Pyridylethanol(phenylethyl)amines are non-azole, highly selective Candida albicans sterol 14α-demethylase inhibitors.

Sterol 14α-demethylase (CYP51) is the main drug target for the treatment of fungal infections. The worldwide increase in the incidence of opportunistic fungal infections and the emerging resistance to available azole-based antifungal drugs, raise the need to develop structurally distinct and selective fungal CYP51 inhibitors. In this work we have, for the first time, investigated the binding of pyridylethanol(phenylethyl)amines to any fungal CYP51. The comparison of the binding to Candida albicans and human CYP51 studied by spectroscopic and modeling methods revealed moieties decisive for selectivity and potency and resulted in the development of highly selective derivatives with significantly increased inhibitory potency. The structure-based insight into the selectivity requirements of this new chemical class of fungal CYP51 inhibitors, their unique binding properties and the low molecular weight of lead derivatives offer novel directions for the targeted development of antifungal clinical candidates.

Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization.

Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/D-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/D-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8-11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.

Hydroxypropyl methylcellulose mediated precipitation inhibition of sirolimus: from a screening campaign to a proof-of-concept human study.

The aim of this study was to develop a sirolimus (BCS class II drug substance) solid oral dosage form containing a precipitation inhibitor, which would result in an improved sirolimus absorption in humans compared to the formulation containing nanosized sirolimus without a precipitation inhibitor, i.e., Rapamune. The selection of the precipitation inhibitor was based on the results of a screening campaign that identified two "hit" excipients: HPMC 603 (i.e., Pharmacoat 603) and Poloxamer 407. However, in a confirmatory precipitation inhibitor study using biorelevant media (Fa/FeSSIF) HPMC 603 more effectively inhibited sirolimus precipitation than Poloxamer 407. In the PAMPA assay, HPMC 603, but not Poloxamer 407, significantly increased the flux of the sirolimus across the membrane lipid layer. Additionally, a differential scanning calorimetry (DSC) and an infrared (IR) spectroscopy study revealed that interactions between the sirolimus and HPMC 603 were developed that could lead to the observed precipitation inhibition effect. Based on the above data, two formulations with HPMC 603-coated sirolimus particles were developed, namely, formulation A (d (0.5) = 0.21 µm) and formulation B (d (0.5) = 1.7 µm). A human pharmacokinetic study outlined that significantly higher AUC and Cmax were obtained for formulations A and B in comparison to Rapamune. This result could be attributed to the HPMC 603 (Pharmacoat 603) mediated sirolimus precipitation inhibition resulting in improved sirolimus absorption from the gastrointestinal tract in humans.

Inhibition of human sterol Δ7-reductase and other postlanosterol enzymes by LK-980, a novel inhibitor of cholesterol synthesis.

Novel potential inhibitors of the postsqualene portion of cholesterol synthesis were screened in HepG2 cells. 2-(4-Phenethylpiperazin-1-yl)-1-(pyridine-3-yl)ethanol (LK-980) was identified as a prospective compound and was characterized further in cultures of human primary hepatocytes from seven donors. In vitro kinetic measurements show that the half-life of LK-980 is at least 4.3 h. LK-980 does not induce CYP3A4 mRNA nor enzyme activity. Target prediction was performed by gas chromatography-mass spectrometry, allowing simultaneous separation and quantification of nine late cholesterol intermediates. Experiments indicated that human sterol Δ(7)-reductase (DHCR7) is the major target of LK-980 (34-fold increase of 7-dehydrocholesterol), whereas human sterol Δ(14)-reductase (DHCR14), human sterol Δ(24)-reductase (DHCR24), and human sterol C5-desaturase (SC5DL) represent minor targets. In the absence of purified enzymes, we used the mathematical model of cholesterol synthesis to evaluate whether indeed more than a single enzyme is inhibited. In silico inhibition of only DHCR7 modifies the flux of cholesterol intermediates, resulting in a sterol profile that does not support experimental data. Partial inhibition of the DHCR14, DHCR24, and SC5DL steps, in addition to DHCR7, supports the experimental sterol profile. In conclusion, we provide experimental and computational evidence that LK-980, a novel inhibitor from the late portion of cholesterol synthesis, inhibits primarily DHCR7 and to a lesser extent three other enzymes from this pathway.

Drug interaction potential of 2-((3,4-dichlorophenethyl)(propyl)amino)-1-(pyridin-3-yl)ethanol (LK-935), the novel nonstatin-type cholesterol-lowering agent.

The widely prescribed lipid-lowering statins are considered to be relatively safe drugs. However, the risk of severe myopathy and drug interactions as a consequence of statin therapy provides a challenge for development of novel cholesterol-lowering agents, targeting enzymes other than HMG-CoA reductase. The novel pyridylethanol-(phenylethyl)amine derivative, (2-((3,4-dichlorophenethyl)(propyl)-amino)-1-(pyridin-3-yl)ethanol (LK-935), blocking lanosterol 14alpha-demethylase, was demonstrated to efficiently reduce cholesterol biosynthesis. The drug interaction potential of LK-935 was investigated and compared with that of atorvastatin and rosuvastatin in primary human hepatocytes. Clear evidence was provided for the induction of CYP3A4 by LK-935. LK-935 was proved to be a potent human pregnane X receptor (hPXR) activator as a prerequisite for the transcriptional activation of CYP3A4 gene; however, the rapid metabolism of LK-935 in primary hepatocytes prevented maximal CYP3A4 induction. Therefore, the induction of CYP3A4 by LK-935 may be prone to mild or negligible drug interactions. However, because CYP3A4 and also CYP2C9 play a significant role in LK-935 metabolism, the inhibition of these cytochromes P450 by coadministered drugs may lead to some increase in the LK-935 concentration required for the potent induction of CYP3A4. Rosuvastatin was found to increase human constitutive androstane receptor (hCAR)-mediated transcription of CYP3A4, CYP2C9, and CYP2B6 genes, predicting the consequent potential for drug interactions with several coadministered drugs. Activation of hCAR and hPXR by atorvastatin and the subsequent induction of not only CYP2B6 and CYP3A4 but also of CYP2C9 present an additional target by which atorvastatin, a widely used cholesterol-lowering drug, can modify the kinetics of numerous drugs.

Novel cholesterol biosynthesis inhibitors targeting human lanosterol 14alpha-demethylase (CYP51).

Novel cholesterol biosynthesis inhibitors, a group of pyridylethanol(phenylethyl)amine derivatives, were synthesized. Sterol profiling assay in the human hepatoma HepG2 cells revealed that compounds target human lanosterol 14alpha-demethylase (CYP51). Structure-activity relationship study of the binding with the overexpressed human CYP51 indicates that the pyridine binds within the heme binding pocket in an analogy with the azoles.

4-Substituted trinems as broad spectrum beta-lactamase inhibitors: structure-based design, synthesis, and biological activity.

A wide variety of pathogens have acquired antimicrobial resistance as an inevitable evolutionary response to the extensive use of antibacterial agents. In particular, one of the most widely used antibiotic structural classes is the beta-lactams, in which the most common and the most efficient mechanism of bacterial resistance is the synthesis of beta-lactamases. Class C beta-lactamase enzymes are primarily cephalosporinases, mostly chromosomally encoded, and are inducible by exposure to some beta-lactam agents and resistant to inhibition by marketed beta-lactamase inhibitors. In an ongoing effort to alleviate this problem a series of novel 4-substituted trinems was designed and synthesized. Significant in vitro inhibitory activity was measured against the bacterial beta-lactamases of class C and additionally against class A. The lead compound LK-157 was shown to be a potent mechanism-based inactivator. Acylation of the active site Ser 64 of the class C enzyme beta-lactamase was observed in the solved crystal structures of two inhibitors complexes to AmpC enzyme from E. cloacae. Structure-activity relationships in the series reveal the importance of the trinem scaffold for inhibitory activity and the interesting potential of the series for further development.

A novel 2-oxoindolinylidene inhibitor of bacterial MurD ligase: Enzyme kinetics, protein-inhibitor binding by NMR and a molecular dynamics study.

N-(5-(5-nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)4-oxo-2-thioxo-1,3-thiazolidin-3-yl)nicotinamide, a 2-oxoindolinylidene derivative with novel structure scaffold, was evaluated for inhibition potency against the MurD enzyme from Escherichia coli using an enzyme steady-state kinetics study. The compound exerted competitive inhibition with respect to UMA, a MurD substrate, and affected bacterial growth. Furthermore, we isolated and purified (13)C selectively labeled MurD enzyme from E. coli and evaluated the binding interactions of the new compound using the (1)H/(13)C-HSQC 2D NMR method. Molecular dynamics calculations showed stable structure for the MurD-inhibitor complex. The binding mode of novel inhibitor was determined and compared to naphthalene-N-sulfonamide-d-Glu derivatives, transition state mimicking inhibitors, UMA and AMP-PCP, an ATP analog. It binds to the UDP/MurNAc binding region. In contrast to transition state mimicking inhibitors, it does not interact with the enzyme's C-terminal domain, which can be beneficial for ligand binding. A pharmacophore pattern was established for the design of novel drugs having a propensity to inhibit a broad spectrum of Mur enzymes.

Isolation and structure determination of oxidative degradation products of atorvastatin.

Methods were developed for the preparation and isolation of four oxidative degradation products of atorvastatin. ATV-FX1 was prepared in the alkaline acetonitrile solution of atorvastatin with the addition of hydrogen peroxide. The exposition of aqueous acetonitrile solution of atorvastatin to sunlight for several hours followed by the alkalization of the solution with potassium hydroxide to pH 8-9 gave ATV-FXA. By the acidification of the solution with phosphoric acid to pH 3 ATV-FXA1 and FXA2 were prepared. The isolation of oxidative degradation products was carried out on a reversed-phase chromatographic column Luna prep C18(2) 10 microm applying several separation steps. The liquid chromatography coupled with a mass spectrometer (LC-MS), high resolution MS (HR-MS), 1D and 2D NMR spectroscopy methods were applied for the structure elucidation. All degradants are due to the oxidation of the pyrrole ring. The most probable reaction mechanism is intermediate endoperoxide formation with subsequent rearrangement and nucleophilic attack by the 5-hydroxy group of the heptanoic fragment. ATV-FX1 is 4-[1b-(4-Fluoro-phenyl)-6-hydroxy-6-isopropyl-1a-phenyl-6a-phenylcarbamoyl-hexahydro-1,2-dioxa-5a-aza-cyclopropa[a]inden-3-yl]-3-(R)-hydroxy-butyric acid and has a molecular mass increased by two oxygen atoms with regard to atorvastatin. ATV-FXA is the regioisomeric compound, 4-[6-(4-Fluoro-phenyl)-6-hydroxy-1b-isopropyl-6a-phenyl-1a-phenylcarbamoyl-hexahydro-1,2-dioxa-5a-aza-cyclopropa[a]inden-3-yl]-3-(R)-hydroxy-butyric acid. Its descendants ATV-FXA1 and FXA2 appeared without the atorvastatin heptanoic fragment and are 3-(4-Fluoro-benzoyl)-2-isobutyryl-3-phenyl-oxirane-2-carboxylic acid phenylamide and 4-(4-Fluoro-phenyl)-2,4-dihydroxy-2-isopropyl-5-phenyl-3,6-dioxa-bicyclo[3.1.0]hexane-1-carboxylic acid phenylamide, respectively. Quantitative NMR spectroscopy was employed for the assay determination of isolated oxidative degradation products. The results obtained were used for the determination of the UV response factors relative to atorvastatin.

NMR and molecular dynamics study of the binding mode of naphthalene-N-sulfonyl-D-glutamic acid derivatives: novel MurD ligase inhibitors.

The presented series of naphthalene-N-sulfonyl-D-glutamic acid derivatives are novel MurD ligase inhibitors with moderate affinity that occupy the D-Glu binding site. We performed an NMR study including transfer NOE to determine the ligand bound conformation, as well as saturation transfer difference experiments to obtain ligand epitope maps. The difference in overall appearance of the epitope maps highlights the importance of hydrophobic interactions and shows the segments of molecular structure that are responsible for them. Transfer NOE experiments indicate the conformational flexibility of bound ligands, which were then further examined by unrestrained molecular dynamics calculations. The results revealed the differing degrees of ligand flexibility and their effect on particular ligand-enzyme contacts. Conformational flexibility not evident in the crystal structures may have an effect on ligand-binding site adaptability, and this is probably one of the important reasons for the only moderate activity of novel derivatives.

Synthesis, conformation, and stereodynamics of a salt of 2-{[2-(3,4-dichlorophenyl)- ethyl]propylamino}-1-pyridin-3-ylethanol.

[reaction: see text] A novel synthetic route was developed for 2-{[2-(3,4-dichlorophenyl)ethyl]propylamino}-1-pyridin-3-ylethanol (4). A dynamic process due to nitrogen inversion at the central amine nitrogen has been identified by NMR spectroscopy for the dihydrobromide salt of this compound. The conformational properties of the diastereomeric pair were determined by analysis of NOE connectivities and MO calculations.

Entropic trapping binding mechanism: its likely role in receptor-ligand and other biochemical systems.

Entropy-driven binding continues to be discovered in receptor-ligand systems as well as in other important biochemical systems. In receptor-ligand systems the "thermodynamic agonist-antagonist discrimination" is often found: in some such receptor types the binding of antagonists is entropy-driven, in others this is a characteristic of the agonist binding. The interpretation of the entropy-driven binding mechanism in the systems in question is still rather elusive. Experimental findings clearly indicate that the entropic binding mechanisms which are usually considered cannot provide a consistent interpretation in these cases. Entropic trapping was therefore proposed in our earlier papers as a possible binding model which does not contradict the experimental facts. It is pointed out here that through the work which has appeared subsequently the existence of such a mechanism has been firmly established and that its role is strongly corroborated by the more recent data from several biochemical systems.

Electrospray ionization mass spectrometric investigation of ammonium ion complexes with anomeric 2,3-O-isopropylidene-1alpha- and -1beta-D-ribofuranosyl azides: anomeric and kinetic isotope effects in ammonium affinities.

[Methanol + ammonium acetate] solutions of anomeric 2,3-O-isopropylidene-1alpha- and 1beta-ribofuranosyl azides were investigated by electrospray ionization mass spectrometry (ESI-MS). The compounds included d6-labeled and/or unlabeled isopropylidene groups that enable the identification of peaks characteristic of the ammonium-attached monomeric (MNH4(+)), ammonium-bound homodimeric ([M]2NH4(+)) and heterodimeric ([MNH4M1](+)) complex ions in ESI mass spectra of solutions of a pair of compounds. The intensities of the product ion peaks obtained by the collisionally activated ammonium-bound dimeric ions are related to the secondary isotope effect k(alpha)/k(alphad6) = 0.88 and k(beta)/k(betad6) = 1.25 or to isotope plus anomeric effects k(alpha)/k(betad6) = 1.43 and k(beta)/k(alphad6) = 0.59 in the ammonium affinities of these compounds. The calculations of solely anomeric effects in the ammonium affinities of alpha and beta anomeric compounds obtained from the data presented previously give two series of values: k(alpha)/k(beta) = (k(alpha)/k(alphad6))(k(alphad6)/k(beta)) = 1.49 and k(alphad6)/k(betad6) = (k(alphad6)/k(beta))(k(beta)/k(betad6)) = 2.12 or k(alpha)/k(beta) = (k(alpha)/k(betad6))(k(betad6)/k(beta)) = 1.14 and k(alphad6)/k(betad6) = (k(alphad6)/k(alpha))(k(alpha)/k(betad6)) = 1.63. The disparities of these results indicate the different structures of hydrogen bonding in ammonium-bound dimeric complexes which decompose to monomeric ions with different rate constants. Comparison of experimental results obtained by the qualitative approach of the kinetic method and ammonium affinities of these compounds calculated by the semi-empirical molecular orbital method (AM1) show that the [MNH4M1](+) dimeric complex ions dissociate to the most stable MNH4(+) and M1NH4(+) monomeric ions. The obtained relative order of ammonium affinities of these compounds is: alphad6 > alpha > beta > betad6.

Protein chemical shifts arising from alpha-helices and beta-sheets depend on solvent exposure.

The NMR chemical shifts of certain atomic nuclei in proteins ((1)H(alpha),(13)C(alpha), and (13)C(beta)) depend sensitively on whether or not the amino acid residue is part of a secondary structure (alpha-helix, beta-sheet), and if so, whether it is helix or sheet. The physical origin of the different chemical shifts of atomic nuclei in alpha-helices versus beta-sheets is a problem of long standing. We report that the chemical shift contributions arising from secondary structure (secondary structure shifts) depend strongly on the extent of exposure to solvent. This behavior is observed for (1)H(alpha), (13)C(alpha), and (13)C(beta) (sheet), but not for(13)C(beta) (helix), whose secondary structure shifts are small. When random coil values are subtracted from the chemical shifts of all(1)H(alpha) nuclei (Pro residues excluded) and the residual chemical shifts are summed to plot the mean values against solvent exposure, the results give a funnel-shaped curve that approaches a small value at full-solvent exposure. When chemical shifts are plotted instead against E(local), the electrostatic contribution to conformational energy produced by local dipole-dipole interactions, a well characterized dependence of (1)H(alpha) chemical shifts on E(local) is found. The slope of this plot varies with both the type of amino acid and the extent of solvent exposure. These results indicate that secondary structure shifts are produced chiefly by the electric field of the protein, which is screened by water dipoles at residues in contact with solvent.