Discovery of a new class of cell-penetrating peptides by novel phage display platform.

The primary hurdles for small interference RNA (siRNA) in clinical use are targeted and cytosolic delivery. To overcome both challenges, we have established a novel platform based on phage display, called NNJA. In this approach, a lysosomal cathepsin substrate is engineered within the flexible loops of PIII, that is displaying a unique random sequence at its N-terminus. NNJA library selection targeting cell-expressed targets should yield specific peptides localized in the cytoplasm. That is because phage internalization and subsequent localization to lysosome, upon peptide binding to the cell expressed target, will result in cleavage of PIII, rendering phage non-infective. Such phage will be eliminated from the selected pool and only peptide-phage that escapes lysosomes will advance to the next round. Proof of concept studies with the NNJA library demonstrated cytosolic localization of selected peptide-phage and peptide-siRNA, confirmed through confocal microscopy. More importantly, conjugation of siHPRT to monomeric or multimeric NNJA peptides resulted in significant reduction in HPRT mRNA in various cell types without significant cytotoxicity. Sequence similarity and clustering analysis from NGS dataset provide insights into sequence composition facilitating cell penetration. NNJA platform offers a highly efficient peptide discovery engine for targeted delivery of oligonucleotides to cytosol.

Fumed-Si-Pr-PNS as a Photoluminescence sensor for the Detection of Hg2+ in Aqueous Media.

Fumed silica was functionalized by piperazine followed by the reaction with 2- naphthalenesulfonyl chloride to prepare Fumed-Si-Pr-Piperazine-Naphthalenesulfonyl chloride (Fumed-Si-Pr-PNS), which was characterized to demonstrate the effective attachment on the surface of fumed silica. The optical sensing ability of Fumed-Si-Pr-PNS was studied via diverse metal ions in H2O solution by photoluminescence spectroscopy. The results showed that Fumed-Si-Pr-PNS detected selectively Hg2+ ions. The prepared sensor showed almost high absorption at different pH for Hg ion. After drawing various diagrams, The detection limits were calculated at about 12.45 × 10-6 M for Hg2+.

PET imaging studies to investigate functional expression of mGluR2 using [11C]mG2P001.

Metabotropic glutamate receptor 2 (mGluR2) has been extensively studied for the treatment of various neurological and psychiatric disorders. Understanding of the mGluR2 function is pivotal in supporting the drug discovery targeting mGluR2. Herein, the positive allosteric modulation of mGluR2 was investigated via the in vivo positron emission tomography (PET) imaging using 2-((4-(2-[11C]methoxy-4-(trifluoromethyl)phenyl)piperidin-1-yl)methyl)-1-methyl-1H-imidazo[4,5-b]pyridine ([11C]mG2P001). Distinct from the orthosteric compounds, pretreatment with the unlabeled mG2P001, a potent mGluR2 positive allosteric modulator (PAM), resulted in a significant increase instead of decrease of the [11C]mG2P001 accumulation in rat brain detected by PET imaging. Subsequent in vitro studies with [3H]mG2P001 revealed the cooperative binding mechanism of mG2P001 with glutamate and its pharmacological effect that contributed to the enhanced binding of [3H]mG2P001 in transfected CHO cells expressing mGluR2. The in vivo PET imaging and quantitative analysis of [11C]mG2P001 in non-human primates (NHPs) further validated the characteristics of [11C]mG2P001 as an imaging ligand for mGluR2. Self-blocking studies in primates enhanced accumulation of [11C]mG2P001. Altogether, these studies show that [11C]mG2P001 is a sensitive biomarker for mGluR2 expression and the binding is affected by the tissue glutamate concentration.

Design, Synthesis, and Characterization of [18F]mG2P026 as a High-Contrast PET Imaging Ligand for Metabotropic Glutamate Receptor 2.

An array of triazolopyridines based on JNJ-46356479 (6) were synthesized as potential positron emission tomography radiotracers for metabotropic glutamate receptor 2 (mGluR2). The selected candidates 8-10 featured enhanced positive allosteric modulator (PAM) activity (20-fold max.) and mGluR2 agonist activity (25-fold max.) compared to compound 6 in the cAMP GloSensor assays. Radiolabeling of compounds 8 and 9 (mG2P026) was achieved via Cu-mediated radiofluorination with satisfactory radiochemical yield, >5% (non-decay-corrected); high molar activity, >180 GBq/µmol; and excellent radiochemical purity, >98%. Preliminary characterization of [18F]8 and [18F]9 in rats confirmed their excellent brain permeability and binding kinetics. Further evaluation of [18F]9 in a non-human primate confirmed its superior brain heterogeneity in mapping mGluR2 and higher affinity than [18F]6. Pretreatment with different classes of PAMs in rats and a primate led to similarly enhanced brain uptake of [18F]9. As a selective ligand, [18F]9 has the potential to be developed for translational studies.

Longitudinal PET studies of mGluR5 in FXS using an FMR1 knockout mouse model.

Fragile X syndrome (FXS) is a monogenic disorder characterized by intellectual disability and behavioral challenges. It is caused by aberrant methylation of the fragile X mental retardation 1 (FMR1) gene. Given the failure of clinical trials in FXS and growing evidence of a role of metabotropic glutamate subtype 5 receptors (mGluR5) in the pathophysiology of the disorder, we investigated mGluR5 function in FMR1 Knockout (FMR1-KO) mice and age- and sex-matched control mice using longitudinal positron emission tomography (PET) imaging to better understand the disorder. The studies were repeated at four time points to examine age- and disease-induced changes in mGluR5 availability using 3-fluoro-[18F]5-(2-pyridinylethynyl)benzonitrile ([18F]FPEB). We found that the binding potential (BP) of [18F]FPEB was significantly lower in the KO mice in mGluR5-implicated brain areas including striatum, cortex, hippocampus, thalamus, and olfactory bulb. The BP also changed with age, regardless of disorder status, increasing in early adulthood in male but not in female mice before decreasing later in both sexes. The difference in mGluR5 availability between the FMR1-KO and control mice and the change in BP in the KO mice as a function of age and sex illustrate the nature of the disorder and its progression, providing mechanistic insights for treatment design.

Synthesis and characterization of a new Positron emission tomography probe for orexin 2 receptors neuroimaging.

The orexin receptors (OXRs) have been involved in multiple physiological and neuropsychiatric functions. Identification of PET imaging probes specifically targeting OXRs enables us to better understand the OX system. Seltorexant (JNJ-42847922) is a potent OX2R antagonist with the potential to be an OX2R PET imaging probe. Here, we describe the synthesis and characterization of [18F]Seltorexant as an OX2R PET probe. The ex vivo autoradiography studies indicated the good binding specificity of [18F]Seltorexant. In vivo PET imaging of [18F]Seltorexant in rodents showed suitable BBB penetration with the highest brain uptake of %ID/cc = 3.4 at 2 min post-injection in mice. The regional brain biodistribution analysis and blocking studies showed that [18F]Seltorexant had good binding selectivity and specificity. However, pretreatment with unlabelled Seltorexant and P-gp competitor CsA observed significantly increased brain uptake of [18F]Seltorexant, indicating [18F]Seltorexant could interact P-gp at the blood-brain barrier. Our findings demonstrated that [18F]Seltorexant is a potential brain OX2R PET imaging probe, which paves the way for new OX2R PET probes development and OX system investigation.

Preliminary studies of an imidazole-based alcohol derivative for imaging of Heme oxygenase 1.

Heme oxygenase-1 (HO-1) has been involved in the pathogenesis of Alzheimer's disease (AD), thus constituting a promising target for AD drug development. Positron emission tomography (PET) is a fully translational imaging technology, which will help us understand the role of HO-1 in the progression of AD, facilitating to validate promising HO-1 inhibitors in clinical trials. To our knowledge, there is no report on PET imaging probe targeting HO-1 in animals and humans. We report herein the synthesis and characterization of a 11C-labeled imidazole-based alcohol derivative ([11C]QC-33) for imaging of HO-1 in the brain. The desired product [11C]QC-33 was afforded with a radiochemical yield of 16 ± 9% (n = 3, decay corrected). The radiochemical purity was greater than 99%, and the molar radioactivity was greater than 185 GBq/µmol. In vitro autoradiography studies indicated specific binding of [11C]QC-33 in the HO-1 rich regions, showing 75%, 75%, and 69% radioactivity binding reductions in cerebellum, brain stem, and midbrain, respectively. PET/CT scanning in C57BL/6 mice showed low brain uptake and poor blood-brain barrier (BBB) penetration of [11C]QC-33. These results suggested that [11C]QC-33 can serve as a lead compound to advance the development of next generation PET tracer with the potential to monitor HO-1 in AD progression.

Synthesis and Characterization of 5-(2-Fluoro-4-[11C]methoxyphenyl)-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridine-7-carboxamide as a PET Imaging Ligand for Metabotropic Glutamate Receptor 2.

Metabotropic glutamate receptor 2 (mGluR2) is a therapeutic target for several neuropsychiatric disorders. An mGluR2 function in etiology could be unveiled by positron emission tomography (PET). In this regard, 5-(2-fluoro-4-[11C]methoxyphenyl)-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridine-7-carboxamide ([11C]13, [11C]mG2N001), a potent negative allosteric modulator (NAM), was developed to support this endeavor. [11C]13 was synthesized via the O-[11C]methylation of phenol 24 with a high molar activity of 212 ± 76 GBq/µmol (n = 5) and excellent radiochemical purity (>99%). PET imaging of [11C]13 in rats demonstrated its superior brain heterogeneity and reduced accumulation with pretreatment of mGluR2 NAMs, VU6001966 (9) and MNI-137 (26), the extent of which revealed a time-dependent drug effect of the blocking agents. In a nonhuman primate, [11C]13 selectively accumulated in mGluR2-rich regions and resulted in high-contrast brain images. Therefore, [11C]13 is a potential candidate for translational PET imaging of the mGluR2 function.

Visualization of Receptor-Interacting Protein Kinase 1 (RIPK1) by Brain Imaging with Positron Emission Tomography.

We report the development of the first positron emission tomography (PET) radiotracer, [18F]CNY-07, based on a highly specific and potent RIPK1 inhibitor, Nec-1s, for RIPK1/necroptosis brain imaging in rodents. [18F]CNY-07 was synthesized through copper-mediated 18F-radiolabeling from an aryl boronic ester precursor and studied in vivo PET imaging in rodents. PET imaging results showed that [18F]CNY-07 can penetrate the blood-brain barrier with a maximum percent injected dose per unit volume of 3 at 10 min postinjection in the brain in vivo. Self-blocking studies of [18F]CNY-07 by pretreating with unlabeled molecules in rodents showed reduced radioactivity in animal brains (30% radioactivity decreased), indicating the binding specificity of our radiotracer. Our studies demonstrate that [18F]CNY-07 has provided a useful PET radioligand enabling brain RIPK1 imaging, which could be a valuable research tool in studying RIPK1-related neurological disorders in animals and potentially humans.

Design, Synthesis, and Evaluation of Thienodiazepine Derivatives as Positron Emission Tomography Imaging Probes for Bromodomain and Extra-Terminal Domain Family Proteins.

To better understand the role of bromodomain and extra-terminal domain (BET) proteins in epigenetic mechanisms, we developed a series of thienodiazepine-based derivatives and identified two compounds, 3a and 6a, as potent BET inhibitors. Further in vivo pharmacokinetic studies and analysis of in vitro metabolic stability of 6a revealed excellent brain penetration and reasonable metabolic stability. Compounds 3a and 6a were radiolabeled with fluorine-18 in two steps and utilized in positron emission tomography (PET) imaging studies in mice. Preliminary PET imaging results demonstrated that [18F]3a and [18F]6a have good brain uptake (with maximum SUV = 1.7 and 2, respectively) and binding specificity in mice brains. These results show that [18F]6a is a potential PET radiotracer that could be applied to imaging BET proteins in the brain. Further optimization and improvement of the metabolic stability of [18F]6a are still needed in order to create optimal PET imaging probes of BET family members.

Synthesis and Characterization of a Positron Emission Tomography Imaging Probe Selectively Targeting the Second Bromodomain of Bromodomain Protein BRD4.

Two tandem bromodomains (BD1 and BD2) of bromodomain and extraterminal domain (BET) family proteins have shown distinct roles in mediating gene transcription and expression. Inhibitors that interact with a specific bromodomain may contribute to a specific therapeutic potential with fewer side effects. However, little is known about this disease-related target. Positron emission tomography (PET) imaging could allow us to achieve in-depth knowledge of the BD2 bromodomain. Herein we describe the radiosynthesis and evaluation of [11C]1 as a BRD4 BD2 bromodomain PET imaging radioligand. Our preliminary PET imaging results in rodents demonstrated that [11C]1 had suitable biodistribution in peripheral organs and tissues. Further blocking studies indicated that [11C]1 had good binding specificity toward the BD2 bromodomain. This study may pave the way for the development of a PET radioligand specifically targeting BD1/2 bromodomains as well as for the biological mechanism investigation of BD1/2 bromodomains.

Therapeutic Advances in Diabetes, Autoimmune, and Neurological Diseases.

Since 2015, 170 small molecules, 60 antibody-based entities, 12 peptides, and 15 gene- or cell-therapies have been approved by FDA for diverse disease indications. Recent advancement in medicine is facilitated by identification of new targets and mechanisms of actions, advancement in discovery and development platforms, and the emergence of novel technologies. Early disease detection, precision intervention, and personalized treatments have revolutionized patient care in the last decade. In this review, we provide a comprehensive overview of current and emerging therapeutic modalities developed in the recent years. We focus on nine diseases in three major therapeutics areas, diabetes, autoimmune, and neurological disorders. The pathogenesis of each disease at physiological and molecular levels is discussed and recently approved drugs as well as drugs in the clinic are presented.

Therapeutic Advances in Oncology.

Around 77 new oncology drugs were approved by the FDA in the past five years; however, most cancers remain untreated. Small molecules and antibodies are dominant therapeutic modalities in oncology. Antibody-drug conjugates, bispecific antibodies, peptides, cell, and gene-therapies are emerging to address the unmet patient need. Advancement in the discovery and development platforms, identification of novel targets, and emergence of new technologies have greatly expanded the treatment options for patients. Here, we provide an overview of various therapeutic modalities and the current treatment options in oncology, and an in-depth discussion of the therapeutics in the preclinical stage for the treatment of breast cancer, lung cancer, and multiple myeloma.

Synthesis and Characterization of [18F]JNJ-46356479 as the First 18F-Labeled PET Imaging Ligand for Metabotropic Glutamate Receptor 2.

PURPOSE: Metabotropic glutamate receptor 2 (mGluR2) has been implicated in various psychiatric and neurological disorders, such as schizophrenia and Alzheimer's disease. We have previously developed [11C]7 as a PET radioligand for imaging mGluR2. Herein, [18F]JNJ-46356479 ([18F]8) was synthesized and characterized as the first 18F-labeled mGluR2 imaging ligand to enhance diagnostic approaches for mGluR2-related disorders. PROCEDURES: JNJ-46356479 (8) was radiolabeled via the copper (I)-mediated radiofluorination of organoborane 9. In vivo PET imaging experiments with [18F]8 were conducted first in C57BL/6 J mice and Sprague-Dawley rats to obtain whole body biodistribution and brain uptake profile. Subsequent PET studies were done in a cynomolgus monkey (Macaca fascicularis) to investigate the uptake of [18F]8 in the brain, its metabolic stability, as well as pharmacokinetic properties. RESULTS: JNJ-46356479 (8) exhibited excellent selectivity against other mGluRs. In vivo PET imaging studies showed reversible and specific binding characteristic of [18F]8 in rodents. In the non-human primate, [18F]8 displayed good in vivo metabolic stability, excellent brain permeability, fast and reversible kinetics with moderate heterogeneity across brain regions. Pre-treatment studies with compound 7 revealed time-dependent decrease of [18F]8 accumulation in mGluR2 rich regions based on SUV values with the highest decrease in the nucleus accumbens (18.7 ± 5.9%) followed by the cerebellum (18.0 ± 7.9%), the parietal cortex (16.9 ± 7.8%), and the hippocampus (16.8 ± 6.9%), similar to results obtained in the rat studies. However, the volume of distribution (VT) results derived from 2T4k model showed enhanced VT from a blocking study with compound 7. This is probably because of the potentiating effect of compound 7 as an mGluR2 PAM as well as related non-specific binding in the tissue data. CONCLUSIONS: [18F]8 readily crosses the blood-brain barrier and demonstrates fast and reversible kinetics both in rodents and in a non-human primate. Further investigation of [18F]8 on its binding specificity would warrant translational study in human.

A novel phage display vector for selection of target-specific peptides.

Intrinsic low display level of polypeptides on phage is a fundamental and limiting hurdle in successful isolation of target-specific binders by phage display technology. To circumvent this challenge, we optimized the copy number of peptides displayed on the phage surface using type 33 phage vector. We randomized the first 67 amino acids of the wild type PIII to identify mutants that would result in its reduced expression. Consequently, the display level was improved by 30-fold due to higher incorporation of the synthetic PIII-peptide fusion protein on the phage surface. Utilization of this novel phage vector should provide a solid basis for the discovery of therapeutic peptides.

Design, Synthesis, and Characterization of Benzimidazole Derivatives as Positron Emission Tomography Imaging Ligands for Metabotropic Glutamate Receptor 2.

Three benzimidazole derivatives (13-15) have been synthetized as potential positron emission tomography (PET) imaging ligands for mGluR2 in the brain. Of these compounds, 13 exhibits potent binding affinity (IC50 = 7.6 ± 0.9 nM), positive allosteric modulator (PAM) activity (EC50 = 51.2 nM), and excellent selectivity against other mGluR subtypes (>100-fold). [11C]13 was synthesized via O-[11C]methylation of its phenol precursor 25 with [11C]methyl iodide. The achieved radiochemical yield was 20 ± 2% (n = 10, decay-corrected) based on [11C]CO2 with a radiochemical purity of >98% and molar activity of 98 ± 30 GBq/µmol EOS. Ex vivo biodistribution studies revealed reversible accumulation of [11C]13 and hepatobiliary and urinary excretions. PET imaging studies in rats demonstrated that [11C]13 accumulated in the mGluR2-rich brain regions. Pre-administration of mGluR2-selective PAM, 17 reduced the brain uptake of [11C]13, indicating a selective binding. Therefore, [11C]13 is a potential PET imaging ligand for mGluR2 in different central nervous system-related conditions.

Impact of non-proteinogenic amino acids in the discovery and development of peptide therapeutics.

With the development of modern chemistry and biology, non-proteinogenic amino acids (NPAAs) have become a powerful tool for developing peptide-based drug candidates. Drug-like properties of peptidic medicines, due to the smaller size and simpler structure compared to large proteins, can be changed fundamentally by introducing NPAAs in its sequence. While peptides composed of natural amino acids can be used as drug candidates, the majority have shown to be less stable in biological conditions. The impact of NPAA incorporation can be extremely beneficial in improving the stability, potency, permeability, and bioavailability of peptide-based therapies. Conversely, undesired effects such as toxicity or immunogenicity should also be considered. The impact of NPAAs in the development of peptide-based therapeutics is reviewed in this article. Further, numerous examples of peptides containing NPAAs are presented to highlight the ongoing development in peptide-based therapeutics.

Peptides to combat viral infectious diseases.

Viral infectious diseases have resulted in millions of deaths throughout history and have created a significant public healthcare burden. Tremendous efforts have been placed by the scientific communities, health officials and government organizations to detect, treat, and prevent viral infection. However, the complicated life cycle and rapid genetic mutations of viruses demand continuous development of novel medicines with high efficacy and safety profiles. Peptides provide a promising outlook as a tool to combat the spread and re-emergence of viral infection. This article provides an overview of five viral infectious diseases with high global prevalence: influenza, chronic hepatitis B, acquired immunodeficiency syndrome, severe acute respiratory syndrome, and coronavirus disease 2019. The current and potential peptide-based therapies, vaccines, and diagnostics for each disease are discussed.

In Vitro Assays: Friends or Foes of Cell-Penetrating Peptides.

The cell membrane is a complex and highly regulated system that is composed of lipid bilayer and proteins. One of the main functions of the cell membrane is the regulation of cell entry. Cell-penetrating peptides (CPPs) are defined as peptides that can cross the plasma membrane and deliver their cargo inside the cell. The uptake of a peptide is determined by its sequence and biophysicochemical properties. At the same time, the uptake mechanism and efficiency are shown to be dependent on local peptide concentration, cell membrane lipid composition, characteristics of the cargo, and experimental methodology, suggesting that a highly efficient CPP in one system might not be as productive in another. To better understand the dependence of CPPs on the experimental system, we present a review of the in vitro assays that have been employed in the literature to evaluate CPPs and CPP-cargos. Our comprehensive review suggests that utilization of orthogonal assays will be more effective for deciphering the true ability of CPPs to translocate through the membrane and enter the cell cytoplasm.

Integration of phage and yeast display platforms: A reliable and cost effective approach for binning of peptides as displayed on-phage.

Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.