-IVS2-654 PPNA synthesized with this instrument presented threefold activity compared to transfected PNA in a splice-correction assay. We demonstrated the utility of this approach by chemically synthesizing eight anti-SARS-CoV-2 PPNAs in 1 day. A PPNA targeting the 5' untranslated region of SARS-CoV-2 genomic RNA reduced the viral titer by over 95% in a live virus infection assay (IC50 = 0.8 µM). Our technology can deliver PPNA candidates to further investigate their potential as antiviral agents.

Cell-Penetrating d-Peptides Retain Antisense Morpholino Oligomer Delivery Activity.

Cell-penetrating peptides (CPPs) can cross the cell membrane to enter the cytosol and deliver otherwise nonpenetrant macromolecules such as proteins and oligonucleotides. For example, recent clinical trials have shown that a CPP attached to phosphorodiamidate morpholino oligomers (PMOs) resulted in higher muscle concentration, increased exon skipping, and dystrophin production relative to another study of the PMO alone in patients of Duchenne muscular dystrophy. Therefore, effective design and the study of CPPs could help enhance therapies for difficult-to-treat diseases. So far, the study of CPPs for PMO delivery has been restricted to predominantly canonical l-peptides. We hypothesized that mirror-image d-peptides could have similar PMO delivery activity as well as enhanced proteolytic stability, facilitating their characterization and quantification from biological milieu. We found that several enantiomeric peptide sequences could deliver a PMO-biotin cargo with similar activities while remaining stable against serum proteolysis. The biotin label allowed for affinity capture of fully intact PMO-peptide conjugates from whole-cell and cytosolic lysates. By profiling a mixture of these constructs in cells, we determined their relative intracellular concentrations. When combined with PMO activity, these concentrations provide a new metric for delivery efficiency, which may be useful for determining which peptide sequence to pursue in further preclinical studies.

Sex, species and age: Effects of rodent demographics on the pharmacology of ∆9-tetrahydrocanabinol.

Cannabis edibles are becoming more common in an increasingly diverse population of users, and the impact of first pass metabolism on cannabis's pharmacological profile across age and sex is not well understood. The present study examined the impact of age, sex and rodent species on the effects of intraperitoneal (i.p.) delta-9-tetrahydrocannabinol (THC) and its primary psychoactive metabolite, 11-OH-THC, in rodent models of psychoactivity and molecular assays of cannabinoid receptor type-1 (CB1) pharmacology. Like oral THC, i.p. THC also undergoes first pass metabolism. In both species and sexes, 11-OH-THC exhibited marginally higher affinity (~1.5 fold) than THC and both served as partial agonists in [35S]GTPγS binding with equivalent potency; 11-OH-THC exhibited slightly greater efficacy in rat brain tissue. In ICR mice, 11-OH-THC exhibited greater potency than THC in assays of catalepsy (7- to 15-fold) and hypothermia (7- to 31-fold). Further, 11-OH-THC was more potent in THC drug discrimination (7- to 9-fold) in C57Bl/6 J mice, with THC-like discriminative stimulus effects being CB1-, but not CB2-, mediated. THC's discriminative stimulus also was stable across age in mice, as its potency did not change over the course of the experiment (~17 months). While sex differences in THC's effects were not revealed in mice, THC was significantly more potent in females Sprague-Dawley rats than in males trained to discriminate THC from vehicle. This study demonstrates a cross-species in the psychoactive effects of i.p. THC across sex that may be related to differential metabolism of THC into its psychoactive metabolite 11-OH-THC, suggesting that species is a crucial design consideration in the preclinical study of phytocannabinoids.

Secondary Amino Alcohols: Traceless Cleavable Linkers for Use in Affinity Capture and Release.

Capture and release of peptides is often a critical operation in the pathway to discovering materials with novel functions. However, the best methods for efficient capture impede facile release. To overcome this challenge, we report linkers based on secondary amino alcohols for the release of peptides after capture. These amino alcohols are based on serine (seramox) or isoserine (isoseramox) and can be incorporated into peptides during solid-phase peptide synthesis through reductive amination. Both linkers are quantitatively cleaved within minutes under NaIO4 treatment. Cleavage of isoseramox produced a native peptide N-terminus. This linker also showed broad substrate compatibility; incorporation into a synthetic peptide library resulted in the identification of all sequences by nanoLC-MS/MS. The linkers are cell compatible; a cell-penetrating peptide that contained this linker was efficiently captured and identified after uptake into cells. These findings suggest that such secondary amino alcohol based linkers might be suitable tools for peptide-discovery platforms.

Sex, THC, and hormones: Effects on density and sensitivity of CB1 cannabinoid receptors in rats.

BACKGROUND: The recent NIH mandate to consider sex as a biological variable in preclinical research has focused attention on delineation of sex differences in behavior. To investigate mechanisms underlying sex differences in Δ9-tetrahydrocannabinol (THC) effects, we examined the effects of sex and gonadal hormones on CB1 receptors in cerebellum, hippocampus, prefrontal cortex, and striatum. METHODS: Adult Sprague-Dawley rats underwent gonadectomy (GDX) or sham-GDX. Half of the GDX females and males received estradiol or testosterone replacement (GDX+H), respectively. All rats were injected with vehicle or 30 mg/kg THC twice daily for 1 week before brain collection. CP55,940-stimulated [35S]GTPγS and [3H]SR141716A saturation binding assays were performed. RESULTS: With exception of enhanced receptor activation in the hippocampi of female rats compared to males, vehicle-treated rats exhibited minimal sex differences in CB1 receptor densities or G-protein coupling. Repeated treatment with THC resulted in pronounced CB1 receptor desensitization and downregulation in both sexes in all brain regions with a greater magnitude of change in females. CONCLUSIONS: These results suggest that sex differences in the density and G-protein coupling of brain CB1 receptors may play a limited role in sex differences in acute THC effects not mediated by the hippocampus. In contrast, sex differences after repeated THC were common, with females (intact, GDX, and GDX+H) showing greater downregulation or desensitization in all four brain regions compared to the respective male groups. This result is consistent with a finding that women tend to progress to tolerance and dependence quicker than men after initiation of cannabis use.

Diarylureas Containing 5-Membered Heterocycles as CB1 Receptor Allosteric Modulators: Design, Synthesis, and Pharmacological Evaluation.

Allosteric modulators have attracted significant interest as an alternate strategy to modulate CB1 receptor signaling for therapeutic benefits that may avoid the adverse effects associated with orthosteric ligands. Here we extended our previous structure-activity relationship studies on the diarylurea-based CB1 negative allosteric modulators (NAMs) by introducing five-membered heterocycles to replace the 5-pyrrolidinylpyridinyl group in PSNCBAM-1 (1), one of the first generation CB1 allosteric modulators. Many of these compounds had comparable potency to 1 in blocking the CB1 agonist CP55,940 stimulated calcium mobilization and [35S]GTP-γ-S binding. Similar to 1, most compounds showed positive cooperativity by increasing [3H]CP55,940 binding, consistent with the positive allosteric modulator (PAM)-antagonist mechanism. Interestingly, these compounds exhibited differences in ability to increase specific binding of [3H]CP55,940 and decrease binding of the antagonist [3H]SR141716. In saturation binding studies, only increases in [3H]CP55,940 Bmax, but not Kd, were observed, suggesting that these compounds stabilize low affinity receptors into a high affinity state. Among the series, the 2-pyrrolyl analogue (13) exhibited greater potency than 1 in the [35S]GTP-γ-S binding assay and significantly enhanced the maximum binding level in the [3H]CP5,5940 binding assay, indicating greater CB1 receptor affinity and/or cooperativity.

Synthetic Cannabinoid Hydroxypentyl Metabolites Retain Efficacy at Human Cannabinoid Receptors.

Synthetic cannabinoids (SCs) are novel psychoactive substances that are easily acquired, widely abused as a substitute for cannabis, and associated with cardiotoxicity and seizures. Although the structural bases of these compounds are scaffolds with known affinity and efficacy at the human cannabinoid type-1 receptor (hCB1), upon ingestion or inhalation they can be metabolized to multiple chemical entities of unknown pharmacological activity. A large proportion of these metabolites are hydroxylated on the pentyl chain, a key substituent that determines receptor affinity and selectivity. Thus, the pharmacology of SC metabolites may be an important component in understanding the in vivo effects of SCs. We examined nine SCs (AB-PINACA, 5F-AB-PINACA, ADB/MDMB-PINACA, 5F-ADB, 5F-CUMYL-PINACA, AMB-PINACA, 5F-AMB, APINACA, and 5F-APINACA) and their hydroxypentyl (either 4-OH or 5-OH) metabolites in [3H]CP55,940 receptor binding and the [35S]GTPγS functional assay to determine the extent to which these metabolites retain activity at cannabinoid receptors. All of the SCs tested exhibited high affinity (<10 nM) and efficacy for hCB1 and hCB2 The majority of the hydroxypentyl metabolites retained full efficacy at hCB1 and hCB2, albeit with reduced affinity and potency, and exhibited greater binding selectivity for hCB2 These data suggest that phase I metabolites may be contributing to the in vivo pharmacology and toxicology of abused SCs. Considering this and previous reports demonstrating that metabolites retain efficacy at the hCB1 receptor, the full pharmacokinetic profiles of the parent compounds and their metabolites need to be considered in terms of the pharmacological effects and time course associated with these drugs.

Molecular and Behavioral Pharmacological Characterization of Abused Synthetic Cannabinoids MMB- and MDMB-FUBINACA, MN-18, NNEI, CUMYL-PICA, and 5-Fluoro-CUMYL-PICA.

Synthetic cannabinoids are a class of novel psychoactive substances that exhibit high affinity at the cannabinoid type-1 (CB1) receptor and produce effects similar to those of Δ-9-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis. Illicit drug manufacturers are continually circumventing laws banning the sale of synthetic cannabinoids by synthesizing novel structures and doing so with little regard for the potential impact on pharmacological and toxicological effects. Synthetic cannabinoids produce a wide range of effects that include cardiotoxicity, seizure activity, and kidney damage, and they can cause death. Six synthetic cannabinoids, recently detected in illicit preparations, MMB-FUBINACA, MDMB-FUBINACA, CUMYL-PICA, 5F-CUMYL-PICA, NNEI, and MN-18 were assessed for: 1) receptor binding affinity at the human CB1 and human CB2 receptors, 2) function in [35S]GTPγS and cAMP signaling, and 3) THC-like effects in a mouse drug discrimination assay. All six synthetic cannabinoids exhibited high affinity for human cannabinoid receptors type-1 and type-2 and produced greater maximal effects than THC in [35S]GTPγS and cAMP signaling. Additionally, all six synthetic cannabinoids substituted for THC in drug discrimination, suggesting they probably possess subjective effects similar to those of cannabis. Notably, MDMB-FUBINACA, a methylated analog of MMB-FUBINACA, had higher affinity for CB1 than the parent, showing that minor structural modifications being introduced can have a large impact on the pharmacological properties of these drugs. This study demonstrates that novel structures being sold and used illicitly as substitutes for cannabis are retaining high affinity at the CB1 receptor, exhibiting greater efficacy than THC, and producing THC-like effects in models relevant to subjective effects in humans.

Novel Diarylurea Based Allosteric Modulators of the Cannabinoid CB1 Receptor: Evaluation of Importance of 6-Pyrrolidinylpyridinyl Substitution.

Allosteric modulators of the cannabinoid CB1 receptor have recently been reported as an alternative approach to modulate the CB1 receptor for therapeutic benefits. In this study, we report the design and synthesis of a series of diarylureas derived from PSNCBAM-1 (2). Similar to 2, these diarylureas dose-dependently inhibited CP55,940-induced intracellular calcium mobilization and [35S]GTP-γ-S binding while enhancing [3H]CP55,940 binding to the CB1 receptor. Structure-activity relationship studies revealed that the pyridinyl ring of 2 could be replaced by other aromatic rings and the pyrrolidinyl ring is not required for CB1 allosteric modulation. 34 (RTICBM-74) had similar potencies as 2 in all in vitro assays but showed significantly improved metabolic stability to rat liver microsomes. More importantly, 34 was more effective than 2 in attenuating the reinstatement of extinguished cocaine-seeking behavior in rats, demonstrating the potential of this diarylurea series as promising candidates for the development of relapse treatment of cocaine addiction.

The great divide: Separation between in vitro and in vivo effects of PSNCBAM-based CB1 receptor allosteric modulators.

While allosteric modulators of the cannabinoid type-1 receptor (CB1) continue to be developed and characterized, the gap between the in vitro and in vivo data is widening, raising questions regarding translatability of their effects and biological relevance. Among the CB1 allosteric modulators, PSNCBAM-1 has received little attention regarding its effects in vivo. Recently, pregnenolone was reported to act as an allosteric modulator of CB1, blocking THC's effects in vitro and in vivo, highlighting the potential of CB1 allosteric modulators for treatment of cannabis intoxication. We investigated the pharmacological effects of PSNCBAM-1 and two structural analogs, RTICBM-15 and -28, as well as pregnenolone, in both signaling and behavioral assays including [35S]GTPγS binding, the cannabinoid tetrad and drug discrimination. While the CB1 allosteric modulator PSNCBAM-1 attenuated THC-induced anti-nociception and its structural analog RTICBM-28 reduced THC's potency in drug discrimination, most cannabinoid effects in mice were unaffected. In contrast to the mouse studies, PSNCBAM-1 and analogs insurmountably antagonized CP55,940- and THC-stimulated [35S]GTPγS binding and exhibited negative binding cooperativity with [3H]SR141716 with similar apparent affinities. Notably, RTICBM-28, which contains a cyano substitution at the 4-chlorophenyl position of PSNCBAM-1, exhibited enhanced binding cooperativity with CP55,940. In contrast to previous findings, pregnenolone did not block THC's effects in drug discrimination or [35S]GTPγS. These data further highlight the difficulty in translating pharmacological effects of CB1 allosteric modulators in vivo but confirm the established pharmacology of PSNCBAM-1 and analogs in molecular assays of CB1 receptor function.