Development and pharmacological evaluation of a new chemical series of potent pan-ERR agonists, identification of SLU-PP-915.

Estrogen-related receptors (ERR) are an orphan nuclear receptor sub-family that play a critical role in regulating gene transcription for several physiological processes including mitochondrial function, cellular energy utilization and homeostasis. They have also been implicated to play a role in several pathological conditions. Herein, we report the identification, synthesis, structure-activity relationships and pharmacological evaluation of a new chemical series of potent pan-ERR agonists. This template was designed for ERRγ starting from the known acyl hydrazide template and compounds such as agonist GSK-4716 employing a structure-based drug design approach. This led to the preparation of a series of 2,5-disubstituted thiophenes from which several were found to be potent agonists of ERRγ in cell-based co-transfection assays. Additionally, direct binding to ERRγ was established through 1H NMR protein-ligand binding experiments. Compound optimization revealed that the phenolic or aniline groups could be replaced with a boronic acid moiety, which was able to maintain activity and demonstrated improved metabolic stability in microsomal in vitro assays. Further pharmacological evaluation of these compounds showed that they had roughly equivalent agonist activity on ERR isoforms α and ß representing an ERR pan-agonist profile. One potent agonist, SLU-PP-915 (10s), which contained a boronic acid moiety was profiled in gene expression assays and found to significantly upregulate the expression of ERR target genes such as peroxisome-proliferator activated receptor γ co-activators-1α, lactate dehydrogenase A, DNA damage inducible transcript 4 and pyruvate dehydrogenase kinase 4 both in vitro and in vivo.

Utilizing directed evolution to interrogate and optimize CRISPR/Cas guide RNA scaffolds.

CRISPR-based editing has revolutionized genome engineering despite the observation that many DNA sequences remain challenging to target. Unproductive interactions formed between the single guide RNA's (sgRNA) Cas9-binding scaffold domain and DNA-binding antisense domain are often responsible for such limited editing resolution. To bypass this limitation, we develop a functional SELEX (systematic evolution of ligands by exponential enrichment) approach, termed BLADE (binding and ligand activated directed evolution), to identify numerous, diverse sgRNA variants that bind Streptococcus pyogenes Cas9 and support DNA cleavage. These variants demonstrate surprising malleability in sgRNA sequence. We also observe that particular variants partner more effectively with specific DNA-binding antisense domains, yielding combinations with enhanced editing efficiencies at various target sites. Using molecular evolution, CRISPR-based systems could be created to efficiently edit even challenging DNA sequences making the genome more tractable to engineering. This selection approach will be valuable for generating sgRNAs with a range of useful activities.

Mechanistic Duality of Bacterial Efflux Substrates and Inhibitors: Example of Simple Substituted Cinnamoyl and Naphthyl Amides.

Antibiotic resistance poses an immediate and growing threat to human health. Multidrug efflux pumps are promising targets for overcoming antibiotic resistance with small-molecule therapeutics. Previously, we identified a diaminoquinoline acrylamide, NSC-33353, as a potent inhibitor of the AcrAB-TolC efflux pump in Escherichia coli. This inhibitor potentiates the antibacterial activities of novobiocin and erythromycin upon binding to the membrane fusion protein AcrA. It is also a substrate for efflux and lacks appreciable intrinsic antibacterial activity of its own in wild-type cells. Here, we have modified the substituents of the cinnamoyl group of NSC-33353, giving rise to analogs that retain the ability to inhibit efflux, lost the features of the efflux substrates, and gained antibacterial activity in wild-type cells. The replacement of the cinnamoyl group with naphthyl isosteres generated compounds that lack antibacterial activity but are both excellent efflux pump inhibitors and substrates. Surprisingly, these inhibitors potentiate the antibacterial activity of novobiocin but not erythromycin. Surface plasmon resonance experiments and molecular docking suggest that the replacement of the cinnamoyl group with naphthyl shifts the affinity of the compounds away from AcrA to the AcrB transporter, making them better efflux substrates and changing their mechanism of inhibition. These results provide new insights into the duality of efflux substrate/inhibitor features in chemical scaffolds that will facilitate the development of new efflux pump inhibitors.

Predictive Rules of Efflux Inhibition and Avoidance in Pseudomonas aeruginosa.

Antibiotic-resistant bacteria rapidly spread in clinical and natural environments and challenge our modern lifestyle. A major component of defense against antibiotics in Gram-negative bacteria is a drug permeation barrier created by active efflux across the outer membrane. We identified molecular determinants defining the propensity of small peptidomimetic molecules to avoid and inhibit efflux pumps in Pseudomonas aeruginosa, a human pathogen notorious for its antibiotic resistance. Combining experimental and computational protocols, we mapped the fate of the compounds from structure-activity relationships through their dynamic behavior in solution, permeation across both the inner and outer membranes, and interaction with MexB, the major efflux transporter of P. aeruginosa We identified predictors of efflux avoidance and inhibition and demonstrated their power by using a library of traditional antibiotics and compound series and by generating new inhibitors of MexB. The identified predictors will enable the discovery and optimization of antibacterial agents suitable for treatment of P. aeruginosa infections.IMPORTANCE Efflux pump avoidance and inhibition are desired properties for the optimization of antibacterial activities against Gram-negative bacteria. However, molecular and physicochemical interactions defining the interface between compounds and efflux pumps remain poorly understood. We identified properties that correlate with efflux avoidance and inhibition, are predictive of similar features in structurally diverse compounds, and allow researchers to distinguish between efflux substrates, inhibitors, and avoiders in P. aeruginosa The developed predictive models are based on the descriptors representative of different clusters comprising a physically intuitive combination of properties. Molecular shape (represented by acylindricity), amphiphilicity (anisotropic polarizability), aromaticity (number of aromatic rings), and the partition coefficient (LogD) are physicochemical predictors of efflux inhibitors, whereas interactions with Pro668 and Leu674 residues of MexB distinguish between inhibitors/substrates and efflux avoiders. The predictive models and efflux rules are applicable to compounds with unrelated chemical scaffolds and pave the way for development of compounds with the desired efflux interface properties.

A Selective ERRα/γ Inverse Agonist, SLU-PP-1072, Inhibits the Warburg Effect and Induces Apoptosis in Prostate Cancer Cells.

The estrogen related receptors (ERRs) are a subgroup of nuclear receptors that play a role in regulation of cellular metabolism. Prostate cancer (PCa) cells display altered metabolic signatures, such as the Warburg effect, and the ERRs have been implicated in driving this phenotype. Despite the lack of a known endogenous ligand, synthetic ligands that target the ERRs have been discovered. For example, the ERRα inverse agonist XCT790 modulates metabolic pathways in PCa cells, but it also functions as a mitochondrial uncoupler independent of targeting ERRα. Here, we describe a novel dual ERRα/γ inverse agonist, SLU-PP-1072, derived from the GSK4716 ERRγ agonist scaffold that is distinct from the XCT790 scaffold. SLU-PP-1072 alters PCa cell metabolism and gene expression, resulting in cell cycle dysregulation and increased apoptosis without acute mitochondrial uncoupling activity. Our data suggest that inhibition of ERRα/γ may be beneficial in treatment of PCa, and SLU-PP-1072 provides a unique chemical tool to evaluate the pharmacology of ERRα and ERRγ.

Identification and Structure-Activity Relationships of Novel Compounds that Potentiate the Activities of Antibiotics in Escherichia coli.

In Gram-negative bacteria, efflux pumps are able to prevent effective cellular concentrations from being achieved for a number of antibiotics. Small molecule adjuvants that act as efflux pump inhibitors (EPIs) have the potential to reinvigorate existing antibiotics that are currently ineffective due to efflux mechanisms. Through a combination of rigorous experimental screening and in silico virtual screening, we recently identified novel classes of EPIs that interact with the membrane fusion protein AcrA, a critical component of the AcrAB-TolC efflux pump in Escherichia coli. Herein, we present initial optimization efforts and structure-activity relationships around one of those previously described hits, NSC 60339 (1). From these efforts we identified two compounds, SLUPP-225 (17h) and SLUPP-417 (17o), which demonstrate favorable properties as potential EPIs in E. coli cells including the ability to penetrate the outer membrane, improved inhibition of efflux relative to 1, and potentiation of the activity of novobiocin and erythromycin.

Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump.

Antibiotic resistance is a major threat to human welfare. Inhibitors of multidrug efflux pumps (EPIs) are promising alternative therapeutics that could revive activities of antibiotics and reduce bacterial virulence. Identification of new druggable sites for inhibition is critical for the development of effective EPIs, especially in light of constantly emerging resistance. Here, we describe EPIs that interact with periplasmic membrane fusion proteins, critical components of efflux pumps that are responsible for the activation of the transporter and the recruitment of the outer-membrane channel. The discovered EPIs bind to AcrA, a component of the prototypical AcrAB-TolC pump, change its structure in vivo, inhibit efflux of fluorescent probes, and potentiate the activities of antibiotics in Escherichia coli and other Gram-negative bacteria. Our findings expand the chemical and mechanistic diversity of EPIs, suggest the mechanism for regulation of the efflux pump assembly and activity, and provide a promising path for reviving the activities of antibiotics in resistant bacteria.

Tuning Interfacial Electron Transfer in Nanostructured Cuprous Oxide Photoelectrochemical Cells with Charge-Selective Molecular Coatings.

The coating of nanostructured films of cuprous oxide with electroactive molecules strongly affects their photoelectrochemical performance in nonaqueous photocells, with photocurrent density increased up to an order of magnitude relative to bare cuprous oxide films or almost completely suppressed, depending on the choice of molecular adsorbant. Among adsorbants that enhance photocurrent, a strong variance of photoelectrochemical behavior is observed with changes in the molecular structure of the sensitizer, associated with differences in the reorganization energy and molecular size, which are interpreted to enhance forward electron transport and impede electrolyte/photocathode recombination, respectively. These results demonstrate that nanostructured cuprous oxide is a promising cathode material for p-type dye-sensitized solar cells.

Templated electrodeposition and photocatalytic activity of cuprous oxide nanorod arrays.

Cuprous oxide (Cu2O) nanorod arrays have been prepared via a novel templated electrodeposition process and were characterized for their photocatalytic behavior in nonaqueous photoelectrochemical cells. Zinc oxide (ZnO) nanorod films serve as sacrificial templates for the in situ formation of polymer nanopore membranes on transparent conductive oxide substrates. Nitrocellulose and poly(lactic acid) are effective membrane-forming polymers that exhibit different modes of template formation, with nitrocellulose forming conformal coatings on the ZnO surface while poly(lactic acid) acts as an amorphous pore-filling material. Robust template formation is sensitive to the seeding method used to prepare the precursor ZnO nanorod films. Photoelectrochemical cells prepared from electrodeposited Cu2O films using methyl viologen as a redox shuttle in acetonitrile electrolyte exhibit significant charge recombination that can be partially suppressed by a combination of surface passivation methods. Surface-passivated nanostructured Cu2O films show enhanced photocurrent relative to planar electrodeposited Cu2O films of similar thickness. We have obtained the highest photocurrent ever reported for electrodeposited Cu2O in a nonaqueous photoelectrochemical cell.

Electron transport in acceptor-sensitized polymer-oxide solar cells: the importance of surface dipoles and electron cascade effects.

Fullerene and acenequinone compounds have been examined as electron mediators between a p-type semiconductive polymer and two n-type oxide semiconductors. Composite interlayer materials and photovoltaic test cells were assembled and studied for their fluorescence quenching, current-voltage, and quantum efficiency behavior to characterize the efficacy of the acceptor-sensitizers as electron-selective interlayers. The sensitizers are generally more effective with titanium dioxide than with zinc oxide, due to the difference in magnitude of dipole-induced vacuum level shifts at the respective oxide interfaces. In titanium dioxide-based solar cells, where dipole effects are weak, photovoltage and fill factor increase in a trend that matches the increase in the first reduction potential of the acceptor-sensitizers. Photosensitization of the oxide semiconductor by the acceptor-sensitizers is observed to operate either in parallel with the polymer as an alternate photosensitizer or in series with the polymer in a two-photon process, according to an acceptor-sensitizer's first reduction potential. In zinc oxide-based solar cells, where dipole effects are stronger, the acceptor-sensitizers impaired most devices, which is attributed to an upward shift of the oxide's conduction band edge caused by dipole-induced vacuum level shifts. These results have broad implications for designing electron-selective interlayers and solid-state photocells using sensitized oxide semiconductors.