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.

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.

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.

Identification of Bis-Cyclic Guanidines as Antiplasmodial Compounds from Positional Scanning Mixture-Based Libraries.

The screening of more than 30 million compounds derived from 81 small molecule libraries built on 81 distinct scaffolds identified pyrrolidine bis-cyclic guanidine library (TPI-1955) to be one of the most active and selective antiplasmodial libraries. The screening of the positional scanning library TPI-1955 arranged on four sets of sublibraries (26 + 26 + 26 + 40), totaling 120 samples for testing provided information about the most important groups of each variable position in the TPI-1955 library containing 738,192 unique compounds. The parallel synthesis of the individual compounds derived from the deconvolution of the positional scanning library led to the identification of active selective antiplasmodial pyrrolidine bis-cyclic guanidines.

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.

Identification of a small molecule inhibitor of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] using mixture-based combinatorial libraries.

The aminoglycoside, 6'-N-acetyltransferase type Ib [AAC(6')-Ib] is the most widely distributed enzyme among AAC(6')-I-producing Gram-negative pathogens and confers resistance to clinically relevant aminoglycosides, including amikacin. This enzyme is therefore an ideal target for enzymatic inhibitors that could overcome resistance to aminoglycosides. The search for inhibitors was carried out using mixture-based combinatorial libraries, the scaffold ranking approach, and the positional scanning strategy. A library with high inhibitory activity had pyrrolidine pentamine scaffold and was selected for further analysis. This library contained 738,192 compounds with functionalities derived from 26 different amino acids (R1, R2 and R3) and 42 different carboxylic acids (R4) in four R-group functionalities. The most active compounds all contained S-phenyl (R1 and R3) and S-hydromethyl (R2) functionalities at three locations and differed at the R4 position. The compound containing 3-phenylbutyl at R4 (compound 206) was a robust enzymatic inhibitor in vitro, in combination with amikacin it potentiated the inhibition of growth of three resistant bacteria in culture, and it improved survival when used as treatment of Galleria mellonella infected with aac(6')-Ib-harboring Klebsiella pneumoniae and Acinetobacter baumannii strains.

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.

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, 18 [Ac-Arg-Arg-(pI)DPhe-Tic-NH2], 1 [Ac-His-Arg-(pI)DPhe-Tic-NH2], and 41 [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.

A key role for the N/OFQ-NOP receptor system in modulating nicotine taking in a model of nicotine and alcohol co-administration.

Alcohol and nicotine are often co-abused. Although the N/OFQ-NOP receptor system is considered a potential target for development of drug abuse pharmacotherapies, especially for alcoholism, little is known about the role of this system in nicotine dependence. Furthermore, the effect of prior history of nicotine dependence on subsequent nicotine and alcohol taking is understudied. Using an operant co-administration paradigm, in which rats concurrently self-administer nicotine and alcohol, we found that nicotine dependent rats increased nicotine self-administration over time as compared to non-dependent animals, while patterns of alcohol lever pressing did not change between groups. Pretreatment with the potent NOP receptor agonist AT-202 (0.3-3 mg/kg) increased nicotine lever pressing of both dependent and non-dependent groups, whereas the selective antagonist SB612111 (1-10 mg/kg) elicited a clear reduction of nicotine responses, in both dependent and non-dependent rats. In parallel, AT-202 only produced minor changes on alcohol responses and SB612111 reduced alcohol taking at a dose that also reduced locomotor behavior. Results indicate that a history of nicotine dependence affects subsequent nicotine- but not alcohol-maintained responding, and that NOP receptor antagonism, rather than agonism, blocks nicotine self-administration, which strongly suggests a critical role for the endogenous N/OFQ in the modulation of nicotine reinforcement processes.

Synthesis and biological evaluations of novel endomorphin analogues containing α-hydroxy-ß-phenylalanine (AHPBA) displaying mixed µ/δ opioid receptor agonist and δ opioid receptor antagonist activities.

A novel series of endomorphin-1 (EM-1) and endomorphin-2 (EM-2) analogues was synthesized, incorporating chiral α-hydroxy-ß-phenylalanine (AHPBA), and/or Dmt(1)-Tic(2) at different positions. Pharmacological activity and metabolic stability of the series was assessed. Consistent with earlier studies of ß-amino acid substitution into endomorphins, multiple analogues incorporation AHPBA displayed high affinity for µ and δ opioid receptors (MOR and DOR, respectively) in radioligand competition binding assays, and an increased stability in rat brain membrane homogenates, notably Dmt-Tic-(2R,3S)AHPBA-Phe-NH2 (compound 26). Intracerebroventricular (i.c.v.) administration of 26 produced antinociception (ED50 value (and 95% confidence interval) = 1.98 (0.79-4.15) nmol, i.c.v.) in the mouse 55 °C warm-water tail-withdrawal assay, equivalent to morphine (2.35 (1.13-5.03) nmol, i.c.v.), but demonstrated DOR-selective antagonism in addition to non-selective opioid agonism. The antinociception of 26 was without locomotor activity or acute antinociceptive tolerance. This novel class of peptides adds to the potentially therapeutically relevant collection of previously reported EM analogues.

Identification of tetrapeptides from a mixture based positional scanning library that can restore nM full agonist function of the L106P, I69T, I102S, A219V, C271Y, and C271R human melanocortin-4 polymorphic receptors (hMC4Rs).

Human obesity has been linked to genetic factors and single nucleotide polymorphisms (SNPs). Melanocortin-4 receptor (MC4R) SNPs have been associated with up to 6% frequency in morbidly obese children and adults. A potential therapy for individuals possessing such genetic modifications is the identification of molecules that can restore proper receptor signaling and function. These compounds could serve as personalized medications improving quality of life issues as well as alleviating diseases symptoms associated with obesity including type 2 diabetes. Several hMC4 SNP receptors have been pharmacologically characterized in vitro to have a decreased, or a lack of response, to endogenous agonists such as α-, ß-, and γ2-melanocyte stimulating hormones (MSH) and adrenocorticotropin hormone (ACTH). Herein we report the use of a mixture based positional scanning combinatorial tetrapeptide library to discover molecules with nM full agonist potency and efficacy to the L106P, I69T, I102S, A219V, C271Y, and C271R hMC4Rs. The most potent compounds at all these hMC4R SNPs include Ac-His-(pI)DPhe-Tic-(pNO2)DPhe-NH2, Ac-His-(pCl)DPhe-Tic-(pNO2)DPhe-NH2, Ac-His-(pCl)DPhe-Arg-(pI)Phe-NH2, and Ac-Arg-(pCl)DPhe-Tic-(pNO2)DPhe-NH2, revealing new ligand pharmacophore models for melanocortin receptor drug design strategies.

Libraries from Libraries: A Series of Sulfonamide Linked Heterocycles Derived from the Same Scaffold.

A libraries from libraries approach is described for the synthesis of five different sulfonamide linked scaffolds. Four of the scaffolds are sulfonamides linked to heterocycles; piperazine, thiourea, cyclic guanidine, and dimethyl cyclic guanidine. The fifth scaffold is a polyamine linked sulfonamide. Three different diversity positions were effectively incorporated into each scaffold providing a number of different compounds with good yields and purity.

Fluorescent mu selective opioid ligands from a mixture based cyclic peptide library.

A positional scanning cyclic peptide library was generated using a penta-peptide thioester scaffold. Glycine was fixed at position R(1). Diaminopropionic acid was fixed at position R(3), with its γ-amino attaching to an anthraniloyl group. Positions R(2) and R(4) contained 36 L- and D- amino acids and position R(5) contained 19 L- amino acids. Cyclization was performed in a mixture of acetonitrile and 1.5 M aqueous imidazole solution (7:1 v/v) at room temperature for 5 days. No significant cross-oligomerization was detected under the cyclization conditions. The library was screened in a binding assay for mu opioid receptor, identifying the active amino acid mixture at each position. A total of 40 individual cyclic peptides were identified and synthesized by the combinations of the most active amino acid mixtures found at three positions 5 × 4 × 2. Two cyclic peptides exhibited high binding affinities to opioid receptor. The most active cyclic peptide in the library was yielded to have Tyr at R(2), D-Lys at R(4), and Tyr at R(5). Further investigation on this compound revealed the side chain-to-tail isomer to have greater binding affinity (14 nM) than the head-to-tail isomer (39 nM). Both isomers were selective for the mu-opioid receptor.

A novel method for the determination of isokinetic ratios and its application in the synthesis of two new positional scanning libraries.

A novel method for the direct evaluation of the equimolarity of the compounds contained in a mixture is presented. We applied the method toward calculating isokinetic ratios for the reaction between the amine termini of a resin bound peptide fragment and a sulfonyl chloride to produce equal molar mixtures of sulfonamides. The results of this study and the application of the method to the synthesis of two new positional scanning synthetic combinatorial libraries (PS-SCL) are discussed.