Piplartine attenuates aminoglycoside-induced TRPV1 activity and protects from hearing loss in mice.

Hearing loss is a major health concern in our society, affecting more than 400 million people worldwide. Among the causes, aminoglycoside therapy can result in permanent hearing loss in 40% to 60% of patients receiving treatment, and despite these high numbers, no drug for preventing or treating this type of hearing loss has yet been approved by the US Food and Drug Administration. We have previously conducted high-throughput screenings of bioactive compounds, using zebrafish as our discovery platform, and identified piplartine as a potential therapeutic molecule. In the present study, we expanded this work and characterized piplartine's physicochemical and therapeutic properties. We showed that piplartine had a wide therapeutic window and neither induced nephrotoxicity in vivo in zebrafish nor interfered with aminoglycoside antibacterial activity. In addition, a fluorescence-based assay demonstrated that piplartine did not inhibit cytochrome C activity in microsomes. Coadministration of piplartine protected from kanamycin-induced hair cell loss in zebrafish and protected hearing function, outer hair cells, and presynaptic ribbons in a mouse model of kanamycin ototoxicity. Last, we investigated piplartine's mechanism of action by phospho-omics, immunoblotting, immunohistochemistry, and molecular dynamics experiments. We found an up-regulation of AKT1 signaling in the cochleas of mice cotreated with piplartine. Piplartine treatment normalized kanamycin-induced up-regulation of TRPV1 expression and modulated the gating properties of this receptor. Because aminoglycoside entrance to the inner ear is, in part, mediated by TRPV1, these results suggested that by regulating TRPV1 expression, piplartine blocked aminoglycoside's entrance, thereby preventing the long-term deleterious effects of aminoglycoside accumulation in the inner ear compartment.

Enhanced Hydrogen Bonding by Urea Functionalization Tunes the Stability and Biological Properties of Peptide Amphiphiles.

Self-assembled nanostructures such as those formed by peptide amphiphiles (PAs) are of great interest in biological and pharmacological applications. Herein, a simple and widely applicable chemical modification, a urea motif, was included in the PA's molecular structure to stabilize the nanostructures by virtue of intermolecular hydrogen bonds. Since the amino acid residue nearest to the lipid tail is the most relevant for stability, we decided to include the urea modification at that position. We prepared four groups of molecules (13 PAs in all), with varying levels of intermolecular cohesion, using amino acids with distinct ß-sheet promoting potential and/or containing hydrophobic tails of distinct lengths. Each subset contained one urea-modified PA and nonmodified PAs, all with the same peptide sequence. The varied responses of these PAs to variations in pH, temperature, counterions, and biologically related proteins were examined using microscopic, X-ray, spectrometric techniques, and molecular simulations. We found that the urea group contributes to the stabilization of the morphology and internal arrangement of the assemblies against environmental stimuli for all peptide sequences. In addition, microbiological and biological studies were performed with the cationic PAs. These assays reveal that the addition of urea linkages affects the PA-cell membrane interaction, showing the potential to increase the selectivity toward bacteria. Our data indicate that the urea motif can be used to tune the stability of a wide range of PA nanostructures, allowing flexibility on the biomaterial's design and opening a myriad of options for clinical therapies.

Structure-Activity Relationship Study to Develop Peptide Amphiphiles as Species-Specific Antimicrobials.

Antimicrobial peptide amphiphiles (PAs) are a promising class of molecules that can disrupt the bacterial membrane or act as drug nanocarriers. In this study, we prepared 33 PAs to establish supramolecular structure-activity relationships. We studied the morphology and activity of the nanostructures against different Gram-positive and Gram-negative bacterial strains (such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii). Next, we used principal component analysis (PCA) to determine the key contributors to activity. We found that for S. aureus, the zeta potential was the major contributor to the activity while Gram-negative bacteria were more influenced by the partition coefficient (LogP) with the following order P. aeruginosa>E. coli>A. baumannii. We also performed a study of the mechanism of action of selected PAs on the bacterial membrane assessing the membrane permeability and depolarization, changes in zeta potential and overall integrity. We studied the toxicity of the nanostructures against mammalian cells. Finally, we performed an in vivo study using the wax moth larvae to determine the therapeutic efficacy of the active PAs. This study shows cationic PA nanostructures can be an intriguing platform for the development of nanoantibacterials.

Urea-Modified Self-Assembling Peptide Amphiphiles That Form Well-Defined Nanostructures and Hydrogels for Biomedical Applications.

Hydrogen bonding plays a critical role in the self-assembly of peptide amphiphiles (PAs). Herein, we studied the effect of replacing the amide linkage between the peptide and lipid portions of the PA with a urea group, which possesses an additional hydrogen bond donor. We prepared three PAs with the peptide sequence Phe-Phe-Glu-Glu (FFEE): two are amide-linked with hydrophobic tails of different lengths and the other possesses an alkylated urea group. The differences in the self-assembled structures formed by these PAs were assessed using diverse microscopies, nuclear magnetic resonance (NMR), and dichroism techniques. We found that the urea group influences the morphology and internal arrangement of the assemblies. Molecular dynamics simulations suggest that there are about 50% more hydrogen bonds in nanostructures assembled from the urea-PA than those assembled from the other PAs. Furthermore, in silico studies suggest the presence of urea-π stacking interactions with the phenyl group of Phe, which results in distinct peptide conformations in comparison to the amide-linked PAs. We then studied the effect of the urea modification on the mechanical properties of PA hydrogels. We found that the hydrogel made of the urea-PA exhibits increased stability and self-healing ability. In addition, it allows cell adhesion, spreading, and growth as a matrix. This study reveals that the inclusion of urea bonds might be useful in controlling the morphology, mechanical, and biological properties of self-assembled nanostructures and hydrogels formed by the PAs.

Control of Peptide Amphiphile Supramolecular Nanostructures by Isosteric Replacements.

Supramolecular nanostructures with tunable properties can have applications in medicine, pharmacy, and biotechnology. In this work, we show that the self-assembly behavior of peptide amphiphiles (PAs) can be effectively tuned by replacing the carboxylic acids exposed to the aqueous media with isosteres, functionalities that share key physical or chemical properties with another chemical group. Transmission electron microscopy, atomic force microscopy, and small-angle X-ray scattering studies indicated that the nanostructure's morphologies are responsive to the ionization states of the side chains, which are related to their pKa values. Circular dichroism studies revealed the effect of the isosteres on the internal arrangement of the nanostructures. The interactions between diverse surfaces and the nanostructures and the effect of salt concentration and temperature were assessed to further understand the properties of these self-assembled systems. These results indicate that isosteric replacements allow the pH control of supramolecular morphology by manipulating the pKa of the charged groups located on the nanostructure's surface. Theoretical studies were performed to understand the morphological transitions that the nanostructures underwent in response to pH changes, suggesting that the transitions result from alterations in the Coulomb forces between PA molecules. This work provides a strategy for designing biomaterials that can maintain or change behaviors based on the pH differences found within cells and tissues.

Targeting SARS-CoV-2 Receptor Binding Domain with Stapled Peptides: An In Silico Study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into a pandemic of unprecedented scale. This coronavirus enters cells by the interaction of the receptor binding domain (RBD) with the human angiotensin-converting enzyme 2 receptor (hACE2). In this study, we employed a rational structure-based design to propose 22-mer stapled peptides using the structure of the hACE2 α1 helix as a template. These peptides were designed to retain the α-helical character of the natural structure, to enhance binding affinity, and to display a better solubility profile compared to other designed peptides available in the literature. We employed different docking strategies (PATCHDOCK and ZDOCK) followed by a double-step refinement process (FIBERDOCK) to rank our peptides, followed by stability analysis/evaluation of the interaction profile of the best docking predictions using a 500 ns molecular dynamics (MD) simulation, and a further binding affinity analysis by molecular mechanics with generalized Born and surface area (MM/GBSA) method. Our most promising stapled peptides presented a stable profile and could retain important interactions with the RBD in the presence of the E484K RBD mutation. We predict that these peptides can bind to the viral RBD with similar potency to the control NYBSP-4 (a 30-mer experimentally proven peptide inhibitor). Furthermore, our study provides valuable information for the rational design of double-stapled peptide as inhibitors of SARS-CoV-2 infection.

The ClpX and ClpP2 Orthologs of Chlamydia trachomatis Perform Discrete and Essential Functions in Organism Growth and Development.

Chlamydia trachomatis is an obligate intracellular bacterium that undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms, the elementary body (EB) and reticulate body (RB), each of which expresses its own specialized repertoire of proteins. Both primary (EB to RB) and secondary (RB to EB) differentiations require protein turnover, and we hypothesize that proteases are critical for mediating differentiation. The Clp protease system is well conserved in bacteria and important for protein turnover. Minimally, the system relies on a serine protease subunit, ClpP, and an AAA+ ATPase, such as ClpX, that recognizes and unfolds substrates for ClpP degradation. In Chlamydia, ClpX is encoded within an operon 3' to clpP2 We present evidence that the chlamydial ClpX and ClpP2 orthologs are essential to organism viability and development. We demonstrate here that chlamydial ClpX is a functional ATPase and forms the expected homohexamer in vitro Overexpression of a ClpX mutant lacking ATPase activity had a limited impact on DNA replication or secondary differentiation but, nonetheless, reduced EB viability with observable defects in EB morphology noted. Conversely, overexpression of a catalytically inactive ClpP2 mutant significantly impacted developmental cycle progression by reducing the overall number of organisms. Blocking clpP2X transcription using CRISPR interference led to a decrease in bacterial growth, and this effect was complemented in trans by a plasmid copy of clpP2 Taken together, our data indicate that ClpX and the associated ClpP2 serve distinct functions in chlamydial developmental cycle progression and differentiation.IMPORTANCEChlamydia trachomatis is the leading cause of infectious blindness globally and the most reported bacterial sexually transmitted infection both domestically and internationally. Given the economic burden, the lack of an approved vaccine, and the use of broad-spectrum antibiotics for treatment of infections, an understanding of chlamydial growth and development is critical for the advancement of novel targeted antibiotics. The Clp proteins comprise an important and conserved protease system in bacteria. Our work highlights the importance of the chlamydial Clp proteins to this clinically important bacterium. Additionally, our study implicates the Clp system playing an integral role in chlamydial developmental cycle progression, which may help establish models of how Chlamydia spp. and other bacteria progress through their respective developmental cycles. Our work also contributes to a growing body of Clp-specific research that underscores the importance and versatility of this system throughout bacterial evolution and further validates Clp proteins as drug targets.

Protecting Groups in Peptide Synthesis.

The protection of amino acid reactive functionalities including the α-amino group, the side chain (amines, carboxylic acids, alcohols, and thiols), or the carboxylic acid terminus is an essential strategy in peptide chemistry. This is mandatory to prevent polymerization of the amino acids and to minimize undesirable side reactions during the synthetic process. Proper protecting group manipulation strategies can maximize the yield of the desired product or allow the construction of complex peptide-based structures. Thus, the compatibility and orthogonality of each protecting group are key to achieve the proper control of molecular structure. Herein, we describe some common protecting groups and their general unmasking methods, in order to mask and expose amine, carboxylic acid, alcohol, and thiol functionalities to achieve the synthesis of peptides and related molecules.

A review of the molecular design and biological activities of RXR agonists.

An attractive approach to combat disease is to target theregulation of cell function. At the heart of this task are nuclear receptors (NRs); which control functions such as gene transcription. Arguably, the key player in this regulatory machinery is the retinoid X receptor (RXR). This NR associates with a third of the NRs found in humans. Scientists have hypothesized that controlling the activity of RXR is an attractive approach to control cellular functions that modulate diseases such as cancer, diabetes, Alzheimer's disease and Parkinson's disease. In this review, we will describe the key features of the RXR, present a historic perspective of the first RXR agonists, and discuss various templates that have been reported to activate RXR with a focus on their molecular structure, biological activity, and limitations. Finally, we will present an outlook of the field and future directions and considerations to synthesize or modulate RXR agonists to make these compounds a clinical reality.

Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each.

Members of Chlamydia are obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other. Chlamydia spp. have five uncharacterized clp genes, clpX, clpC, two clpP paralogs, and clpB In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactive clpP mutants in Chlamydia spp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detected in vitro This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen.IMPORTANCEChlamydia trachomatis is the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression in Chlamydia spp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials.

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Developing Polyamine-Based Peptide Amphiphiles with Tunable Morphology and Physicochemical Properties.

The ability to tune supramolecular properties such as size, morphology, or metabolic stability is of paramount importance in the field of supramolecular chemistry. Peptide amphiphiles (PAs) are a family of functional self-assembling biomaterials that have garnered widespread attention due to their broad applicability in medicine. PAs are generally comprised of an amino acid sequence connected to lipid tail(s) allowing them to self-assemble into supramolecular structures with diverse morphologies. Herein, this study describes the synthesis of a new class of polyamine-based "hybrid" PAs (PPAs) as novel self-assembling systems. The described molecules possess diverse polyamine head groups with the goal of tuning physicochemical properties. The findings indicate that small changes in the polyamine head groups result in altered PPA morphologies (nanofibers, micelles, nanoworms). The PPAs present a wide range of physicochemical characteristics, show superior resistance to aggregation, a diverse metabolic profile, and varied assembling kinetics. Most of the PPAs do not show toxicity in the human cells lines evaluated. The PPAs described herein hold promising potential as a safe and nontoxic option for drug delivery, targeting, and tissue engineering applications.

Design and synthesis of new piperidone grafted acetylcholinesterase inhibitors.

Alzheimer's disease (AD) is a neurodegenerative disorder affecting 35million people worldwide. A common strategy to improve the well-being of AD patients consists on the inhibition of acetylcholinesterase with the concomitant increase of the neurotransmitter acetylcholine at cholinergic synapses. Two series of unreported N-benzylpiperidines 5(a-h) and thiazolopyrimidines 9(a-q) molecules were synthesized and evaluated in vitro for their acetylcholinesterase (AChE) inhibitory activities. Among the newly synthesized compounds, 5h, 9h, 9j, and 9p displayed higher AChE enzyme inhibitory activities than the standard drug, galantamine, with IC50 values of 0.83, 0.98, and 0.73µM, respectively. Cytotoxicity studies of 5h, 9h, 9j, 9n and 9p on human neuroblastoma cells SH-SY5Y, showed no toxicity up to 40µM concentration. Molecular docking simulations of the active compounds 5h and 9p disclosed the crucial role of π-π-stacking in their binding interaction to the active site AChE enzyme. The presented compounds have potential as AChE inhibitors and potential AD drugs.

Esterase-activated release of naproxen from supramolecular nanofibres.

Nanofibre forming peptide amphiphiles were conjugated to naproxen through an esterase-sensitive linker. The amount of naproxen released, in the presence of enzymes, was influenced by the linker conjugating the drug to the supramolecular assembly. In vitro studies showed the anti-inflammatory activity of the released drug was maintained.

Design, synthesis, and biological evaluation of indenoisoquinoline rexinoids with chemopreventive potential.

Nuclear receptors, such as the retinoid X receptor (RXR), are proteins that regulate a myriad of cellular processes. Molecules that function as RXR agonists are of special interest for the prevention and control of carcinogenesis. The majority of these ligands possess an acidic moiety that is believed to be key for RXR activation. This communication presents the design, synthesis, and biological evaluation of both acidic and nonacidic indenoisoquinolines as new RXR ligands. In addition, a comprehensive structure-activity relationship study is presented that identifies the important features of the indenoisoquinoline rexinoids. The ease of modification of the indenoisoquinoline core and the lack of the necessity of a carboxyl group for activity make them an attractive and unusual family of RXR agonists. This work establishes a structural foundation for the design of new and novel rexinoid cancer chemopreventive agents.

Synthesis and biological evaluation of indenoisoquinolines that inhibit both tyrosyl-DNA phosphodiesterase I (Tdp1) and topoisomerase I (Top1).

Tyrosyl-DNA phosphodiesterase I (Tdp1) plays a key role in the repair of damaged DNA resulting from the topoisomerase I (Top1) inhibitor camptothecin and a variety of other DNA-damaging anticancer agents. This report documents the design, synthesis, and evaluation of new indenoisoquinolines that are dual inhibitors of both Tdp1 and Top1. Enzyme inhibitory data and cytotoxicity data from human cancer cell cultures were used to establish structure-activity relationships. The potencies of the indenoisoquinolines against Tdp1 ranged from 5 µM to 111 µM, which places the more active compounds among the most potent known inhibitors of this target. The cytotoxicity mean graph midpoints ranged from 0.02 to 2.34 µM. Dual Tdp1-Top1 inhibitors are of interest because the Top1 and Tdp1 inhibitory activities could theoretically work synergistically to create more effective anticancer agents.

Identification, synthesis, and biological evaluation of the metabolites of 3-amino-6-(3'-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11-(6H)dione (AM6-36), a promising rexinoid lead compound for the development of cancer chemotherapeutic and chemopreventive agents.

Activation of the retinoid X receptor (RXR), which is involved in cell proliferation, differentiation, and apoptosis, is a strategy for cancer chemotherapy and chemoprevention, and 3-amino-6-(3'-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11-(6H)dione (AM6-36) (3) is among the few RXR ligands known. The presently reported studies of 3 include its binding to human plasma proteins, metabolic stability using human liver microsomes, metabolism by human liver microsomes and hepatocytes, and in vivo disposition in rat serum, liver, and mammary tissue. Compound 3 was 75% bound to human plasma proteins, and its metabolic stability was much greater than propranolol. One phase I metabolite was formed by human liver microsomes, seven phase I and II metabolites were formed by human hepatocytes, and five metabolites were detected in rat serum and liver after oral administration. The putative metabolites predicted using LC-MS-MS were synthesized to confirm their structures and to provide sufficient material for investigation of induction of RXRE transcriptional activity and inhibition of NFκB.

Synthesis and biological evaluation of the first dual tyrosyl-DNA phosphodiesterase I (Tdp1)-topoisomerase I (Top1) inhibitors.

Substances with dual tyrosyl-DNA phosphodiesterase I-topoisomerase I inhibitory activity in one low molecular weight compound would constitute a unique class of anticancer agents that could potentially have significant advantages over drugs that work against the individual enzymes. The present study demonstrates the successful synthesis and evaluation of the first dual Top1-Tdp1 inhibitors, which are based on the indenoisoquinoline chemotype. One bis(indenoisoquinoline) had significant activity against human Tdp1 (IC(50) = 1.52 ± 0.05 µM), and it was also equipotent to camptothecin as a Top1 inhibitor. Significant insights into enzyme-drug interactions were gained via structure-activity relationship studies of the series. The present results also document the failure of the previously reported sulfonyl ester pharmacophore to confer Tdp1 inhibition in this indenoisoquinoline class of inhibitors even though it was demonstrated to work well for the steroid NSC 88915 (7). The current study will facilitate future efforts to optimize dual Top1-Tdp1 inhibitors.

Induction of apoptosis by 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline via modulation of MAPKs (p38 and c-Jun N-terminal kinase) and c-Myc in HL-60 human leukemia cells.

Recently, we reported that 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (AM6-36), sharing structural similarity with naturally occurring isoquinolines, induced activities mediated by retinoid X receptor (RXR) response element accompanied by antiproliferative effects on breast cancer cells. To further characterize the biologic potential of AM6-36, we currently report studies conducted with HL-60 human leukemia cells. AM6-36 significantly inhibited cellular proliferation in a dose- and time-dependent manner with an IC(50) value of 86 nM. When evaluated at low test concentrations (≤0.25 µM), AM6-36 induced arrest in the G2/M phase of the cell cycle. At higher concentrations (1 and 2 µM), the response shifted to apoptosis, which was consistent with the effect of AM6-36 on other apoptotic signatures including an increase of apoptotic annexin V(+) 7-AAD(-) cells, loss of mitochondrial membrane potential, induction of poly(ADP-ribose) polymerase cleavage, and activation of several caspases. These apoptotic effects are potentially due to up-regulation of p38 MAPK and JNK phosphorylation and down-regulation of c-Myc oncogene expression. Taken together, AM6-36 might serve as an effective anticancer agent by inducing G2/M cell cycle arrest and apoptosis through the activation of MAPKs and inhibition of c-Myc.

N-Heterocyclic Carbene Bound Nickel(I) Complexes and Their Roles in Catalysis.

New paramagnetic Ni(I)(IMes)(2)X (IMes: 1,3-bis-(2,4,6-trimethylphenyl)-imidazol-2-ylidene) were prepared from the reaction of Ni(IMes)(2) with aryl halides. Products that would arise from oxidative addition were not observed. In contrast, Ni(II)(tmiy)(2)(X)(Ar) was formed from the oxidative addition of aryl halides to Ni bound by a sterically-less hindered NHC ligand, tmiy (tetramethylimidazol-2-ylidene). The paramagnetic Ni(I)(IMes)(2)X complexes were compared to known Ni(0) and Ni(II) catalysts for Kumada and Suzuki coupling reactions. Stoichiometric reactions between the Ni(I)(IMes)(2)X complexes with aryl halides and transmetallating agents were also evaluated.