Structure-activity relationship of pyrrolidine pentamine derivatives as inhibitors of the aminoglycoside 6'- N -acetyltransferase type Ib.

Resistance to amikacin and other major aminoglycosides is commonly due to enzymatic acetylation by aminoglycoside 6'- N -acetyltransferase type I enzyme, of which type Ib [AAC(6')-Ib] is the most widespread among Gram-negative pathogens. Finding enzymatic inhibitors could be an effective way to overcome resistance and extend the useful life of amikacin. Small molecules possess multiple properties that make them attractive compounds to be developed as drugs. Mixture-based combinatorial libraries and positional scanning strategy led to the identification of a chemical scaffold, pyrrolidine pentamine, that, when substituted with the appropriate functionalities at five locations (R1 - R5), inhibits AAC(6')-Ib-mediated inactivation of amikacin. Structure-activity relationship (SAR) studies showed that while truncations to the molecule result in loss of inhibitory activity, modifications of functionalities and stereochemistry have different effects on the inhibitory properties. In this study, we show that alterations at position R1 of the two most active compounds, 2700.001 and 2700.003 , reduced inhibition levels, demonstrating the essential nature not only of the presence of an S -phenyl moiety at this location but also the distance to the scaffold. On the other hand, modifications on the R3, R4, and R5 positions have varied effects, demonstrating the potential for optimization. A correlation analysis between molecular docking values (ΔG) and the dose required for two-fold potentiation of compounds described in this and the previous studies showed a significant correlation between ΔG values and inhibitory activity. Highlights: Amikacin resistance in Gram-negatives is mostly caused by the AAC(6')-Ib enzymeAAC(6')-Ib has been identified in most Gram-negative pathogensInhibitors of AAC(6')-Ib could be used to treat resistant infectionsCombinatorial libraries and positional scanning identified an inhibitorThe lead compound can be optimized by structure activity relationship studies.

The Parallel Structure-Activity Relationship Screening of Three Compounds Identifies the Common Agonist Pharmacophore of Pyrrolidine Bis-Cyclic Guanidine Melanocortin-3 Receptor (MC3R) Small-Molecule Ligands.

The melanocortin receptors are involved in numerous physiological pathways, including appetite, skin and hair pigmentation, and steroidogenesis. In particular, the melanocortin-3 receptor (MC3R) is involved in fat storage, food intake, and energy homeostasis. Small-molecule ligands developed for the MC3R may serve as therapeutic lead compounds for treating disease states of energy disequilibrium. Herein, three previously reported pyrrolidine bis-cyclic guanidine compounds with five sites for molecular diversity (R1-R5) were subjected to parallel structure-activity relationship studies to identify the common pharmacophore of this scaffold series required for full agonism at the MC3R. The R2, R3, and R5 positions were required for full MC3R efficacy, while truncation of either the R1 or R4 positions in all three compounds resulted in full MC3R agonists. Two additional fragments, featuring molecular weights below 300 Da, were also identified that possessed full agonist efficacy and micromolar potencies at the mMC5R. These SAR experiments may be useful in generating new small-molecule ligands and chemical probes for the melanocortin receptors to help elucidate their roles in vivo and as therapeutic lead compounds.

Identification of B Cell and T Cell Epitopes Using Synthetic Peptide Combinatorial Libraries.

This article presents a combinatorial library method that consists of the synthesis and screening of mixture-based synthetic combinatorial libraries of peptide molecules to identify B and T cell epitopes. The protocols employ peptide libraries to identify peptides recognized by MAbs and T cells. The first protocol uses a positional scanning peptide library made up of hexapeptides to identify antigenic determinants recognized by MAbs. The 120 mixtures in the hexapeptide library are tested for their inhibitory activity in a competitive ELISA. The second protocol uses a decapeptide library to identify T cell peptide ligands. The 200 mixtures of the decapeptide library are tested for their ability to induce T cell activation. Support protocols cover optimization of the assay conditions for each MAb or T cell, to achieve the best level of sensitivity and reproducibility, and preparation of a hexapeptide library, along with deconvolution approaches. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Screening peptide library for antibody inhibition Basic Protocol 2: Screening a peptide library to identify CD4+ Or CD8+ T cell ligands Support Protocol 1: Optimizing antigen and antibody concentrations for screening assay Support Protocol 2: Preparing a positional scanning peptide library.

Identification of SARS-CoV-2 Spike Palmitoylation Inhibitors That Results in Release of Attenuated Virus with Reduced Infectivity.

The spike proteins of enveloped viruses are transmembrane glycoproteins that typically undergo post-translational attachment of palmitate on cysteine residues on the cytoplasmic facing tail of the protein. The role of spike protein palmitoylation in virus biogenesis and infectivity is being actively studied as a potential target of novel antivirals. Here, we report that palmitoylation of the first five cysteine residues of the C-terminal cysteine-rich domain of the SARS-CoV-2 S protein are indispensable for infection, and palmitoylation-deficient spike mutants are defective in membrane fusion. The DHHC9 palmitoyltransferase interacts with and palmitoylates the spike protein in the ER and Golgi and knockdown of DHHC9 results in reduced fusion and infection of SARS-CoV-2. Two bis-piperazine backbone-based DHHC9 inhibitors inhibit SARS-CoV-2 S protein palmitoylation and the resulting progeny virion particles released are defective in fusion and infection. This establishes these palmitoyltransferase inhibitors as potential new intervention strategies against SARS-CoV-2.

Biomanufacturing in low Earth orbit for regenerative medicine.

Research in low Earth orbit (LEO) has become more accessible. The 2020 Biomanufacturing in Space Symposium reviewed space-based regenerative medicine research and discussed leveraging LEO to advance biomanufacturing for regenerative medicine applications. The symposium identified areas where financial investments could stimulate advancements overcoming technical barriers. Opportunities in disease modeling, stem-cell-derived products, and biofabrication were highlighted. The symposium will initiate a roadmap to a sustainable market for regenerative medicine biomanufacturing in space. This perspective summarizes the 2020 Biomanufacturing in Space Symposium, highlights key biomanufacturing opportunities in LEO, and lays the framework for a roadmap to regenerative medicine biomanufacturing in space.

Identification and characterization of an atypical Gαs-biased ß2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis.

G protein-coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For ß2-adrenergic receptors (ß2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and ß-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable ß-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling-a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: ß2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from ß-arrestin, in contrast to albuterol and C5-S C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from ß-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of ß2AR actions favorable for treating obstructive lung disease.

Functional Mixture-Based Positional Scan Identifies a Library of Antagonist Tetrapeptide Sequences (LAtTeS) with Nanomolar Potency for the Melanocortin-4 Receptor and Equipotent with the Endogenous AGRP(86-132) Antagonist.

The melanocortin-4 receptor (MC4R) plays an important role in appetite. Agonist ligands that stimulate the MC4R decrease appetite, while antagonist compounds increase food consumption. Herein, a functional mixture-based positional scan identified novel MC4R antagonist sequences. Mixtures comprising a library of 12,960,000 tetrapeptides were screened in the presence and absence of the NDP-MSH agonist. These results led to the synthesis of 48 individual tetrapeptides, of which 40 were screened for functional activity at the melanocortin receptors. Thirteen compounds were found to possess nanomolar antagonist potency at the MC4R, with the general tetrapeptide sequence Ac-Aromatic-Basic-Aromatic-Basic-NH2. The most notable results include the identification of tetrapeptide 48 [COR1-25, Ac-DPhe(pI)-Arg-Nal(2')-Arg-NH2], an equipotent MC4R antagonist to agouti-related protein [AGRP(86-132)], more potent than miniAGRP(87-120), and possessing 15-fold selectivity for the MC4R versus the MC3R. These tetrapeptides may serve as leads for novel appetite-inducing therapies to treat states of negative energy balance, such as cachexia and anorexia.

Inhibition of Aminoglycoside 6'-N-acetyltransferase Type Ib (AAC(6')-Ib): Structure-Activity Relationship of Substituted Pyrrolidine Pentamine Derivatives as Inhibitors.

The aminoglycoside 6'-N-acetyltransferase type Ib (AAC(6')-Ib) is a common cause of resistance to amikacin and other aminoglycosides in Gram-negatives. Utilization of mixture-based combinatorial libraries and application of the positional scanning strategy identified an inhibitor of AAC(6')-Ib. This inhibitor's chemical structure consists of a pyrrolidine pentamine scaffold substituted at four locations (R1, R3, R4, and R5). The substituents are two S-phenyl groups (R1 and R4), an S-hydroxymethyl group (R3), and a 3-phenylbutyl group (R5). Another location, R2, does not have a substitution, but it is named because its stereochemistry was modified in some compounds utilized in this study. Structure-activity relationship (SAR) analysis using derivatives with different functionalities, modified stereochemistry, and truncations was carried out by assessing the effect of the addition of each compound at 8 µM to 16 µg/mL amikacin-containing media and performing checkerboard assays varying the concentrations of the inhibitor analogs and the antibiotic. The results show that: (1) the aromatic functionalities at R1 and R4 are essential, but the stereochemistry is essential only at R4; (2) the stereochemical conformation at R2 is critical; (3) the hydroxyl moiety at R3 as well as stereoconformation are required for full inhibitory activity; (4) the phenyl functionality at R5 is not essential and can be replaced by aliphatic groups; (5) the location of the phenyl group on the butyl carbon chain at R5 is not essential; (6) the length of the aliphatic chain at R5 is not critical; and (7) all truncations of the scaffold resulted in inactive compounds. Molecular docking revealed that all compounds preferentially bind to the kanamycin C binding cavity, and binding affinity correlates with the experimental data for most of the compounds evaluated. The SAR results in this study will serve as the basis for the design of new analogs in an effort to improve their ability to induce phenotypic conversion to susceptibility in amikacin-resistant pathogens.

Discovery of Nanomolar Melanocortin-3 Receptor (MC3R)-Selective Small Molecule Pyrrolidine Bis-Cyclic Guanidine Agonist Compounds Via a High-Throughput "Unbiased" Screening Campaign.

The central melanocortin-3 and melanocortin-4 receptors (MC3R, MC4R) are key regulators of body weight and energy homeostasis. Herein, the discovery and characterization of first-in-class small molecule melanocortin agonists with selectivity for the melanocortin-3 receptor over the melanocortin-4 receptor are reported. Identified via "unbiased" mixture-based high-throughput screening approaches, pharmacological evaluation of these pyrrolidine bis-cyclic guanidines resulted in nanomolar agonist activity at the melanocortin-3 receptor. The pharmacological profiles at the remaining melanocortin receptor subtypes tested indicated similar agonist potencies at both the melanocortin-1 and melanocortin-5 receptors and antagonist or micromolar agonist activities at the melanocortin-4 receptor. This group of small molecules represents a new area of chemical space for the melanocortin receptors with mixed receptor pharmacology profiles that may serve as novel lead compounds to modulate states of dysregulated energy balance.

The value of antimicrobial peptides in the age of resistance.

Accelerating growth and global expansion of antimicrobial resistance has deepened the need for discovery of novel antimicrobial agents. Antimicrobial peptides have clear advantages over conventional antibiotics which include slower emergence of resistance, broad-spectrum antibiofilm activity, and the ability to favourably modulate the host immune response. Broad bacterial susceptibility to antimicrobial peptides offers an additional tool to expand knowledge about the evolution of antimicrobial resistance. Structural and functional limitations, combined with a stricter regulatory environment, have hampered the clinical translation of antimicrobial peptides as potential therapeutic agents. Existing computational and experimental tools attempt to ease the preclinical and clinical development of antimicrobial peptides as novel therapeutics. This Review identifies the benefits, challenges, and opportunities of using antimicrobial peptides against multidrug-resistant pathogens, highlights advances in the deployment of novel promising antimicrobial peptides, and underlines the needs and priorities in designing focused development strategies taking into account the most advanced tools available.

Tissue Chips in Space: Modeling Human Diseases in Microgravity.

PURPOSE: Microphysiological systems (MPS), also known as "organs-on-chips" or "tissue chips," leverage recent advances in cell biology, tissue engineering, and microfabrication to create in vitro models of human organs and tissues. These systems offer promising solutions for modeling human physiology and disease in vitro and have multiple applications in areas where traditional cell culture and animal models fall short. Recently, the National Center for Advancing Translational Sciences (NCATS) at the National Institutes of Health (NIH) and the International Space Station (ISS) U.S. National Laboratory have coordinated efforts to facilitate the launch and use of these MPS platforms onboard the ISS. Here, we provide an introduction to the NIH Tissue Chips in Space initiative and an overview of the coordinated efforts between NIH and the ISS National Laboratory. We also highlight the current progress in addressing the scientific and technical challenges encountered in the development of these ambitious projects. Finally, we describe the potential impact of the Tissue Chips in Space program for the MPS field as well as the wider biomedical and health research communities.

Discovery of Polypharmacological Melanocortin-3 and -4 Receptor Probes and Identification of a 100-Fold Selective nM MC3R Agonist versus a µM MC4R Partial Agonist.

The centrally expressed melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R, respectively) are established targets to treat diseases of positive- and negative-energy homeostasis. We previously reported [ Doering , S. R. ; J. Med. Chem. 2017 , 60 , 4342 - 4357 ] mixture-based positional scanning approaches to identify dual MC3R agonist and MC4R antagonist tetrapeptides. Herein, 46 tetrapeptides were chosen for MC3R agonist screening selectivity profiles, synthesized, and pharmacologically characterized at the mouse melanocortin-1, -3, -4, and -5 receptors. Substitutions to the tetrapeptide template were selected solely based on MC3R agonist potency from the mixture-based screen. This study resulted in the discovery of compound 42 (Ac-Val-Gln-(pI)DPhe-DTic-NH2), a full MC3R agonist that is 100-fold selective for the MC3R over the µM MC4R partial agonist pharmacology. This compound represents a first-in-class MC3R selective agonist. This ligand will serve as a useful in vivo molecular probe for the investigation of the roles of the MC3R and MC4R in diseases of dysregulated energy homeostasis.

Scaffold Ranking and Positional Scanning Identify Novel Neurite Outgrowth Promoters with Nanomolar Potency.

Central nervous system (CNS) neurons typically fail to regrow their axons after injury. Injuries or neuropathies that damage CNS axons and disrupt neuronal circuitry often result in permanent functional deficits. Axon regeneration is therefore an intensely pursued therapeutic strategy for numerous CNS disorders. Phenotypic screens utilizing primary neurons have proven successful at identifying agents that promote axon regeneration in vivo. Here, we report the screening of mixture-based combinatorial small molecule libraries in a phenotypic assay utilizing primary CNS neurons and the discovery of neurite outgrowth promoters with low nanomolar potency.

Identification of a Novel Polyamine Scaffold With Potent Efflux Pump Inhibition Activity Toward Multi-Drug Resistant Bacterial Pathogens.

We have previously reported the use of combinatorial chemistry to identify broad-spectrum antibacterial agents. Herein, we extend our analysis of this technology toward the discovery of anti-resistance molecules, focusing on efflux pump inhibitors. Using high-throughput screening against multi-drug resistant Pseudomonas aeruginosa, we identified a polyamine scaffold that demonstrated strong efflux pump inhibition without possessing antibacterial effects. We determined that these molecules were most effective with an amine functionality at R1 and benzene functionalities at R2 and R3. From a library of 188 compounds, we studied the properties of 5 lead agents in detail, observing a fivefold to eightfold decrease in the 90% effective concentration of tetracycline, chloramphenicol, and aztreonam toward P. aeruginosa isolates. Additionally, we determined that our molecules were not only active toward P. aeruginosa, but toward Acinetobacter baumannii and Staphylococcus aureus as well. The specificity of our molecules to efflux pump inhibition was confirmed using ethidium bromide accumulation assays, and in studies with strains that displayed varying abilities in their efflux potential. When assessing off target effects we observed no disruption of bacterial membrane polarity, no general toxicity toward mammalian cells, and no inhibition of calcium channel activity in human kidney cells. Finally, combination treatment with our lead agents engendered a marked increase in the bactericidal capacity of tetracycline, and significantly decreased viability within P. aeruginosa biofilms. As such, we report a unique polyamine scaffold that has strong potential for the future development of novel and broadly active efflux pump inhibitors targeting multi-drug resistant bacterial infections.

Differential regulation of alcohol taking and seeking by antagonism at α4ß2 and α3ß4 nAChRs.

RATIONALE: Alcoholism is a serious public health problem throughout the world. Current pharmacotherapies for the treatment of this disorder are poorly effective. Preclinical and clinical findings point to nicotinic acetylcholine receptors (nAChRs) as a promising target for the development of novel and effective medications. Assuage Pharmaceuticals, in collaboration with Torrey Pines Institute for Molecular Studies, has discovered a new class of potent and selective α4ß2 nAChR antagonists. OBJECTIVE: Here, it was hypothesized that α4ß2 nAChR antagonism is a viable approach for treatment of alcohol use disorders. RESULTS: When tested in rats, one lead compound, AP-202, attenuated both operant alcohol and nicotine self-administration in a paradigm in which the two reinforcers were concurrently available. The conotoxin TP2212-59, a selective α3ß4 nAChR antagonist, was only effective in reducing nicotine self-administration. AP-202 also reduced alcohol but not food responding when alcohol was presented as the only reinforcer, whereas the commercially available α4ß2 nAChR antagonist dihydro-ß-erythroidine failed to alter alcohol self-administration. AP-202 did not block relapse-like behavior induced by previously alcohol-associated stimuli or yohimbine stress. In a reinstatement paradigm, in which alcohol seeking was triggered by a nicotine challenge, a behavior successfully inhibited by the nonselective nAChR antagonist mecamylamine, AP-202 was not effective, while pretreatment with TP2212-59 abolished nicotine-induced reinstatement of alcohol seeking. CONCLUSIONS: These findings suggest differential roles for α4ß2 and α3ß4 nAChR on alcohol taking and seeking with selective blockade of α4ß2 nAChR being more implicated in modulating alcohol taking while selective blockade of α3ß4 nAChR is involved in nicotine-induced alcohol seeking.

Highly Selective and Potent α4ß2 nAChR Antagonist Inhibits Nicotine Self-Administration and Reinstatement in Rats.

The α4ß2 nAChR is the most predominant subtype in the brain and is a well-known culprit for nicotine addiction. Previously we presented a series of α4ß2 nAChR selective compounds that were discovered from a mixture-based positional-scanning combinatorial library. Here we report further optimization identified highly potent and selective α4ß2 nAChR antagonists 5 (AP-202) and 13 (AP-211). Both compounds are devoid of in vitro agonist activity and are potent inhibitors of epibatidine-induced changes in membrane potential in cells containing α4ß2 nAChR, with IC50 values of approximately 10 nM, but are weak agonists in cells containing α3ß4 nAChR. In vivo studies show that 5 can significantly reduce operant nicotine self-administration and nicotine relapse-like behavior in rats at doses of 0.3 and 1 mg/kg. The pharmacokinetic data also indicate that 5, via sc administration, is rapidly absorbed into the blood, reaching maximal concentration within 10 min with a half-life of less than 1 h.

A one-pot multicomponent approach to a new series of morphine derivatives and their biological evaluation.

A novel and facile domino reaction has been developed to synthesize a variety of new derivatives from hydromorphone, amines and paraformaldehyde in good yields in a catalyst-free fashion with high atom efficiency. The products show a mixed MOR/DOR biological characteristic which makes them valuable for further study as opioid analgesics.

Identification of a Hit Series of Antileishmanial Compounds through the Use of Mixture-Based Libraries.

From a screening campaign that included mixture-based libraries containing more than 6 million compounds, a lead series of bis-cyclic guanidines was identified as the most promising. Lead optimization resulted in the identification of potent (IC50 < 500 nM) and selective compounds within this series as well as potent and selective monoguanidines.