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.

Restoring susceptibility to aminoglycosides: identifying small molecule inhibitors of enzymatic inactivation.

Growing resistance to antimicrobial medicines is a critical health problem that must be urgently addressed. Adding to the increasing number of patients that succumb to infections, there are other consequences to the rise in resistance like the compromise of several medical procedures and dental work that are heavily dependent on infection prevention. Since their introduction in the clinics, aminoglycoside antibiotics have been a critical component of the armamentarium to treat infections. Still, the increase in resistance and their side effects led to a decline in their utilization. However, numerous current factors, like the urgent need for antimicrobials and their favorable properties, led to renewed interest in these drugs. While efforts to design new classes of aminoglycosides refractory to resistance mechanisms and with fewer toxic effects are starting to yield new promising molecules, extending the useful life of those already in use is essential. For this, numerous research projects are underway to counter resistance from different angles, like inhibition of expression or activity of resistance components. This review focuses on selected examples of one aspect of this quest, the design or identification of small molecule inhibitors of resistance caused by enzymatic modification of the aminoglycoside. These compounds could be developed as aminoglycoside adjuvants to overcome resistant infections.

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.

Identification of small molecules by screening a mixture-based scaffold compound library for treatment of alpha-1 antitrypsin deficiency.

This study aimed to identify small molecules that have the potential to treat alpha1-antitrypsin deficiency (AATD) by screening compounds available from a mixture-based scaffold library. 93 scaffold libraries (total diversity of >30 million compounds in mixture format) were screened using a cell model of AATD in order to identify samples that could either reduce intracellular aggregation of Z-form AAT protein, increase extracellular secretion of Z-AAT or both. Mixture libraries containing compounds with in vitro activity, for example library 1295, were screened further to identify individual active compounds. The mixture format of the scaffold library allowed for some preliminary structure-activity relationships to be developed and also enabled the rapid selection of a promising scaffold. Utilizing this scaffold, 1295, a collection of individual "control" compounds contained in the 1295 mixture sample were then screened. A sub-library of individual "control" compounds featuring structural diversity at position R1 (1295.R1), was screened and 7 compounds were found to reduce the intracellular accumulation of Z-AAT without affecting cell viability at a concentration of 25ug/ml (about 50 µM). Screening sub-libraries featuring structural diversity at R2 and R3 (1295.R2 and 1295.R3) identified an additional 15 active compounds. Titration experiments identified 3 compounds from the 1295.R2 library that retained activity at 5ug/ml (approx. 10uM). One compound (1295.263) from 1295.R2 decreased intracellular levels of Z-AAT without affecting cell viability and wild-type AAT levels at the concentration of 5ug/ml. Molecular docking of this compound to the Z-AAT crystal structure identified a potential binding site near the C-terminal domain, an identified polymerization site. Our results indicate that screening large mixture-based compound libraries can be used to identify small molecules that may have the potential to treat AATD and other disease.

A Robust and Cost-Effective Luminescent-Based High-Throughput Assay for Fructose-1,6-Bisphosphate Aldolase A.

Hypoxic solid tumors induce the stabilization of hypoxia-inducible factor 1 alpha (HIF1α), which stimulates the expression of many glycolytic enzymes and hypoxia-responsive genes. A high rate of glycolysis supports the energetic and material needs for tumors to grow. Fructose-1,6-bisphosphate aldolase A (ALDOA) is an enzyme in the glycolytic pathway that promotes the expression of HIF1α. Therefore, inhibition of ALDOA activity represents a potential therapeutic approach for a range of cancers by blocking two critical cancer survival mechanisms. Here, we present a luminescence-based strategy to determine ALDOA activity. The assay platform was developed by integrating a previously established ALDOA activity assay with a commercial NAD/NADH detection kit, resulting in a significant (>12-fold) improvement in signal/background (S/B) compared with previous assay platforms. A screening campaign using a mixture-based compound library exhibited excellent statistical parameters of Z' (>0.8) and S/B (~20), confirming its robustness and readiness for high-throughput screening (HTS) application. This assay platform provides a cost-effective method for identifying ALDOA inhibitors using a large-scale HTS campaign.

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.

A Novel Probe for Spliceosomal Proteins that Induces Autophagy and Death of Melanoma Cells Reveals New Targets for Melanoma Drug Discovery.

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets. We have previously reported a discovery of novel anti-melanoma compound 2155-14 (Onwuha-Ekpete et al., J Med Chem. 2014 Feb 27; 57(4):1599-608). In the report presented herein we aim to identify its target(s) and mechanism of action. METHODS: We utilized biotinylated analog of 2155-14 to pull down its targets from melanoma cells. Proteomics in combination with western blot were used to identify the targets. Mechanism of action of 2155-14 was determined using flow cytometry, RT-PCR, microscopy, western blot, and enzymatic activity assays. Where applicable, one-way analysis of variance (ANOVA) was used followed by Dunnett post hoc test. RESULTS: In the present study, we identified ATP-dependent RNA helicase DDX1 and heterogeneous nuclear ribonucleoproteins (hnRNPs) H1, H2 and A2/B1 as targets of anti-melanoma compound 215514. To the best of our knowledge, this is a first report suggesting that these proteins could be targeted for melanoma therapy. Mechanistic investigations showed that 2155-14 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. CONCLUSION: Identification of mode of action of 2155-14 may provide insight into novel therapies against a broad range of melanoma subtypes. These studies were enabled by the novel probe derived from a mixture-based library, an important class of chemical biology tools for discovering novel targets.

A Novel Class of Common Docking Domain Inhibitors That Prevent ERK2 Activation and Substrate Phosphorylation.

Extracellular signal-regulated kinases (ERK1/2) are mitogen-activated protein kinases (MAPKs) that play a pro-tumorigenic role in numerous cancers. ERK1/2 possess two protein-docking sites that are distinct from the active site: the D-recruitment site (DRS) and the F-recruitment site. These docking sites facilitate substrate recognition, intracellular localization, signaling specificity, and protein complex assembly. Targeting these sites on ERK in a therapeutic context may overcome many problems associated with traditional ATP-competitive inhibitors. Here, we identified a new class of inhibitors that target the ERK DRS by screening a synthetic combinatorial library of more than 30 million compounds. The screen detects the competitive displacement of a fluorescent peptide from the DRS of ERK2. The top molecular scaffold from the screen was optimized for structure-activity relationship by positional scanning of different functional groups. This resulted in 10 compounds with similar binding affinities and a shared core structure consisting of a tertiary amine hub with three functionalized cyclic guanidino branches. Compound 2507-1 inhibited ERK2 from phosphorylating a DRS-targeting substrate and prevented the phosphorylation of ERK2 by a constitutively active MEK1 (MAPK/ERK kinase 1) mutant. Interaction between an analogue, 2507-8, and the ERK2 DRS was confirmed by nuclear magnetic resonance and X-ray crystallography. 2507-8 forms critical interactions at the common docking domain residue Asp319 via an arginine-like moiety that is shared by all 10 hits, suggesting a common binding mode. The structural and biochemical insights reported here provide the basis for developing new ERK inhibitors that are not ATP-competitive but instead function by disrupting critical protein-protein interactions.

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.