Data_Sheet_1_Identification of a Novel Polyamine Scaffold With Potent Efflux Pump Inhibition Activity Toward Multi-Drug Resistant Bacterial Pathogens.PDF

<p>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.</p

Combinatorial Libraries As a Tool for the Discovery of Novel, Broad-Spectrum Antibacterial Agents Targeting the ESKAPE Pathogens

Mixture based synthetic combinatorial libraries offer a tremendous enhancement for the rate of drug discovery, allowing the activity of millions of compounds to be assessed through the testing of exponentially fewer samples. In this study, we used a scaffold-ranking library to screen 37 different libraries for antibacterial activity against the ESKAPE pathogens. Each library contained between 10000 and 750000 structural analogues for a total of >6 million compounds. From this, we identified a bis-cyclic guanidine library that displayed strong antibacterial activity. A positional scanning library for these compounds was developed and used to identify the most effective functional groups at each variant position. Individual compounds were synthesized that were broadly active against all ESKAPE organisms at concentrations <2 μM. In addition, these compounds were bactericidal, had antibiofilm effects, showed limited potential for the development of resistance, and displayed almost no toxicity when tested against human lung cells and erythrocytes. Using a murine model of peritonitis, we also demonstrate that these agents are highly efficacious in vivo

Rapid Scanning Structure–Activity Relationships in Combinatorial Data Sets: Identification of Activity Switches

We present a general approach to describe the structure–activity relationships (SAR) of combinatorial data sets with activity for two biological endpoints with emphasis on the rapid identification of substitutions that have a large impact on activity and selectivity. The approach uses dual-activity difference (DAD) maps that represent a visual and quantitative analysis of all pairwise comparisons of one, two, or more substitutions around a molecular template. Scanning the SAR of data sets using DAD maps allows the visual and quantitative identification of activity switches defined as specific substitutions that have an opposite effect on the activity of the compounds against two targets. The approach also rapidly identifies single- and double-target R-cliffs, i.e., compounds where a single or double substitution around the central scaffold dramatically modifies the activity for one or two targets, respectively. The approach introduced in this report can be applied to any analogue series with two biological activity endpoints. To illustrate the approach, we discuss the SAR of 106 pyrrolidine bis-diketopiperazines tested against two formylpeptide receptors obtained from positional scanning deconvolution methods of mixture-based libraries

Scaffold Ranking and Positional Scanning Utilized in the Discovery of nAChR-Selective Compounds Suitable for Optimization Studies

Nicotine binds to nicotinic acetylcholine receptors (nAChR), which can exist as many different subtypes. The α4β2 nAChR is the most prevalent subtype in the brain and possesses the most evidence linking it to nicotine seeking behavior. Herein we report the use of mixture based combinatorial libraries for the rapid discovery of a series of α4β2 nAChR selective compounds. Further chemistry optimization provided compound <b>301</b>, which was characterized as a selective α4β2 nAChR antagonist. This compound displayed no agonist activity but blocked nicotine-induced depolarization of HEK cells with an IC₅₀ of approximately 430 nM. <b>301</b> demonstrated nearly 500-fold selectivity for binding and 40-fold functional selectivity for α4β2 over α3β4 nAChR. In total over 5 million compounds were assessed through the use of just 170 samples in order to identify a series of structural analogues suitable for future optimization toward the goal of developing clinically relevant smoking cessation medications

Discovery of Mixed Pharmacology Melanocortin‑3 Agonists and Melanocortin‑4 Receptor Tetrapeptide Antagonist Compounds (TACOs) Based on the Sequence Ac-Xaa¹‑Arg-(pI)DPhe-Xaa⁴‑NH₂

The centrally expressed melanocortin-3 and -4 receptors (MC3R/MC4R) have been studied as possible targets for weight management therapies, with a preponderance of studies focusing on the MC4R. Herein, a novel tetrapeptide scaffold [Ac-Xaa¹-Arg-(pI)­DPhe-Xaa⁴-NH₂] is reported. The scaffold was derived from results obtained from a MC3R mixture-based positional scanning campaign. From these results, a set of 48 tetrapeptides were designed and pharmacologically characterized at the mouse melanocortin-1, -3, -4, and -5 receptors. This resulted in the serendipitous discovery of nine compounds that were MC3R agonists (EC₅₀ < 1000 nM) and MC4R antagonists (5.7 < pA₂ < 7.8). The three most potent MC3R agonists, <b>18</b> [Ac-Arg-Arg-(pI)­DPhe-Tic-NH₂], <b>1</b> [Ac-His-Arg-(pI)­DPhe-Tic-NH₂], and <b>41</b> [Ac-Arg-Arg-(pI)­DPhe-DNal(2′)-NH₂] were more potent (EC₅₀ < 73 nM) than the melanocortin tetrapeptide Ac-His-DPhe-Arg-Trp-NH₂. This template contains a sequentially reversed “Arg-(pI)­DPhe” motif with respect to the classical “Phe-Arg” melanocortin signaling motif, which results in pharmacology that is first-in-class for the central melanocortin receptors