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Methionine aminopeptidase (MetAp) enzymes catalyze the post-translational removal of the initiator methionine residue in newly synthesized proteins, a process that is often essential in the maturation of proteins. Consequently, these enzymes serve as important targets for drug development. Rickettsia prowazekii (Rp) is an obligate coccobacillus and the causative agent of the louse-borne epidemic typhus and despite adequate treatment causes a latent infection. This research aimed to identify potential anti-rickettsial agents by screening 400 compounds from the MMV Pandemic Response Box against RpMetAp1. Overall, 19 compounds were identified that possessed IC 50 values from 10 µM to 340 nM. The most potent inhibitor was MMV 1580488 (17), which was observed to have an IC 50 of 340 nM. The selected hits serve as chemical leads that can be used for the development of potent inhibitors of the RpMetAp1 enzyme., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Timothy Hagen reports equipment, drugs, or supplies was provided by Medicines for Malaria Venture. Timothy Hagen reports equipment, drugs, or supplies was provided by Seattle Structural Genomics Center for Infectious Disease. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The methylerythritol phosphate (MEP) pathway is a metabolic pathway that produces the isoprenoids isopentyl pyrophosphate and dimethylallyl pyrophosphate. Notably, the MEP pathway is present in bacteria and not in mammals, which makes the enzymes of the MEP pathway attractive targets for discovering new anti-infective agents due to the reduced chances of off-target interactions leading to side effects. There are seven enzymes in the MEP pathway, the third of which is IspD. Two crystal structures of Burkholderia thailandensis IspD (BtIspD) were determined: an apo structure and that of a complex with cytidine triphosphate (CTP). Comparison of the CTP-bound BtIspD structure with the apo structure revealed that CTP binding stabilizes the loop composed of residues 13-19. The apo structure of Mycobacterium paratuberculosis IspD (MpIspD) is also reported. The melting temperatures of MpIspD and BtIspD were evaluated by circular dichroism. The moderate T m values suggest that a thermal shift assay may be feasible for future inhibitor screening. Finally, the binding affinity of CTP for BtIspD was evaluated by isothermal titration calorimetry. These structural and biophysical data will aid in the discovery of IspD inhibitors.

The compound ethyl-adenosyl monophosphate ester (ethyl-AMP) has been shown to effectively inhibit acetyl-CoA synthetase (ACS) enzymes and to facilitate the crystallization of fungal ACS enzymes in various contexts. In this study, the addition of ethyl-AMP to a bacterial ACS from Legionella pneumophila resulted in the determination of a co-crystal structure of this previously elusive structural genomics target. The dual functionality of ethyl-AMP in both inhibiting ACS enzymes and promoting crystallization establishes its significance as a valuable resource for advancing structural investigations of this class of proteins.

Methionine aminopeptidases (MetAp) are dinuclear metalloenzymes found in both prokaryotes and eukaryotes that catalyze the hydrolysis of the N-terminal methionine residue from nascent proteins, an important post-translational modification, which makes it an attractive target for drug discovery. Rickettsia prowazekii (Rp) is an obligate pathogen and causative agent of epidemic typhus and typhus fever. In our ongoing search for anti-rickettsial agents we screened 400 compounds from the Malaria Box for inhibition of RpMetAp1 and discovered 12 compounds that inhibited the enzyme with IC 50 values ranging from 800 nM to 22 μM. These inhibitors are from eleven different chemical series and represent leads that can be used to discover more potent and efficacious anti-rickettsial agents., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Background: Low blood pressure (BP) in acute ischaemic stroke (AIS) is associated with poor functional outcome, death, or severe disability. Increasing BP might benefit patients with post-stroke hypotension including those with potentially salvageable ischaemic penumbra. This updated systematic review considers the present evidence regarding the use of vasopressors in AIS., Methods: We searched the Cochrane Database of Systematic Reviews, MEDLINE, EMBASE and trial databases using a structured search strategy. We examined reference lists of relevant publications for additional studies examining BP elevation in AIS., Results: We included 27 studies involving 1886 patients. Nine studies assessed increasing BP during acute reperfusion therapy (intravenous thrombolysis, mechanical thrombectomy, intra-arterial thrombolysis or combined). Eighteen studies tested BP elevation alone. Phenylephrine was the most commonly used agent to increase BP (n = 16 studies), followed by norepinephrine (n = 6), epinephrine (n = 3) and dopamine (n = 2). Because of small patient numbers and study heterogeneity, a meta-analysis was not possible. Overall, BP elevation was feasible in patients with fluctuating or worsening neurological symptoms, large vessel occlusion with labile BP, sustained post-stroke hypotension and ineligible for intravenous thrombolysis or after acute reperfusion therapy. The effects on functional outcomes were largely unknown and close monitoring is advised if such intervention is undertaken., Conclusion: Although theoretical arguments support increasing BP to improve cerebral blood flow and sustain the ischaemic penumbra in selected AIS patients, the data are limited and results largely inconclusive. Large, randomised controlled trials are needed to identify the optimal BP target, agent, duration of treatment and effects on clinical outcomes., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© European Stroke Organisation 2022.)

Praziquantel (PZQ) remains the only drug of choice for the treatment of schistosomiasis, caused by parasitic flatworms. The widespread use of PZQ in schistosomiasis endemic areas for about four decades raises concerns about the emergence of resistance of Schistosoma spp. to PZQ under drug selection pressure. This reinforces the urgency in finding alternative therapeutic options that could replace or complement PZQ. We explored the potential of medicinal plants commonly used by indigenes in Kenya for the treatment of various ailments including malaria, pneumonia, and diarrhoea for their antischistosomal properties. Employing the Soxhlet extraction method with different solvents, seven medicinal plants Artemisia annua, Ajuga remota, Bredilia micranta, Cordia africana, Physalis peruviana, Prunus africana and Senna didymobotrya were extracted. Qualitative phytochemical screening was performed to determine the presence of various phytochemicals in the plant extracts. Extracts were tested against Schistosoma mansoni newly transformed schistosomula (NTS) and adult worms and the schistosomicidal activity was determined by using the adenosine triphosphate quantitation assay. Phytochemical analysis of the extracts showed different classes of compounds such as alkaloids, tannins, terpenes, etc., in plant extracts active against S. mansoni worms. Seven extracts out of 22 resulted in <20% viability against NTS in 24 h at 100 μg/ml. Five of the extracts with inhibitory activity against NTS showed >69.7% and ≥72.4% reduction in viability against adult worms after exposure for 24 and 48 h, respectively. This study provides encouraging preliminary evidence that extracts of Kenyan medicinal plants deserve further study as potential alternative therapeutics that may form the basis for the development of the new treatments for schistosomiasis.

The enzyme 2-methylerythritol 2,4-cyclodiphosphate synthase, IspF, is essential for the biosynthesis of isoprenoids in most bacteria, some eukaryotic parasites, and the plastids of plant cells. The development of inhibitors that target IspF may lead to novel classes of anti-infective agents or herbicides. Enantiomers of tryptophan hydroxamate were synthesized and evaluated for binding to Burkholderia pseudomallei (Bp) IspF. The L-isomer possessed the highest potency, binding BpIspF with a K D of 36 µM and inhibited BpIspF activity 55% at 120 µM. The high-resolution crystal structure of the L-tryptophan hydroxamate (3)/BpIspF complex revealed a non-traditional mode of hydroxamate binding where the ligand interacts with the active site zinc ion through the primary amine. In addition, two hydrogen bonds are formed with active site groups, and the indole group is buried within the hydrophobic pocket composed of side chains from the 60 s/70 s loop. Along with the co-crystal structure, STD NMR studies suggest the methylene group and indole ring are potential positions for optimization to enhance binding potency., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Acetyl CoA synthetases (ACSs) are A cyl-CoA/ N RPS/ L uciferase (ANL) superfamily enzymes that couple acetate with CoA to generate acetyl CoA, a key component of central carbon metabolism in eukaryotes and prokaryotes. Normal mammalian cells are not dependent on ACSs, while tumor cells, fungi, and parasites rely on acetate as a precursor for acetyl CoA. Consequently, ACSs have emerged as a potential drug target. As part of a program to develop antifungal ACS inhibitors, we characterized fungal ACSs from five diverse human fungal pathogens using biochemical and structural studies. ACSs catalyze a two-step reaction involving adenylation of acetate followed by thioesterification with CoA. Our structural studies captured each step of these two half-reactions including the acetyl-adenylate intermediate of the first half-reaction in both the adenylation conformation and the thioesterification conformation and thus provide a detailed picture of the reaction mechanism. We also used a systematic series of increasingly larger alkyl adenosine esters as chemical probes to characterize the structural basis of the exquisite ACS specificity for acetate over larger carboxylic acid substrates. Consistent with previous biochemical and genetic data for other enzymes, structures of fungal ACSs with these probes bound show that a key tryptophan residue limits the size of the alkyl binding site and forces larger alkyl chains to adopt high energy conformers, disfavoring their efficient binding. Together, our analysis provides highly detailed structural models for both the reaction mechanism and substrate specificity that should be useful in designing selective inhibitors of eukaryotic ACSs as potential anticancer, antifungal, and antiparasitic drugs.

Enzymes in the methylerythritol phosphate pathway make attractive targets for antibacterial activity due to their importance in isoprenoid biosynthesis and the absence of the pathway in mammals. The fifth enzyme in the pathway, 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), contains a catalytically important zinc ion in the active site. A series of de novo designed compounds containing a zinc binding group was synthesized and evaluated for antibacterial activity and interaction with IspF from Burkholderia pseudomallei, the causative agent of Whitmore's disease. The series demonstrated antibacterial activity as well as protein stabilization in fluorescence-based thermal shift assays. Finally, the binding of one compound to Burkholderia pseudomallei IspF was evaluated through group epitope mapping by saturation transfer difference NMR., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Novel pyridine- and pyrimidine-based allosteric inhibitors are reported that achieve PDE4D subtype selectivity through recognition of a single amino acid difference on a key regulatory domain, known as UCR2, that opens and closes over the catalytic site for cAMP hydrolysis. The design and optimization of lead compounds was based on iterative analysis of X-ray crystal structures combined with metabolite identification. Selectivity for the activated, dimeric form of PDE4D provided potent memory enhancing effects in a mouse model of novel object recognition with improved tolerability and reduced vascular toxicity over earlier PDE4 inhibitors that lack subtype selectivity. The lead compound, 28 (BPN14770), has entered midstage, human phase 2 clinical trials for the treatment of Fragile X Syndrome.

Filth flies, including house flies, Musca domestica L., develop in animal manure. Adult house flies often are controlled with pesticides such as imidacloprid. How imidacloprid disseminates and persists after it contaminates manure was measured at a dairy farm. A week after application of imidacloprid via fly bait to cattle manure, a mean of approximately 4 ppm of imidacloprid, and as high as 15 ppm, was quantifiable up to 12 cm from the application site, but not farther. Laboratory experiments addressed the impact of 15 ppm of imidacloprid in manure on egg-to-adult development of house flies and on the biological control ability of a house fly pupal parasitoid, Spalangia endius Walker. In uncontaminated manure, 93% of eggs developed to adults, versus 7% in contaminated manure. In the parasitoid experiment, fly pupae were placed in contaminated or uncontaminated manure with or without S. endius. In the absence of S. endius, nearly 100% of flies emerged, with or without imidacloprid. In the presence of S. endius, only 11% of flies emerged from uncontaminated manure, versus 36% from contaminated manure; and parasitoids emerged from 82% of hosts in uncontaminated manure versus 53% in contaminated manure. These results suggest that realistic concentrations of imidacloprid in filth fly breeding habitat may interfere with house flies developing to the pupal stage, but also with parasitoids locating and utilizing house flies. However, after 1 wk, the effects on parasitoids will be low 12 cm beyond where bait was applied., (© The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: Therapies that recapitulate the health benefits of caloric restriction in older adults are needed. Phosphodiesterase 4 inhibitors demonstrate such promise. We examined their effects on body weight and composition, physical and cognitive function in aged mice using Compound D159687 (D159687)., Methods: Nineteen 18-months old mice were randomized to receive either control (DMSO) or D159687 for seven weeks. We assessed food intake, body weight and body composition over time and performed once the following tests: treadmill, inverted grip strength, rotarod, spontaneous Y maze tests and skeletal muscle mitochondrial biogenesis., Results: Four of the D159687 treated mice died in the first week. Necropsy suggests acute lung injury. D159687 treated mice weighed more than control mice at baseline. After controlling for baseline weight, D159687 treated mice lost 4.2 grams(g) more weight than control mice, mainly from fat mass loss (p value < 0.001). Muscle mass was unchanged between the two mice groups. D159587 mice ate significantly more food than the control mice. We found no difference between the two groups in the results of treadmill, rotarod and spontaneous Y maze tests and in mitochondrial biogenesis., Conclusion: Compound D159687 induced weight loss, predominantly fat mass loss and increased food intake in aged mice. The caloric restriction and lean mass preservation potential of PDE4D inhibitors deserve further verification. Findings may have major therapeutic implications when translated to the older adult population. Although physical and cognitive parameters were unchanged in this study, further studies would be needed to verify these results. The high death rate in the D159687 treated mice may have been due to the technical aspects of oral gavage., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Methionine aminopeptidase (MetAP) is a dinuclear metalloprotease responsible for the cleavage of methionine initiator residues from nascent proteins. MetAP activity is necessary for bacterial proliferation and is therefore a projected novel antibacterial target. A compound library consisting of 294 members containing metal-binding functional groups was screened against Rickettsia prowazekii MetAP to determine potential inhibitory motifs. The compounds were first screened against the target at a concentration of 10 µM and potential hits were determined to be those exhibiting greater than 50% inhibition of enzymatic activity. These hit compounds were then rescreened against the target in 8-point dose-response curves and 11 compounds were found to inhibit enzymatic activity with IC 50 values of less than 10 µM. Finally, compounds (1-5) were docked against RpMetAP with AutoDock to determine potential binding mechanisms and the results were compared with crystal structures deposited within the PDB., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Drug design and discovery remains a popular topic of study to many students interested in visible, real-world applications of the chemical sciences. It is important that laboratory experiments detailing the early stages of drug discovery incorporate both compound design and an exploration of ligand/receptor interactions. Molecular modeling is widely employed in research endeavors seeking to predict the activity of potential compounds prior to synthesis and can therefore be used to illustrate these concepts. The following activity therefore details the use of AutoDock to predict the binding affinity and docked pose of a series of CDK2 inhibitors. Students can then compare their docking output to experimentally determined inhibitory activities and crystal structures. Finally, the AutoDock workflow detailed in this activity can be used in research settings, provided the receptor crystal structure is known.

Methionine aminopeptidase (MetAP) is a class of ubiquitous enzymes essential for the survival of numerous bacterial species. These enzymes are responsible for the cleavage of N-terminal formyl-methionine initiators from nascent proteins to initiate post-translational modifications that are often essential to proper protein function. Thus, inhibition of MetAP activity has been implicated as a novel antibacterial target. We tested this idea in the present study by targeting the MetAP enzyme in the obligate intracellular pathogen Rickettsia prowazekii. We first identified potent RpMetAP inhibitory species by employing an in vitro enzymatic activity assay. The molecular docking program AutoDock was then utilized to compare published crystal structures of inhibited MetAP species to docked poses of RpMetAP. Based on these in silico and in vitro screens, a subset of 17 compounds was tested for inhibition of R. prowazekii growth in a pulmonary vascular endothelial cell (EC) culture infection model system. All compounds were tested over concentration ranges that were determined to be non-toxic to the ECs and 8 of the 17 compounds displayed substantial inhibition of R. prowazekii growth. These data highlight the therapeutic potential for inhibiting RpMetAP as a novel antimicrobial strategy and set the stage for future studies in pre-clinical animal models of infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The title compound, C 19 H 12 Cl 2 N 2 O 4 S 3 , is related to a ditosylated 2-iminobenzothiazole with the two methyl groups on the two phenyl rings replaced by chlorine. There is a weak intramolecular π - π contact between the two phenyl rings, with a centroid-to-centroid distance of 4.004 (2) Å. The dihedral angle between the rings is 9.96 (13)°. An intramolecular C-H⋯O hydrogen bond stabilizes the molecular conformation.

Methionine aminopeptidases (MetAPs) are metalloenzymes that cleave the N-terminal methionine from newly synthesized peptides and proteins. These MetAP enzymes are present in bacteria, and knockout experiments have shown that MetAP activity is essential for cell life, suggesting that MetAPs are good antibacterial drug targets. MetAP enzymes are also present in the human host and selectivity is essential. There have been significant structural biology efforts and over 65 protein crystal structures of bacterial MetAPs are deposited into the PDB. This review highlights the available crystallographic data for bacterial MetAPs. Structural comparison of bacterial MetAPs with human MetAPs highlights differences that can lead to selectivity. In addition, this review includes the chemical diversity of molecules that bind and inhibit the bacterial MetAP enzymes. Analysis of the structural biology and chemical space of known bacterial MetAP inhibitors leads to a greater understanding of this antibacterial target and the likely development of potential antibacterial agents.

The fragment FOL7185 (compound 17) was found to be a hit against IspD and IspE enzymes isolated from bacteria, and a series of analogs containing the pyrazolopyrimidine core were synthesized. The majority of these compounds inhibited the growth of Burkholderia thailandensis (Bt) and Pseudomonas aeruginosa (Pa) in the Kirby–Bauer disk diffusion susceptibility test. Compound 29 shows inhibitory activity at 0.1 mM (32.2 lg/mL), which is comparable to the control compound kanamycin (48.5 lg/mL). Compound 29 also shows inhibitory activity at 0.5 mM against kanamycin resistant P. aeruginosa. Saturation transfer difference NMR (STD-NMR) screening of these compounds against BtIspD and BtIspE indicated that most of these compounds significantly interact with BtIspE, suggesting that the compounds may inhibit the growth of Bt by disrupting isoprenoid biosynthesis. Ligand epitope mapping of compound 29 with BtIspE indicated that hydrogens on 2,4-dichlorophenyl group have higher proximity to the surface of the enzyme than hydrogens on the pyrazolopyrimidine ring.

A series of novel aminoalkylated quercetin analogs, prepared via the Mannich reaction of various primary and secondary amines with formaldehyde, were tested for antimalarial activity. The compounds were screened against three drug resistant malarial strains (D6, C235 and W2) and were found to exhibit sub-micromolar activity across all three strains (0.065-13.0μM). The structure-activity relationship determined from the antimalarial activity data suggests the inclusion of phenethyl amine sidechains on the quercetin scaffolding is necessary for potent activity. Additionally, the most active compounds ((5) and (6)) were tested for both early and late stage anti-gametocytocidal activity. Finally, the antimalarial activity data were utilized to construct comparative molecular field analysis (CoMFA) models to be used for further compound refinement., (Copyright © 2014 Elqsevier Ltd. All rights reserved.)

In this study we report a series of triazine derivatives that are potent inhibitors of PDE4B. We also provide a series of structure activity relationships that demonstrate the triazine core can be used to generate subtype selective inhibitors of PDE4B versus PDE4D. A high resolution co-crystal structure shows that the inhibitors interact with a C-terminal regulatory helix (CR3) locking the enzyme in an inactive 'closed' conformation. The results show that the compounds interact with both catalytic domain and CR3 residues. This provides the first structure-based approach to engineer PDE4B-selective inhibitors., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Evaluation of a series of MetAP inhibitors in an in vitro enzyme activity assay led to the first identification of potent molecules that show significant growth inhibition against Burkholderia pseudomallei. Nitroxoline analogs show excellent inhibition potency in the BpMetAP1 enzyme activity assay with the lowest IC 50 of 30 nM, and inhibit the growth of B. pseudomallei and B. thailandensis at concentrations ≥ 31 μM.

5(S)-Fluoro-N6-(iminoethyl)-l-lysine (14), an analogue of the potent, selective induced nitric oxide synthase (iNOS) inhibitor iminoethyl-l-lysine (1), was synthesized and found to be a selective iNOS inhibitor.

(3S,4S,5R)-2-Imino-4-methyl-5-pentyl-3-pyrrolidinol hydrochloride (1) is a potent inducible nitric oxide synthase (i-NOS) inhibitor that has three times the selectivity of its parent, (+)-cis-4-methyl-5-pentylpyrrolidin-2-imine hydrochloride (2).

8-Chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid, 2-[1-oxo-3-(4-pyridinyl)propyl]hydrazide, monohydrochloride (1, SC-51089) is a functional PGE2 antagonist selective for the EP1 receptor subtype with antinociceptive activity. Analogues of SC-51089, in which the diacylhydrazine moiety has been replaced with 2,4-disubstituted-oxazoles and-thiazoles, are described.

A series of substituted 2-iminopyrrolidines has been prepared and shown to be potent and selective inhibitors of the human inducible nitric oxide synthase (hiNOS) isoform versus the human endothelial nitric oxide synthase (heNOS) and the human neuronal nitric oxide synthase (hnNOS). Simple substitutions at the 3-, 4-, or 5-position afforded more potent analogues than the parent 2-iminopyrrolidine 1. The effect of ring substitutions on both potency and selectivity for the different NOS isoforms is described. Substitution at the 4- and 5-positions of the 2-iminopyrrolidine yielded both potent and selective inhibitors of hiNOS. In particular, (+)-cis-4-methyl-5-pentylpyrrolidin-2-imine, monohydrochloride (20), displayed potent inhibition of hiNOS (IC50 = 0.25 microM) and selectivities of 897 (heNOS IC50/hiNOS IC50) and 13 (hnNOS IC50/hiNOS IC50). Example 20 was shown to be an efficacious inhibitor of NO production in the mouse endotoxin assay. Furthermore, 20 displayed in vivo selectivity, versus heNOS isoform, by not elevating blood pressure at multiples of the effective dose in the mouse.

A series of analogues of 2-iminopiperidine have been prepared and shown to be potent inhibitors of the human nitric oxide synthase (NOS) isoforms. Methyl substitutions on the 4-position (3) or 4- and 6-positions (8) afforded the most potent analogues. These compounds exhibited IC50 values of 0.1 and 0.08 microM, respectively, for hiNOS inhibition. Substitution with cyclohexylmethyl at the 6-position (13) afforded an inhibitor that showed the best selectivity for hiNOS versus heNOS (heNOS IC50/hiNOS IC50 = 64). Following oral administration, inhibitors were found to decrease serum nitrite/nitrate levels in an in vivo rat endotoxin assay. This series of 2-iminopiperidines were prepared via the described synthetic methodologies. The effect of ring substitutions on potency and selectivity for this class of cyclic amidines as NOS inhibitors is described.

8-Chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid, 2-[1-oxo-3-(4-pyridinyl)propyl]hydrazide, monohydrochloride (1, SC-51089) is a functional PGE2 antagonist selective for the EP1 receptor subtype with antinociceptive activity. During metabolism in cultured rat hepatocytes, SC-51089, which contains a diacylhydrazine moiety, has been shown to release hydrazine. Analogs of SC-51089, in which the diacylhydrazine functionality has been replaced by isosteric and isoelectronic groups, have been synthesized and have been shown to be analgesics and PGE2 antagonists of the EP1 subtype. This report discusses the structure-activity relationships within these series.

8-Chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid, 2-acetylhydrazide (1, SC-19220) has been previously reported by us and others to be a PGE2 antagonist selective for the EP1 receptor subtype with antinociceptive activities. Analogs of SC-19220, in which the acetyl moiety has been replaced with pyridylpropionyl groups and their homologs, have been synthesized as illustrated by compounds 13 and 29. These and other members of this series have been shown to be efficacious analgesics and PGE2 antagonists of the EP1 subtype. This report discusses the structure activity relationships within this series.

The synthesis and affinities of six new 3-substituted beta-carbolines (6-10, 12) for the benzodiazepine receptor (BzR) are described. These analogs were used both to probe the dimensions of the hydrophobic pocket in the benzodiazepine receptor and to test the predictive ability of a previously reported 3D-QSAR regression model. Of the new analogs synthesized, the gamma-branched derivatives (isobutoxy, 7, IC50 = 93 nM; isopentoxy, 9, IC50 = 104 nM) display significantly higher affinity for the BzR than either the beta-branched (sec-butoxy, 6, IC50 = 471 nM; tert-butyl ketone, 12, IC50 = 358 nM) or delta-branched (isopentoxy, 8, IC50 = 535 nM) analogs. An exception to this rule is the gamma-branched 3-benzyloxy derivative 10 (IC50 > 1000 nM) which appears to have a chain length that is too long to be accommodated by the BzR. The standard error of prediction for these six new beta-carbolines using the original regression model is significantly lower than the standard error estimate of the cross validation runs on the training set, hence the predictions made using this model are much better than expected. In order to obtain more credible predictions, a new procedure called GOLPE (generating optimal linear PLS estimates) was used to eliminate irrelevant electrostatic and steric descriptors from the regression equation. A substantial reduction in the standard error estimate resulted. The predictions from this new regression equation were somewhat less accurate than the ones obtained with the original regression equation; however the standard error of prediction and the standard error estimate are in much closer agreement. Finally, to probe the effect that the quality of the steric and electrostatic potentials has on 3D-QSAR analyses, the semiempirical MNDO parallel PRDDOE geometries and Mulliken charges used in the original analyses were replaced with ab initio 3-21G parallel 6-31G* geometries and electrostatic potential fit charges. A modest decrease in the standard error estimate and increase in cross validated R2 resulted.

In order to employ rational drug design in the discovery of selective benzodiazepine receptor agonists and inverse agonists, pharmacophore/receptor models for both these activities must first be established. Recently, a pharmacophore for the inverse agonist site has been formulated employing the most recent receptor mapping techniques (22). The continuation of this approach to the pharmacophore for agonist ligands has permitted a definition of this site independently of the inverse agonist model. The agonist pharmacophore/receptor contains two hydrogen bond donating sites of interaction (H1 and H2) located about 6.5 A from each other, as well as three areas of lipophilic interaction (L1-L3). The areas L1 and L2 are critical for agonist activity; moreover, some ligands also require an interaction in a third lipophilic area termed L3. This is in agreement with previous work (12-23). In addition, an area of negative steric interaction (S1) between the ligand and receptor-binding protein is defined. In regard to the pharmacophore, it was established that the alignment rule for agonist beta-carbolines is different from that which elicits inverse agonist activity. Consideration of the pharmacophore has resulted in the synthesis of a new beta-carboline 16 which elicits agonist activity. This ligand 16 not only satisfied the requirements of the pharmacophore, but more importantly it elicited both anticonvulsant and anxiolytic activity, but was devoid of the myorelaxant/ataxic properties associated with the benzodiazepines.

The structural requirements for ligand binding to the benzodiazepine receptor (BzR) inverse agonist site were probed through the synthesis and in vitro evaluation of 3-substituted beta-carbolines 6, 7, 11, 12, gamma-carboline 13, and diindoles 18-21, 23-25, 27, 28, and 34. On the basis of the apparent binding affinities of these and other analogues, a hydrogen bond acceptor site (A2) on the receptor is proposed to interact with the N(9) hydrogen atom of the beta-carbolines or the N(7) hydrogen nuclei of the diindoles. Likewise, a proposed hydrogen bond donating site (H1) interacts with the N(2) nitrogen atom of the beta-carbolines or the N(5) nitrogen atom of the diindoles. It appears that interaction with both sites is a prerequisite for high affinity since analogues which have either one or both of these positions blocked exhibit substantial reduction in affinity. Moreover, H1 appears to be capable of engaging in a three-centered hydrogen bond with appropriately functionalized ligands, which explains the increase in potency observed in the following series of 3-substituted beta-carbolines: the n-butyl (12, IC50 = 245 nM), n-propoxy (9, IC50 = 11 nM), and propyl ketone (11, IC50 = 2.8 nM) congeners. In addition to H1 and A2, there appears to be a relatively narrow hydrophobic pocket in the binding cleft that can accommodate substituents at the 3-position of the beta-carbolines which have chain lengths less than or equal to C5. There is a 1 order of magnitude decrease in affinity between n-propoxy analogue 9 (IC50 = 11 nM, chain length = 4) and n-butoxy derivative 7 (IC50 = 98 nM, chain length = 5). Furthermore, alpha- and gamma-branching [e.g. ethoxycarbonyl (2), IC50 = 5 nM and tert-butoxycarbonyl (31) IC50 = 10 nM] but not beta- and delta-branching [e.g. isopropoxy (6), IC50 = 500 nM and (neopentyloxy) carbonyl (48), IC50 = 750 nM] at position 3 are tolerated. Occupation of this hydrophobic pocket is clearly important for high affinity as evidenced by the relatively low affinity of 30, a beta-carboline which possesses a hydrogen atom at the 3-position. This same hydrophobic pocket is partially filled by the D and E rings of the diindoles, which accounts for the high affinity of several members of this series. An excluded volume analysis using selected 3-substituted beta-carbolines and ring-E substituted pyridodiindoles is consistent with the presence of this hydrophobic pocket (see Figure 1).(ABSTRACT TRUNCATED AT 400 WORDS)

The csg locus of Myxococcus xanthus appears to control the production of an intercellular signal that is essential for development. The complete nucleotide sequence of a clone containing the csg locus was determined by the dideoxy-chain termination method. Pattern recognition analyses of the DNA sequence revealed the presence of two protein-coding regions that are convergently oriented and separated by only 8 nucleotides. Tn5 lac insertions into this clone detected two transcriptional units that are transcribed in a convergent fashion and whose expression increases during development. The two genes represented by these protein-coding regions and transcriptional units have been designated csgA and fprA. Northern (RNA) blot analyses detected an 800-nucleotide RNA specific to the csgA gene and a 900-nucleotide RNA specific to the fprA gene. Our results, along with mutational studies, identify csgA as the gene involved in cell communication. The function of the fprA gene is described in an accompanying paper (L. J. Shimkets, J. Bacteriol. 172:24-30, 1990).

Using Northern blot analysis we have studied the transcription of the CM4-184 Ga. strain of cauliflower mosaic virus (CaMV) DNA. This analysis reveals that this CaMV strain, like the Cabb-BS and the Cabb B-JI strains, produces both a genomic length transcript and a 1900-nucleotide (nt) transcript during infection. In addition, we detect an 1800-nt PA+ transcript mapping primarily to the EcoRI-c region of the virus, and three apparent minor viral-specific PA + RNAs of 4900, 4500, and 4300 nt. We also report the presence of two small viral single-stranded DNAs produced during infection, and show that both DNAs are derived from the EcoRI-b region of the virus.

Rats were trained to discriminate between saline and 1.0 mg/kg of diazepam in a two-choice procedure where responding was maintained under a fixed-ratio, 5-response schedule of stimulus shock termination. beta-Carboline-3-carboxylate-methyl ester (beta CCM), beta-carboline-3-carboxylate-ethyl ester (beta CCE) and beta-carboline-3-carboxylate-t-butyl ester (beta CCtB), compounds with alkylcarboxy substitutions on the 3-position of the beta-carboline ring structure, were effective antagonists of the discriminative effects of diazepam. The 3-hydroxymethyl-substituted compound (3HMC) was relatively ineffective in antagonizing the discriminative effects of diazepam. The order of potency in antagonizing the 1.0 mg/kg training dose of diazepam was beta CCtB greater than beta CCM greater than beta CCE much greater than 3 HMC. The greater potency of beta CCtB likely reflects its resistance to metabolism in vivo. beta CCE and beta CCtB produced dose-related, parallel shifts in the dose-response curve for the discriminative effects of diazepam, but the magnitude of the shifts was limited: the two highest doses of beta CCE and beta CCtB produced shifts that were not significantly different in magnitude. These latter results suggest that these beta-carbolines antagonize only a portion of the component(s) of action of diazepam in producing discriminative stimuli. In contrast, the 7-substituted beta-carbolines harmane, harmol and harmine were ineffective in antagonizing the discriminative effects of diazepam up to doses of the beta-carbolines which disrupted the ability of the animals to respond.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Ethoxy-beta-carboline binds with high affinity to benzodiazepine receptors in the central nervous system (Ki approximately equal to 10.1, 15.3, and 25.3 nM in rat cerebellum, cerebral cortex, and hippocampus, respectively). This compound has pharmacological actions reminiscent of benzodiazepine receptor partial inverse agonists such as FG 7142 and 3-carboethoxy-beta-carboline. Thus, while not a convulsant, 3-ethoxy-beta-carboline potentiated the convulsant actions of pentylenetetrazole in mice. Furthermore, this compound reduced both the time spent and the total entries in the open arms of an elevated plus maze and also inhibited stress-induced ulcer formation, effects that are also observed with benzodiazepine receptor inverse agonists. These findings suggest that 3-ethoxy-beta-carboline is a partial inverse agonist at benzodiazepine receptors which may prove useful for in vivo studies since it has a higher affinity for benzodiazepine receptors and better solubility than the commonly used partial inverse agonist FG 7142. Furthermore, 3-ethoxy-beta-carboline appears to be less vulnerable to metabolic degradation than ester analogs with a similar pharmacological profile such as 3-carboethoxy-beta-carboline.

Physostigmine has been reported to reverse the sedation and paradoxical delirium induced by benzodiazepines. Little is known about how these drugs may interact to produce changes in cerebral metabolism and cerebral blood flow (CBF). In the present experiments, the effect of physostigmine on cerebral oxygen consumption (CMRO2) and CBF as well as the ability of physostigmine to reverse the effects of midazolam and 3-carbo-t-butoxy-B-carboline (B-CCT), a benzodiazepine antagonist, was tested in rats. Physostigmine by itself produced dose-dependent increases in blood pressure, CBF, and CMRO2, and it inhibited the decrease in these parameters produced by midazolam. Alone, B-CCT increased CBF and CMRO2, and these changes were potentiated by physostigmine. Thus, physostigmine increases CBF and CMRO2, probably by a direct effect on central cholinergic pathways. The ability of physostigmine to antagonize the metabolic effects of midazolam and to potentiate the stimulation produced by B-CCT suggests an additive effect of the two neurotransmitter systems rather than a direct interaction at the central receptor sites.

The effects of systemic injections of beta-carboline-3-carboxylate-t-butyl ester (beta-CCtB) were investigated with regard to normally occurring sleep and several measures of benzodiazepine receptor occupancy in rats. A dose of 30 mg/kg of beta-CCtB was found to have a long time-course of action as measured by an in vivo assay for benzodiazepine binding, with an 84% depletion of [3H]diazepam binding at one hour after the intraperitoneal injection. This dose of beta-CCtB was shown to delay sleep onset, decrease non-REM and total sleep in the first two hours after the injection, and to delay the appearance of REM sleep after the sleep onset. The dose- and time-dependence of the effects on sleep approximated the dose- and time-dependence of inhibitory effects of an IP injection of beta-CCtB on in vitro measures of benzodiazepine receptor affinity and number.

Three squirrel monkeys were trained to emit one response after IV administration of nicotine (0.1 or 0.18 mg/kg depending on the subject) and a different response after IV administration of saline. Subjects emitted nicotine-appropriate responses with substitutions of higher doses, but only emitted saline-appropriate responses after substitutions of lower doses. Discrimination performance was then maintained at 0.1 mg/kg of nicotine in all subjects. Neither morphine nor cocaine substituted for the effects of nicotine in any subjects across a range of doses up to those that suppressed responding. Ethyl-beta-carboline-3-carboxylate, an inverse agonist at the benzodiazepine receptor, substituted or partially substituted for nicotine in both subjects in which it was studied.

The synthesis of the first beta-carboline, 6-(benzylamino)-beta-carboline (1c), to be devoid of a substituent at the 3-position and that still binds to benzodiazepine receptors with potent affinity is described. Furthermore, 1c proved to be a partial inverse agonist when tested in mice. Addition of the benzylamino group at the 6-position of the beta-carboline nucleus is primarily responsible for the activity of beta-carbolines 1b and 1c. The importance of the Nb-nitrogen atom for binding affinity was also demonstrated since 3-(benzylamino)carbazole (6) exhibited little or no affinity for benzodiazepine receptors in vitro, in contrast to the activity of 1c.

The 3-substituted beta-carbolines 2-4 and 5-7 were prepared from 3-amino-beta-carboline (8) in one step via diazotization, followed by reaction with the appropriate nucleophile in order to determine their binding affinity for benzodiazepine receptors (BzR). All three of the 3-alkoxy-beta-carbolines 2 (IC50 = 124 nM), 3 (IC50 = 24 nM), and 4 (IC50 = 11 nM) have high affinities for BzR. The beta-carbolines substituted with electron-withdrawing groups including 5 (Cl; IC50 = 45 nM), 6 (NO2; IC50 = 125 nM), and 7 (N = C = S; IC50 = 8 nM) also had high affinities for BzR. The affinities of 5-8 clearly indicate that a carbonyl moiety at position 3 of a beta-carboline is not required for high-affinity binding to BzR. These findings have led to the development of a model for the binding of ligands to an inverse agonist domain at BzR. This model is supported by the recent synthesis of 3-ethoxy-beta-carboline (3), a potent, long-lived partial inverse agonist, and 7, an irreversible BzR ligand.