Development of AC265347-Inspired Calcium-Sensing Receptor Ago-Positive Allosteric Modulators.

The calcium-sensing receptor (CaSR) is a clinical target in the treatment of hyperparathyroidism and related diseases. However, clinical use of approved CaSR-targeting drugs such as cinacalcet is limited due to adverse side effects including hypocalcaemia, nausea and vomiting, and in some instances, a lack of efficacy. The CaSR agonist and positive allosteric modulator (ago-PAM), AC265347, is chemically distinct from clinically-approved CaSR PAMs. AC265347 potently suppressed parathyroid hormone (PTH) release in rats with a lower propensity to cause hypocalcaemia compared to cinacalcet and may therefore offer benefits over current CaSR PAMs. Here we report a structure activity relationship (SAR) study seeking to optimise AC265347 as a drug candidate and disclose the discovery of AC265347-like compounds with diverse pharmacology and improved physicochemical and drug-like properties.

Therapeutic Opportunities of Targeting Allosteric Binding Sites on the Calcium-Sensing Receptor.

The CaSR is a class C G protein-coupled receptor (GPCR) that acts as a multimodal chemosensor to maintain diverse homeostatic functions. The CaSR is a clinical therapeutic target in hyperparathyroidism and has emerged as a putative target in several other diseases. These include hyper- and hypocalcaemia caused either by mutations in the CASR gene or in genes that regulate CaSR signaling and expression, and more recently in asthma. The development of CaSR-targeting drugs is complicated by the fact that the CaSR possesses many different binding sites for endogenous and exogenous agonists and allosteric modulators. Binding sites for endogenous and exogenous ligands are located throughout the large CaSR protein and are interconnected in ways that we do not yet fully understand. This review summarizes our current understanding of CaSR physiology, signaling, and structure and how the many different binding sites of the CaSR may be targeted to treat disease.

Negative allosteric modulators of the human calcium-sensing receptor bind to overlapping and distinct sites within the 7-transmembrane domain.

BACKGROUND AND PURPOSE: Negative allosteric modulators (NAMs) that target the calcium-sensing receptor (CaS receptor) were originally developed for the treatment of osteoporosis by stimulating the release of endogenous parathyroid hormone, but failed in human clinical trials. Several chemically and structurally distinct NAM scaffolds have been described, but it is not known how these different scaffolds interact with the CaS receptor to inhibit receptor signalling in response to agonists. EXPERIMENTAL APPROACH: In the present study, we used a mutagenesis approach combined with analytical pharmacology and computational modelling to probe the binding sites of four distinct NAM scaffolds. KEY RESULTS: Although all four scaffolds bind to the 7-transmembrane and/or extracellular or intracellular loops, they occupy distinct regions, as previously shown for positive allosteric modulators of the CaS receptor. Furthermore, different NAM scaffolds mediate negative allosteric modulation via distinct amino acid networks. CONCLUSION AND IMPLICATIONS: These findings aid our understanding of how different NAMs bind to and inhibit the CaS receptor. Elucidation of allosteric binding sites in the CaS receptor has implications for the discovery of novel allosteric modulators.

Substituted Pyridazin-3(2 H)-ones as Highly Potent and Biased Formyl Peptide Receptor Agonists.

Herein we describe the development of a focused series of functionalized pyridazin-3(2 H)-one-based formyl peptide receptor (FPR) agonists that demonstrate high potency and biased agonism. The compounds described demonstrated biased activation of prosurvival signaling, ERK1/2 phosphorylation, through diminution of the detrimental FPR1/2-mediated intracellular calcium (Cai2+) mobilization. Compound 50 showed an EC50 of 0.083 µM for phosphorylation of ERK1/2 and an approximate 20-fold bias away from Cai2+ mobilization at the hFPR1.

Novel RU486 (mifepristone) analogues with increased activity against Venezuelan Equine Encephalitis Virus but reduced progesterone receptor antagonistic activity.

There are currently no therapeutics to treat infection with the alphavirus Venezuelan equine encephalitis virus (VEEV), which causes flu-like symptoms leading to neurological symptoms in up to 14% of cases. Large outbreaks of VEEV can result in 10,000 s of human cases and mass equine death. We previously showed that mifepristone (RU486) has anti-VEEV activity (EC50 = 20 µM) and only limited cytotoxicity (CC50 > 100 µM), but a limitation in its use is its abortifacient activity resulting from its ability to antagonize the progesterone receptor (PR). Here we generate a suite of new mifepristone analogues with enhanced antiviral properties, succeeding in achieving >11-fold improvement in anti-VEEV activity with no detectable increase in toxicity. Importantly, we were able to derive a lead compound with an EC50 of 7.2 µM and no detectable PR antagonism activity. Finally, based on our SAR analysis we propose avenues for the further development of these analogues as safe and effective anti-VEEV agents.

Overcoming P-Glycoprotein-Mediated Drug Resistance with Noscapine Derivatives.

The antitussive agent noscapine has been shown to inhibit the proliferation of cancer cells by disruption of tubulin dynamic. However, the efficacy of several anticancer drugs that inhibit tublin dynamics (vinca alkaloids and taxanes) is reduced by the multidrug resistance phenotype. These compounds are substrates for P-glycoprotein (P-gp)-mediated extrusion from cells. Consequently, the antiproliferative activity of noscapine and a series of derivatives was measured in drug-sensitive and drug-resistant cells that overexpress P-gp. None of the noscapine derivatives displayed lower potency in cells overexpressing P-gp, thereby suggesting a lack of interaction with this pump. However, the cellular efflux of a fluorescent substrate by P-gp was potently inhibited by noscapine and most derivatives. Further investigation with purified, reconstituted P-gp demonstrated that inhibition of P-gp function was due to direct interaction of noscapine derivatives with the transporter. Moreover, coadministration of vinblastine with two of the noscapine derivatives displayed synergistic inhibition of proliferation, even in P-gp-expressing resistant cell lines. Therefore, noscapine derivatives offer a dual benefit of overcoming the significant impact of P-gp in conferring multidrug resistance and synergy with tubulin-disrupting anticancer drugs.

Author Correction: Novel inhibitors targeting Venezuelan equine encephalitis virus capsid protein identified using In Silico Structure-Based-Drug-Design.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Identification of novel antivirals inhibiting recognition of Venezuelan equine encephalitis virus capsid protein by the Importin α/ß1 heterodimer through high-throughput screening.

Although the alphavirus Venezuelan equine encephalitis virus (VEEV) has been the cause of multiple outbreaks resulting in extensive human and equine mortality and morbidity, there are currently no anti-VEEV therapeutics available. VEEV pathogenicity is largely dependent on targeting of the viral capsid protein (CP) to the host cell nucleus through the nuclear transporting importin (Imp) α/ß1 heterodimer. Here we perform a high-throughput screen, combined with nested counterscreens to identify small molecules able to inhibit the Impα/ß1:CP interaction for the first time. Several compounds were able to significantly reduce viral replication in infected cells. Compound G281-1564 in particular could inhibit VEEV replication at low µM concentration, while showing minimal toxicity, with steady state and dynamic quantitative microscopic measurements confirming its ability to inhibit CP nuclear import. This study establishes the principle that inhibitors of CP nucleocytoplasmic trafficking can have potent antiviral activity against VEEV, and represents a platform for future development of safe anti-VEEV compounds with high efficacy and specificity.

8-Mercaptoguanine Derivatives as Inhibitors of Dihydropteroate Synthase.

Dihydropteroate synthase (DHPS) is an enzyme of the folate biosynthesis pathway, which catalyzes the formation of 7,8-dihydropteroate (DHPt) from 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPPP) and para-aminobenzoic acid (pABA). DHPS is the long-standing target of the sulfonamide class of antibiotics that compete with pABA. In the wake of sulfa drug resistance, targeting the structurally rigid (and more conserved) pterin site has been proposed as an alternate strategy to inhibit DHPS in wild-type and sulfa drug resistant strains. Following the work on developing pterin-site inhibitors of the adjacent enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), we now present derivatives of 8-mercaptoguanine, a fragment that binds weakly within both enzymes, and quantify sub-µm binding using surface plasmon resonance (SPR) to Escherichia coli DHPS (EcDHPS). Eleven ligand-bound EcDHPS crystal structures delineate the structure-activity relationship observed providing a structural framework for the rational development of novel, substrate-envelope-compliant DHPS inhibitors.

Novel inhibitors targeting Venezuelan equine encephalitis virus capsid protein identified using In Silico Structure-Based-Drug-Design.

Therapeutics are currently unavailable for Venezuelan equine encephalitis virus (VEEV), which elicits flu-like symptoms and encephalitis in humans, with an estimated 14% of cases resulting in neurological disease. Here we identify anti-VEEV agents using in silico structure-based-drug-design (SBDD) for the first time, characterising inhibitors that block recognition of VEEV capsid protein (C) by the host importin (IMP) α/ß1 nuclear transport proteins. From an initial screen of 1.5 million compounds, followed by in silico refinement and screening for biological activity in vitro, we identified 21 hit compounds which inhibited IMPα/ß1:C binding with IC50s as low as 5 µM. Four compounds were found to inhibit nuclear import of C in transfected cells, with one able to reduce VEEV replication at µM concentration, concomitant with reduced C nuclear accumulation in infected cells. Further, this compound was inactive against a mutant VEEV that lacks high affinity IMPα/ß1:C interaction, supporting the mode of its antiviral action to be through inhibiting C nuclear localization. This successful application of SBDD paves the way for lead optimization for VEEV antivirals, and is an exciting prospect to identify inhibitors for the many other viral pathogens of significance that require IMPα/ß1 in their infectious cycle.

Structural Basis for the Selective Binding of Inhibitors to 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase from Staphylococcus aureus and Escherichia coli.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a member of the folate biosynthesis pathway found in prokaryotes and lower eukaryotes that catalyzes the pyrophosphoryl transfer from the ATP cofactor to a 6-hydroxymethyl-7,8-dihydropterin substrate. We report the chemical synthesis of a series of S-functionalized 8-mercaptoguanine (8MG) analogues as substrate site inhibitors of HPPK and quantify binding against the E. coli and S. aureus enzymes (EcHPPK and SaHPPK). The results demonstrate that analogues incorporating acetophenone-based substituents have comparable affinities for both enzymes. Preferential binding of benzyl-substituted 8MG derivatives to SaHPPK was reconciled when a cryptic pocket unique to SaHPPK was revealed by X-ray crystallography. Differential chemical shift perturbation analysis confirmed this to be a common mode of binding for this series to SaHPPK. One compound (41) displayed binding affinities of 120 nM and 1.76 µM for SaHPPK and EcHPPK, respectively, and represents a lead for the development of more potent and selective inhibitors of SaHPPK.

Progress Toward the Development of Noscapine and Derivatives as Anticancer Agents.

Many nitrogen-moiety containing alkaloids derived from plant origins are bioactive and play a significant role in human health and emerging medicine. Noscapine, a phthalideisoquinoline alkaloid derived from Papaver somniferum, has been used as a cough suppressant since the mid 1950s, illustrating a good safety profile. Noscapine has since been discovered to arrest cells at mitosis, albeit with moderately weak activity. Immunofluorescence staining of microtubules after 24 h of noscapine exposure at 20 µM elucidated chromosomal abnormalities and the inability of chromosomes to complete congression to the equatorial plane for proper mitotic separation ( Proc. Natl. Acad. Sci. U. S. A. 1998 , 95 , 1601 - 1606 ). A number of noscapine analogues possessing various modifications have been described within the literature and have shown significantly improved antiprolific profiles for a large variety of cancer cell lines. Several semisynthetic antimitotic alkaloids are emerging as possible candidates as novel anticancer therapies. This perspective discusses the advancing understanding of noscapine and related analogues in the fight against malignant disease.

Structure-based design and development of functionalized Mercaptoguanine derivatives as inhibitors of the folate biosynthesis pathway enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Staphylococcus aureus.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), an enzyme from the folate biosynthesis pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin and is a yet-to-be-drugged antimicrobial target. Building on our previous discovery that 8-mercaptoguanine (8MG) is an inhibitor of Staphylococcus aureus HPPK (SaHPPK), we have identified and characterized the binding of an S8-functionalized derivative (3). X-ray structures of both the SaHPPK/3/cofactor analogue ternary and the SaHPPK/cofactor analogue binary complexes have provided insight into cofactor recognition and key residues that move over 30 Å upon binding of 3, whereas NMR measurements reveal a partially plastic ternary complex active site. Synthesis and binding analysis of a set of analogues of 3 have identified an advanced new lead compound (11) displaying >20-fold higher affinity for SaHPPK than 8MG. A number of these exhibited low micromolar affinity for dihydropteroate synthase (DHPS), the adjacent, downstream enzyme to HPPK, and may thus represent promising new leads to bienzyme inhibitors.

The synthesis and biological evaluation of multifunctionalised derivatives of noscapine as cytotoxic agents.

Noscapine, a phthalideisoquinoline alkaloid derived from Papaver somniferum, is a well-known antitussive drug that has a relatively safe in vitro toxicity profile. Noscapine is also known to possess weak anticancer efficacy, and since its discovery, efforts have been made to design derivatives with improved potency. Herein, the synthesis of a series of noscapine analogues, which have been modified in the 6', 9', 1 and 7-positions, is described. In a previous study, replacement of the naturally occurring N-methyl group in the 6'-position with an N-ethylaminocarbonyl was shown to promote cell-cycle arrest and cytotoxicity against three cancer cell lines. Here, this modification has been combined with other structural changes that have previously been shown to improve anticancer activity, namely halo substitution in the 9'-position, regioselective O-demethylation to reveal a free phenol in the 7-position, and reduction of the lactone to the corresponding cyclic ether in the 1-position. The incorporation of new aryl substituents in the 9'-position was also investigated. The study identified interesting new compounds able to induce G2/M cell-cycle arrest and that possess cytotoxic activity against the human prostate carcinoma cell line PC3, the human breast adenocarcinoma cell line MCF-7, and the human pancreatic epithelioid carcinoma cell line PANC-1. In particular, the ethyl urea cyclic ether noscapinoids and a compound containing a 6'-ethylaminocarbonyl along with 9'-chloro, 7-hydroxy and lactone moieties exhibited the most promising biological activities, with EC50 values in the low micromolar range against all three cancer cell lines, and these derivatives warrant further investigation.

Synthesis and biological evaluation of N-substituted noscapine analogues.

Noscapine is a phthalideisoquinoline alkaloid isolated from the opium poppy Papaver somniferum. It has long been used as an antitussive agent, but has more recently been found to possess microtubule-modulating properties and anticancer activity. Herein we report the synthesis and pharmacological evaluation of a series of 6'-substituted noscapine derivatives. To underpin this structure-activity study, an efficient synthesis of N-nornoscapine and its subsequent reduction to the cyclic ether derivative of N-nornoscapine was developed. Reaction of the latter with a range of alkyl halides, acid chlorides, isocyanates, thioisocyanates, and chloroformate reagents resulted in the formation of the corresponding N-alkyl, N-acyl, N-carbamoyl, N-thiocarbamoyl, and N-carbamate derivatives, respectively. The ability of these compounds to inhibit cell proliferation was assessed in cell-cycle cytotoxicity assays using prostate cancer (PC3), breast cancer (MCF-7), and colon cancer (Caco-2) cell lines. Compounds that showed activity in the cell-cycle assay were further evaluated in cell viability assays using PC3 and MCF-7 cells.