Interaction of digoxin with antihypertensive drugs via MDR1

The multidrug transporter MDR1 (P-glycoprotein)-mediated interaction between digoxin and 29 antihypertensive drugs of various types was examined by using the MDR1 overexpressing LLC-GA5-COL150 cells, which were established by transfecting MDR1 cDNA into porcine kidney epithelial LLC-PK1 cells. These cells construct monolayers with tight junctions, and enable the evaluation of transcellular transport. The MDR1 was highly expressed on the apical membrane (urine side). The basal-to-apical and apical-to-basal transcellular transport of [3H]digoxin in LLC-GA5-COL150 cells was time- and temperature-dependent. The basal-to-apical transport of [3H]digoxin was markedly increased, whereas the apical-to-basal transport was decreased in LLC-GA5-COL150 cells, compared with the host LLC-PK1 cells, suggesting that [3H]digoxin was a substrate for MDR1. Most of the Ca2+ channel blockers used here markedly inhibited basal-to-apical transport and increased apical-to-basal transport. Exceptions were diltiazem, nifedipine and nitrendipine, which hardly showed inhibitory effects on transcellular transport of [3H]digoxin. Alpha-blocker doxazosin and beta-blocker carvedilol also inhibited transcellular transport of [3H]digoxin, but none of the angiotensin converting enzyme inhibitors and AT1 angiotensin II receptor antagonists used here were active. These observations will promote understanding of the digoxin-drug interactions resulting from their actions on MDR1, and which may aid in avoiding these unexpected effects of digoxin.     

The challenges of developing novel antiparasitic drugs

Only a few novel classes of antiparasitic drugs have emerged over the last few decades, reflecting the difficulties associated with bringing a safe, effective molecule to market. In recent years, the screening paradigm has shifted from empirical whole parasite screening towards mechanism-based high throughput screening. This approach requires investment in molecular parasitology and in understanding the basic biology of parasites, as well as requiring considerable investment in an infrastructure for screening. Add to this the fact that the drug discovery process is iterative with high attrition, the Animal Health industry by necessity must focus on discovering medicines for diseases, which will deliver a return on investment. In recent years the rapid progression of genomics has unlocked a plethora of tools for target identification, validation and screening, revolutionising mechanism-based screening for antiparasitic drug discovery. The challenge still remains; however, to identify novel chemical entities with the properties required to deliver a safe, effective antiparasitic drug.     

Structural basis for retinoic X receptor repression on the tetramer

Retinoic X receptor (RXR) is a master nuclear receptor in the processes of cell development and homeostasis. Unliganded RXR exists in an autorepressed tetramer, and agonists can induce RXR dimerization and coactivator recruitment for activation. However, the molecular mechanisms involving the corepressor recruitment and antagonist-mediated repression of RXR are still elusive. Here we report the crystal structure of RXRα ligand-binding domain (LBD) complexed with silencing mediator for retinoid and thyroid hormone receptors (SMRT) corepressor motif. As the first structural report on the unliganded nuclear receptor bound to the corepressor motif, RXRαLBD-SMRT exhibits a significant structural rearrangement, compared with apoRXRαLBD tetramer. To elucidate further the molecular determinants for RXR repression by its antagonist, we also determine the crystal structure of RXRαLBD-SMRT complexed with the identified antagonist rhein. In the structure, two rhein molecules and two SMRT peptides are in the RXRαLBD tetramer, different from the case in RXRαLBD-SMRT structure, where four SMRT peptides bind to RXRαLBD tetramer. It seems that rhein induces a displacement of SMRT motif by activation function 2 (AF-2) motif binding to the receptor. Combining our current work with the published results, structural superposition of RXRαLBDs in different states reveals that RXR uses an overlapped binding site for coactivator, corepressor, and AF-2 motifs, whereas the AF-2 motif adopts different conformations for agonist or antagonist interaction and coactivator or corepressor recruitment. Taken together, we thus propose a molecular model of RXR repression on the tetramer.     

Methods to identify protein targets of metal-based drugs

Metal-based anticancer agents occupy a distinct chemical space due to their particular coordination geometry and reactivity. Despite the initial DNA-targeting paradigm for this class of compounds, it is now clear that they can also be tuned to target proteins in cells, depending on the metal and ligand scaffold. Since metallodrug discovery is dominated by phenotypic screenings, tailored proteomics strategies were crucial to identify and validate protein targets of several investigative and clinically advanced metal-based drugs. Here, such experimental approaches are discussed, which showed that metallodrugs based on ruthenium, gold, rhenium and even platinum, can selectively and specifically target proteins with clear-cut down-stream effects. Target identification strategies are expected to support significantly the mechanism-driven clinical translation of metal-based drugs.     

Structural re-positioning,in silico molecular modelling,oxidative degradation,and biological screening of linagliptin as adenosine 3 receptor (ADORA3) modulators targeting hepatocellular carcinoma

Chemical entities with structural diversity were introduced as candidates targeting adenosine receptor with different clinical activities, containing 3,7-dihydro-1H-purine-2,6-dione, especially adenosine 3 receptors (ADORA3). Our initial approach started with pharmacophore screening of ADORA3 modulators; to choose linagliptin (LIN), approved anti-diabetic drug as Dipeptidyl peptidase-4 inhibitors, to be studied for its modulating effect towards ADORA3. This was followed by generation, purification, analytical method development, and structural elucidation of oxidative degraded product (DEG). Both of LIN and DEG showed inhibitory profile against hepatocellular carcinoma cell lines with induction of apoptosis at G2/M phase with increase in caspase-3 levels, accompanied by a downregulation in gene and protein expression levels of ADORA3 with a subsequent increase in cAMP. Quantitative in vitro assessment of LIN binding affinity against ADORA3 was also performed to exhibit inhibitory profile at Ki of 37.7?nM. In silico molecular modelling showing binding affinity of LIN and DEG towards ADORA3 was conducted.     

Double evaluations of chemicals using a cocktail of fused recombinant receptors

Some chemicals have multipotential as endocrine-disrupting chemicals (EDCs). For example, some chemicals act as both estrogens and antiandrogens. Numbers of such chemicals should be evaluated from many aspects; however, labor and expenses are generally limited. We have developed two expression systems for the wild type of human estrogen receptor alpha and the wild type of human androgen receptor fused with a maltose binding protein. They are soluble and have binding activities. They showed dose-responses to natural hormones and well-known potential EDCs. After we established each assay condition for a competitive binding assay using each receptor, we found that two assay systems can be carried out simultaneously under limited and harmonized conditions. Under harmonized conditions using a cocktail of two types of receptors, we could estimate natural hormones and potential EDCs. Interference between two assay systems was not observed under these conditions. We believe that some competitive binding assays can be carried out using a cocktail of receptors at the same time if interference among different assay systems can be avoided by choosing ideal conditions.     

Structural requirement of tunicamycin V for MraY inhibition

Elucidating a structure-activity relationship study by evaluating a series of truncated analogues is a simple but important and effective tactic in medicinal chemistry based on natural products with a large and complex chemical structure. In this study, a series of truncated analogues of tunicamycin V were designed and synthesized and their MraY inhibitory activity was investigated in order to gain insight into the effect of these moieties on MraY inhibition.     

1-Malonyl-1,4-dihydropyridine as a novel carrier for specific delivery of drugs to the brain

A group of 1-malonyl-1,4-dihydropyridine derivatives were synthesized as novel carrier systems for site-specific and sustained drug delivery to the brain. Such carriers are expected to be stable against air oxidation due to the presence of the carbonyl group close to nitrogen of the dihydropyridine. These carrier systems were attached to a group of different aldehydes to afford novel quaternary pyridinium derivatives 9a–e, 11a–d, 13 and 18a–b. Reduction of the prepared quaternary pyridinium derivatives with sodium dithionite afforded a novel group of 1-malonyl-1,4-dihydropyridine chemical delivery systems (CDSs) 10a–e, 12a–d, 14 and 19a–b. The synthesized 1-malonyl-1,4-dihydropyridine CDSs were subjected to various chemical and biological investigations to evaluate their ability to cross the blood–brain barrier, and to be oxidized biologically into their corresponding quaternary derivatives. The in vitro oxidation studies showed that most of the 1-malonyl-1,4-dihydropyridine CDSs could be oxidized into their corresponding quaternary derivatives at an adequate rate. The in vivo studies showed that compounds 10c and 14 were able to cross the blood–brain barrier at detectable concentrations. Moreover, the pyridinium quaternary intermediates 9a, 9c, 13, 18a and their corresponding dihydro derivatives 10a, 10c, 14 and 19a were screened for their antidepressant activity using tail suspension behavioral despair test compared to imipramine as a reference at a dose level of 10 mg/kg. The results indicated that compounds 13, 14 and 19a induced remarkable antidepressant activity comparable to imipramine. Compounds 10a, 10c and 18a exhibited good antidepressant activity, their activities nearly equal to 92.8%, 86.7% and 90.20% of the activity of imipramine, respectively. The other derivatives 9a and 9c exhibited moderate antidepressant activity compared with imipramine.     

Structural basis for the regulation of phytohormone receptors

Phytohormones are central players in diverse plant physiological events, such as plant growth, development, and environmental stress and defense responses. The elucidation of their regulatory mechanisms through phytohormone receptors could facilitate the generation of transgenic crops with cultivation advantages and the rational design of growth control chemicals. During the last decade, accumulated structural data on phytohormone receptors have provided critical insights into the molecular mechanisms of phytohormone perception and signal transduction. Here, we review the structural bases of phytohormone recognition and receptor activation. As a common feature, phytohormones regulate the interaction between the receptors and their respective target proteins (also called co-receptors) by two types of regulatory mechanisms, acting as either “molecular glue” or an “allosteric regulator.” However, individual phytohormone receptors adopt specific structural features that are essential for activation. In addition, recent studies have focused on the molecular diversity of redundant phytohormone receptors.     

Structural basis for the activity of drugs that inhibit phosphodiesterases

Phosphodiesterases (PDEs) comprise a large family of enzymes that catalyze the hydrolysis of cAMP or cGMP and are implicated in various diseases. We describe the high-resolution crystal structures of the catalytic domains of PDE4B, PDE4D, and PDE5A with ten different inhibitors, including the drug candidates cilomilast and roflumilast, for respiratory diseases. These cocrystal structures reveal a common scheme of inhibitor binding to the PDEs: (i) a hydrophobic clamp formed by highly conserved hydrophobic residues that sandwich the inhibitor in the active site; (ii) hydrogen bonding to an invariant glutamine that controls the orientation of inhibitor binding. A scaffold can be readily identified for any given inhibitor based on the formation of these two types of conserved interactions. These structural insights will enable the design of isoform-selective inhibitors with improved binding affinity and should facilitate the discovery of more potent and selective PDE inhibitors for the treatment of a variety of diseases.     

DNA-dependent protein kinase (DNA-PK) inhibitors: structure-activity relationships for O-alkoxyphenylchromen-4-one probes of the ATP-binding domain

Introduction of an O-alkoxyphenyl substituent at the 8-position of the 2-morpholino-4H-chromen-4-one pharmacophore enabled regions of the ATP-binding site of DNA-dependent protein kinase (DNA-PK) to be probed further. Structure-activity relationships have been elucidated for inhibition of DNA-PK and PI3K (p110α), with N-(2-(cyclopropylmethoxy)-4-(2-morpholino-4-oxo-4H-chromen-8-yl)phenyl)-2-morpholinoacetamide 11a being identified as a potent and selective DNA-PK inhibitor (IC₅₀ = 8 nM).     

Target identification of covalently binding drugs by activity-based protein profiling (ABPP)

The characterization of the target proteins of drug molecules has become an important goal in understanding its mode of action and origin of side effects due to off-target binding. This is especially important for covalently binding drugs usually containing electrophilic moieties, which potentially can react with nucleophilic residues found in many proteins. This review gives a comprehensive overview of the use of activity-based protein profiling (ABPP) as an efficient tool for the target identification of covalently binding drugs.     

Structure-activity relationship of contractile effects induced by helicokinins in the isolated gut of the lepidopteran caterpillar Spodoptera frugiperda

The diuretic helicokinins YFSPWG-amide (Hez KI), VRFSPWG-amide (Hez KII) and KVKFSAWG-amide (Hez KIII) are potent contractants of the isolated gut of the caterpillar Spodoptera frugiperda at doses ranging from 0.1 to 10 nM. In comparison, the pentapeptide FSPWG-amide was a full agonist with greatly reduced potency while SPWG-amide and PWG-amide were weak partial agonists. Substitution of individual amino acids in Hez KI with alanine revealed that replacement of the [phenylalanine²] residue caused a large fall in potency while replacement of [tryptophan⁵] residue caused complete loss of myogenic activity. The striking fall in potency of YASPWG-amide and the lack of activity of YFSPAG-amide confirm the requirement for aromatic groups in positions 2 and 3 of the core pentapeptide as well as supporting the ideas that the active core of these peptides adopts a β-turn when interacting with receptors, bringing together the [Phe] and [Trp] residues that are critical for activity. Neither the pentapeptide proctolin nor the potent mammalian gut contractant Substance P were able to cause contraction when applied to caterpillar gut tissue. Incubation of isolated gut tissue in the phosphodiesterase inhibitor theophylline (10-100 μM) caused significant potentiation of the response to applied Hez KI. Conversely, in the presence of the L-type Ca²⁺ channel blocker verapamil (10 μM-1 mM) or Co²⁺ (1-50 mM) the contractile effects of Hez KI were attentuated significantly. These data suggest that the gut of S. frugiperda contains G-protein-linked kinin receptors that utilise cyclic AMP as their second messenger system and cause contraction by promoting the entry of extracellular Ca²⁺.     

Structural basis for the substrate specificity of a Bacillus 1,3-1,4-beta-glucanase

Depolymerization of polysaccharides is catalyzed by highly specific enzymes that promote hydrolysis of the scissile glycosidic bond by an activated water molecule. 1,3-1,4-beta-Glucanases selectively cleave beta-1,4 glycosidic bonds in 3-O-substituted glucopyranosyl units within polysaccharides with mixed linkage. The reaction follows a double-displacement mechanism by which the configuration of the anomeric C(1)-atom of the glucosyl unit in subsite -I is retained. Here we report the high-resolution crystal structure of the hybrid 1,3-1,4-beta-glucanase H(A16-M)(E105Q/E109Q) in complex with a beta-glucan tetrasaccharide. The structure shows four beta-d-glucosyl moieties bound to the substrate-binding cleft covering subsites -IV to -I, thus corresponding to the reaction product. The ten active-site residues Asn26, Glu63, Arg65, Phe92, Tyr94, Glu105, Asp107, Glu109, Asn182 and Trp184 form a network of hydrogen bonds and hydrophobic stacking interactions with the substrate. These residues were previously identified by mutational analysis as significant for stabilization of the enzyme-carbohydrate complex, with Glu105 and Glu109 being the catalytic residues. Compared to the Michaelis complex model, the tetrasaccharide moiety is slightly shifted toward that part of the cleft binding the non-reducing end of the substrate, but shows previously unanticipated strong stacking interactions with Phe92 in subsite -I. A number of specific hydrogen-bond contacts between the enzyme and the equatorial O(2), O(3) and O(6) hydroxyl groups of the glucosyl residues in subsites -I, -II and -III are the structural basis for the observed substrate specificity of 1,3-1,4-beta-glucanases. Kinetic analysis of enzyme variants with the all beta-1,3 linked polysaccharide laminarin identified key residues mediating substrate specificity in good agreement with the structural data. The comparison with structures of the apo-enzyme H(A16-M) and a covalent enzyme-inhibitor (E.I) complex, together with kinetic and mutagenesis data, yields new insights into the structural requirements for substrate binding and catalysis. A detailed view of enzyme-carbohydrate interactions is presented and mechanistic implications are discussed.     

Structural basis for inhibition of human PNP by immucillin-H

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. PNP is a target for inhibitor development aiming at T-cell immune response modulation. This work reports on the crystallographic study of the complex of human PNP-immucillin-H (HsPNP-ImmH) solved at 2.6A resolution using synchrotron radiation. Immucillin-H (ImmH) inhibits the growth of malignant T-cell lines in the presence of deoxyguanosine without affecting non-T-cell tumor lines. ImmH inhibits activated normal human T cells after antigenic stimulation in vitro. These biological effects of ImmH suggest that this agent may have utility in the treatment of certain human diseases characterized by abnormal T-cell growth or activation. This is the first structural report of human PNP complexed with immucillin-H. The comparison of the complex HsPNP-ImmH with recent crystallographic structures of human PNP explains the high specificity of immucillin-H for human PNP.