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Melanin Concentrating Hormone (MCH) receptor is a G protein-coupled receptor (GPCR) with two subtypes R1 and R2. MCH-R1 is involved in the control of energy homeostasis, feeding behavior and body weight. Many studies have proved that administration of MCH-R1 antagonists significantly reduces food intake and causes weight loss in animal models. Herein, we report the optimization of our previously reported virtual screening hits into novel MCH-R1 ligands with chiral aliphatic nitrogen-containing scaffolds. The activity was improved from the micromolar range of the initial leads to 7 nM. We also disclose the first MCH-R1 ligands based on a diazaspiro[4.5]decane nucleus with sub-micromolar activity. A potent MCH-R1 antagonist with acceptable pharmacokinetic profile could represent a new hope for the management of obesity., 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.)

Treatment failures with artemisinin combination therapies (ACTs) threaten global efforts to eradicate malaria. They highlight the importance of identifying drug targets and new inhibitors and of studying how existing antimalarial classes work. Here, we report the successful development of a heterologous expression-based compound-screening tool. The validated drug target Plasmodium falciparum ATPase 6 (PfATP6) and a mammalian orthologue (sarco/endoplasmic reticulum calcium ATPase 1a [SERCA1a]) were functionally expressed in Saccharomyces cerevisiae, providing a robust, sensitive, and specific screening tool. Whole-cell and in vitro assays consistently demonstrated inhibition and labeling of PfATP6 by artemisinins. Mutations in PfATP6 resulted in fitness costs that were ameliorated in the presence of artemisinin derivatives when studied in the yeast model. As previously hypothesized, PfATP6 is a target of artemisinins. Mammalian SERCA1a can be mutated to become more susceptible to artemisinins. The inexpensive, low-technology yeast screening platform has identified unrelated classes of druggable PfATP6 inhibitors. Resistance to artemisinins may depend on mechanisms that can concomitantly address multitargeting by artemisinins and fitness costs of mutations that reduce artemisinin susceptibility.

An efficient synthesis of rac -6-desmethyl-5β-hydroxy-d-secoartemisinin 2 , a tricyclic analog of R -(+)-artemisinin 1 , was accomplished and the racemate was resolved into the (+)- 2b and (-)- 2a enantiomers via their Mosher Ester diastereomers. Antimalarial activity resided with only the artemisinin-like enantiomer R -(-)- 2a . Several new compounds 9 - 16 , 19a , 19b , 22 and 29 were synthesized from rac - 2 but the C-5 secondary hydroxyl group was surprisingly unreactive. For example, the formation of carbamates and Mitsunobu reactions were unsuccessful. In order to assess the unusual reactivity of 2 , a single crystal X-ray crystallographic analysis revealed a close intramolecular hydrogen bond from the C-5 alcohol to the oxepane ether oxygen (O-11). All products were tested in vitro against the W-2 and D-6 strains of Plasmodium falciparum . Several of the analogs had moderate activity in comparison to the natural product 1 . Iron (II) bromide-promoted rearrangement of 2 gave, in 50% yield, the ring-contracted tetrahydrofuran 22 , while the 5-ketone 15 provided a monocyclic methyl ketone 29 (50%). Neither 22 nor 29 possessed in vitro antimalarial activity. These results have implications in regard to the antimalarial mechanism of action of artemisinin.

The natural product veranamine was isolated from the marine sponge Verongula rigida . It contains a unique heterocyclic scaffold and demonstrates in vivo antidepressant activity and selective affinity for 5HT2B and sigma-1 receptors. The first total synthesis of veranamine is reported. Our scalable synthesis offers veranamine in six steps and 25% yield via an unprecedented vinylogous Pictet-Gams pyridine formation strategy. Veranamine is a promising new lead compound for antidepressant drug development.

MCH receptor is a G protein-coupled receptor with two subtypes R1 and R2. Many studies have demonstrated the role of MCH-R1 in feeding and energy homeostasis. It has been proven that oral administration of small molecule MCH-R1 antagonists significantly reduces food intake and causes a dose-dependent weight loss. In this study, two ligand-based pharmacophores were developed and validated based on recently published MCH-R1 antagonists with diverse structures. Successful pharmacophores had one hydrogen bond acceptor, one positive ionizable, one ring aromatic and two or three hydrophobic groups. These 3D-QSAR models were used for virtual screening of the ZINC chemical database resulting in the identification of nine compounds with more than 50% displacement of radiolabeled MCH at a 20 μM concentration. Moreover, four of these compounds showed antagonistic activities in Aequorin functional assay, including MH-3 which is the first MCH-R1 antagonist based on a diazaspiro[4.5]decane scaffold. The most active compounds were also docked into our previously published MCH-R1 homology model to gain insights into their binding determinants. These compounds could represent a viable starting scaffold for the design of potent MCH-R1 antagonists with improved pharmacokinetic properties as an effective treatment for obesity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We report here the orchestration of molecular ion networking and a set of computationally assisted structural elucidation approaches in the discovery of a new class of pyrroloiminoquinone alkaloids that possess selective bioactivity against pancreatic cancer cell lines. Aleutianamine represents the first in a new class of pyrroloiminoquinone alkaloids possessing a highly strained multibridged ring system, discovered from Latrunculia ( Latrunculia) austini Samaai, Kelly & Gibbons, 2006 (class Demospongiae, order Poecilosclerida, family Latrunculiidae) recovered during a NOAA deep-water exploration of the Aleutian Islands. The molecule was identified with the guidance of mass spectrometry, nuclear magnetic resonance, and molecular ion networking (MoIN) analysis. The structure of aleutianamine was determined using extensive spectroscopic analysis in conjunction with computationally assisted quantifiable structure elucidation tools. Aleutianamine exhibited potent and selective cytotoxicity toward solid tumor cell lines including pancreatic cancer (PANC-1) with an IC 50 of 25 nM and colon cancer (HCT-116) with an IC 50 of 1 μM, and represents a potent and selective candidate for advanced preclinical studies.

Introduction: This study focuses on a Midwest State's tobacco quitline. The purpose was to understand possible relationships between services provided and cessation rates., Methods: The data examined in this study came from aggregated intake/treatment data and follow-up interview data. The overall response rate was 22.9%. Measures included quit rate, quit duration, length of services, number of services, stage of change, confidence to quit, and source of referral., Results: The dataset included 1452 cases; 77% enrolled in services only once, 17% enrolled twice and 6% enrolled three or more times. Use of medication was higher among those who quit [χ 2 (1)=7.1, p=0.009, Cramer's V=0.07] than among those who did not. Use of e-cigarettes was lower among those who quit at the time of follow-up [χ 2 (1)=31.5, p<0.001, Cramer's V=0.15]. Respondents who had quit at the time of the follow-up were significantly more likely to have reported a higher confidence to quit at intake [χ 2 (1)=24.1, p<0.001, Cramer's V=0.13]. Among those who improved their stage of change during treatment, 35% had quit at follow-up, compared with 18% among those who did not improve., Conclusions: Study findings related to stage of change and associations between confidence and cessation may have meaningful implications. Cessation success may depend on what is accomplished during treatment and the intersection of clients' motivation, satisfaction, confidence, and cessation status at the end of treatment., Competing Interests: Authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none was reported., (© 2018 Cornish D.)

A new series of peptidomimetic N-substituted Cbz-4-Hyp-Hpa-amides were designed, synthesized, and evaluated for inhibition of the Plasmodium falciparum. Substituents on the N-atom of the amide group were selected alkyl-, allyl-, aryl-, 2-hydroxyethyl-, 2-cyanoethyl-, cyanomethyl-, 2-hydroxyethyl-, 2,2-diethoxyethyl-, or 2-ethoxy-2-oxoethylamino groups, and about of 40 new compounds were synthesized and evaluated for antiplasmodial activity in vitro. Antimalarial activity has been investigated as for the final peptide mimetics, and their immediate predecessors, carrying TBDMS or TBDPS protecting groups on 4-hydroxyproline residue and 18 derivatives exhibited toxicity against P. falciparum. Of these agents, compound 23e was shown to have potent antimalarial activity with IC 50 528 ng/ml., (© 2017 Wiley-VHCA AG, Zurich, Switzerland.)

The half structure of the symmetrical macrodiolide aplasmomycin A was synthesized by alkylation of a C3-C10 α-sulfonyl ketone subunit, prepared from (R)-pulegone and protected as a C3 ortholactone with (2R,3R)-butanediol, by a protected 15,16-dihydroxy (12E)-allylic chloride representing C11-C17. The latter was obtained from (2S,3R)-1,2-epoxy-3-butanol and propargyl alcohol. Regio- and stereoselective 5-exo-trig cyclization of the ene diol moiety in this segment, mediated by N-bromosuccinimide, led to the (2R,3S,5R)-tetrahydrofuran substructure of aplasmomycin A. Attachment of an α-acetic ester at the C3 carboxylic acid and esterification of the 3'-hydroxyl group of the tetrahydrofuran as its α-bromoacetate enabled coupling of two aplasmomycin half structures as an α-acyloxy acetate. Mukaiyama macrolactonization of this hydroxy acid afforded a symmetrical 36-membered diolide. Base-mediated double Chan rearrangement of this bis α-acyloxy dilactone caused ring contraction to the 34-membered macrocycle of desboroaplasmomycin A while generating the transannular 2-hydroxy-3-hemiketal motif of the natural product in the correct configuration. Final incorporation of boron into the tetraol core produced aplasmomycin A, isolated as its sodium borate. Extension of this route to the unsymmetrical macrodiolide boromycin was accomplished by modifications that included reversal of C12-C13 olefin geometry to (Z) for the southern half structure along with stereoselective hydride reductions of the C9 ketone that produced (9R) and (9S) alcohols for northern and southern half structures, respectively. Coupling of these half structures was made using an α-acyloxy ester linkage as for aplasmomycin A, but ring closure in this case was orchestrated via a blocked C16 alcohol that left open the C15 hydroxyl group of the southern half for Mukaiyama macrolactonization. A double Chan rearrangement of the resulting 35-membered macrocycle produced the 33-membered diolide of desborodesvalinylboromycin which had been obtained previously by degradation of natural boromycin. Insertion of boron into the tetraol core followed by esterification of the C16 alcohol with a masked d-valine and final deprotection furnished boromycin as its zwitterionic (Böeseken) complex.

The generation of natural product libraries containing column fractions, each with only a few small molecules, using a high-throughput, automated fractionation system, has made it possible to implement an improved dereplication strategy for selection and prioritization of leads in a natural product discovery program. Analysis of databased UPLC-MS-ELSD-PDA information of three leads from a biological screen employing the ependymoma cell line EphB2-EPD generated details on the possible structures of active compounds present. The procedure allows the rapid identification of known compounds and guides the isolation of unknown compounds of interest. Three previously known flavanone-type compounds, homoeriodictyol (1), hesperetin (2), and sterubin (3), were identified in a selected fraction derived from the leaves of Eriodictyon angustifolium. The lignan compound deoxypodophyllotoxin (8) was confirmed to be an active constituent in two lead fractions derived from the bark and leaves of Thuja occidentalis. In addition, two new but inactive labdane-type diterpenoids with an uncommon triol side chain were also identified as coexisting with deoxypodophyllotoxin in a lead fraction from the bark of T. occidentalis. Both diterpenoids were isolated in acetylated form, and their structures were determined as 14S,15-diacetoxy-13R-hydroxylabd-8(17)-en-19-oic acid (9) and 14R,15-diacetoxy-13S-hydroxylabd-8(17)-en-19-oic acid (10), respectively, by spectroscopic data interpretation and X-ray crystallography. This work demonstrates that a UPLC-MS-ELSD-PDA database produced during fractionation may be used as a powerful dereplication tool to facilitate compound identification from chromatographically tractable small-molecule natural product libraries.

A new series of peptidomimetic pseudo-prolyl-homophenylalanylketones were designed, synthesized and evaluated for inhibition of the Plasmodium falciparum cysteine proteases falcipain-2 (FP-2) and falcipain-3 (FP-3). In addition, the parasite killing activity of these compounds in human blood-cultured P. falciparum was examined. Of twenty-two (22) compounds synthesized, one peptidomimetic comprising a homophenylalanine-based α-hydroxyketone linked Cbz-protected hydroxyproline (39) showed the most potency (IC50 80 nM against FP-2 and 60 nM against FP-3). In silico analysis of these peptidomimetic analogs offered important protein-ligand structural insights including the role, by WaterMap, of water molecules in the active sites of these protease isoforms. The pseudo-dipeptide 39 and related compounds may serve as a promising direction forward in the design of competitive inhibitors of falcipains for the effective treatment of malaria., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A series of 1,2,3-triazolylsterols was prepared from pregnenolone through reductive amination and copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry). The newly generated stereocenter of the key propargylamino intermediate provided a mixture of diastereomers which were separated chromatographically, and the configuration of the R isomer was determined by X-ray crystallography. Ten triazolyl sterols were prepared, and the products and intermediates were screened in vitro against different parasites, with some compounds presenting IC50 values in the low micromolar range against Leishmania donovani., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Laurenditerpenol is the first marine natural product shown to inhibit hypoxia-inducible factor 1 (HIF-1) activation. Preclinical studies support that the inhibition of HIF-1 is one of the molecular targets for antitumor drug discovery. The synthetically challenging molecular architecture of laurenditerpenol, its absolute stereostructure, and the biological activity of several diastereoisomers were accomplished by our group in 2007 by diastereoselective synthesis. Herein, we report enantioselective syntheses of both enantiomers of laurenditerpenol involving sequential Michael addition and remote homoallylic hydroxyl group-directed asymmetric hydrogenation at ambient temperature and pressure as key reaction steps. The current approach is elegant and overall more efficient than the ones previously reported in the literature.

Background: The mechanism of action of artemisinins against malaria is unclear, despite their widespread use in combination therapies and the emergence of resistance., Results: Here, we report expression of PfATP6 (a SERCA pump) in yeast and demonstrate its inhibition by artemisinins. Mutations in PfATP6 identified in field isolates (such as S769N) and in laboratory clones (such as L263E) decrease susceptibility to artemisinins, whereas they increase susceptibility to unrelated inhibitors such as cyclopiazonic acid. As predicted from the yeast model, Plasmodium falciparum with the L263E mutation is also more susceptible to cyclopiazonic acid. An inability to knockout parasite SERCA pumps provides genetic evidence that they are essential in asexual stages of development. Thaperoxides are a new class of potent antimalarial designed to act by inhibiting PfATP6. Results in yeast confirm this inhibition., Conclusions: The identification of inhibitors effective against mutated PfATP6 suggests ways in which artemisinin resistance may be overcome.

Stevioside is a naturally occurring diterpenoid glycoside in Stevia rebaudiana Bertoni. The title compound, C38H60O18·4CH3OH, crystallized as its methanol tetrasolvate. Stevioside consists of an aglycone steviol (a tetra-cyclic diterpene in which the four-fused-ring system consists of three six-membered rings and one five-membered ring) and a sugar part (three glucose units). A weak intra-molecular O-H⋯O hydrogen bond occurs. In the crystal, the methanol mol-ecules participate in a two-dimensional hydrogen-bonded network parallel to b axis with the sugars and together they form a hydrophilic tunnel which encloses the lipophilic part of the molecule.

Plasmodium falciparum calcium-ATPase (PfATP6) has been reported to be a target of artemisinin and related endoperoxides. In this study, a series of previously reported guaianolide-endoperoxides (thaperoxides) were docked into a homology model of PfATP6 and also used to develop a pharmacophore model. This combined approach led to useful insights into the binding determinants of thaperoxides to the malarial enzyme. In addition, in silico mutagenesis and molecular dynamics suggested the importance of Phe264 and the electrostatic interactions between Lys260 in helix H3 and Lys1036 and Asp1038 in L6/7 loop for the binding of thaperoxides. These results could help in the design of more potent inhibitors of PfATP6., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Falcipains (FPs) are hemoglobinases of Plasmodium falciparum that are validated targets for the development of antimalarial chemotherapy. A combined ligand- and structure-based virtual screening of commercial databases was performed to identify structural analogs of virtual screening hits previously discovered in our laboratory. A total of 28 low micromolar inhibitors of FP-2 and FP-3 were identified and the structure-activity relationship (SAR) in each series was elaborated. The SAR of the compounds was unusually steep in some cases and could not be explained by a traditional analysis of the ligand-protein interactions (van der Waals, electrostatics, and hydrogen bonds). To gain further insights, a statistical thermodynamic analysis of explicit solvent in the ligand binding domains of FP-2 and FP-3 was carried out to understand the roles played by water molecules in binding of these inhibitors. Indeed, the energetics associated with the displacement of water molecules upon ligand binding explained some of the complex trends in the SAR. Furthermore, low potency of a subset of FP-2 inhibitors that could not be understood by the water energetics was explained in the context of poor chemical reactivity of the reactive centers of these compounds. The present study highlights the importance of considering energetic contributors to binding beyond traditional ligand-protein interactions., (© 2012 American Chemical Society)

Among all the antimalarial agents, artemisinin and its semi synthetic family of analogs are the most potent antimalarials available for the treatment of Plasmodium falciparum infections. But these analogs have a few issues such as shorter half-lives and low oral bioavailability values. In order to overcome these inherent problems, novel artemisinin analogs were synthesized from 7β-hydroxy artemisinin by the Department of Medicinal Chemistry, University of Mississippi using a new synthesis mechanism. Out of all the 7β-hydroxy artemisinin analogs synthesized, 7β-hydroxy artemisinin carbamate (ARB-89) was chosen as a lead compound because of its high in vitro and in vivo activity. In this manuscript, a sensitive and rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) method was developed and validated for the quantification of ARB-89 in rat serum. The analysis was carried out on an Acquity™ UPLC BEH C(18) column (1.7 μm, 2.1 mm × 50 mm) with a flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in positive electrospray ionization (ESI) mode. The selected mass-to-charge (m/z) ratio transitions used in the multiple reaction monitoring (MRM) for ARB-89 and artemisinin (internal standard) were m/z 778.4>253.4 and m/z 283.4>151.1 respectively. The calibration curve was linear from 1.00 ng/mL to 10.0 μg/mL (r(2)=0.999). A simple protein precipitation method was used for extraction. Moreover, the inter-day and intra-day precision values were found to be less than 15%. The recoveries of the method ranged from 94.0% to 96.7% at three concentrations. ARB-89 in rat serum was found to be stable at room temperature for 12h. This method was successfully used to quantitate the novel antimalarial compound ARB-89 after intravenous and oral administration to rats., (Copyright © 2012 Elsevier B.V. All rights reserved.)

SARS-CoV from the coronaviridae family has been identified as the etiological agent of Severe Acute Respiratory Syndrome (SARS), a highly contagious upper respiratory disease that reached epidemic status in 2002. SARS-3CL(pro), a cysteine protease indispensible to the viral life cycle, has been identified as one of the key therapeutic targets against SARS. A combined ligand and structure-based virtual screening was carried out against the Asinex Platinum collection. Multiple low micromolar inhibitors of the enzyme were identified through this search, one of which also showed activity against SARS-CoV in a whole cell CPE assay. Furthermore, multinanosecond explicit solvent simulations were carried out using the docking poses of the identified hits to study the overall stability of the binding site interactions as well as identify important changes in the interaction profile that were not apparent from the docking study. Cumulative analysis of the evaluated compounds and the simulation studies led to the identification of certain protein-ligand interaction patterns which would be useful in further structure based design efforts.

The gum resin of Commiphora wightii [(Hook. ex Stocks) Engl.] is an ayurvedic medicine for the treatment of arthritis, inflammation, obesity, lipid disorders, and cardiovascular diseases and is known as guggul. Morphologically, it is not easy to distinguish guggul from closely related gum resins of other plants. Reliability of the commercially available guggul is critical due to the high risk of adulteration. To check authenticity, a commercial guggul sample was investigated for its chemical markers and 17 metabolites were identified, including three new, 20(S),21-epoxy-3-oxocholest-4-ene (1), 8 β-hydroxy-3,20-dioxopregn-4,6-diene (2), and 5-(13' Z-nonadecenyl)resorcinol (17) from the ethyl acetate soluble part. During the current study, compounds 14- 17 were identified as constituents of Mangifera indica gum, as an adulterant in the commercial guggul sample. This discovery highlighted the common malpractices in the trade of medicinal raw material in the developing world. The structures of the compounds were deduced by the spectroscopic technique and chemical methods, as well as by comparison with the reported data. The structure of 20(S),21-epoxy-3-oxocholest-4-ene (1) was also unambiguously deduced by single-crystal X-ray diffraction technique., (© Georg Thieme Verlag KG Stuttgart · New York.)

Malaria, in particular that caused by Plasmodium falciparum , is prevalent across the tropics, and its medicinal control is limited by widespread drug resistance. Cysteine proteases of P. falciparum , falcipain-2 (FP-2) and falcipain-3 (FP-3), are major hemoglobinases, validated as potential antimalarial drug targets. Structure-based virtual screening of a focused cysteine protease inhibitor library built with soft rather than hard electrophiles was performed against an X-ray crystal structure of FP-2 using the Glide docking program. An enrichment study was performed to select a suitable scoring function and to retrieve potential candidates against FP-2 from a large chemical database. Biological evaluation of 50 selected compounds identified 21 diverse nonpeptidic inhibitors of FP-2 with a hit rate of 42%. Atomic Fukui indices were used to predict the most electrophilic center and its electrophilicity in the identified hits. Comparison of predicted electrophilicity of electrophiles in identified hits with those in known irreversible inhibitors suggested the soft-nature of electrophiles in the selected target compounds. The present study highlights the importance of focused libraries and enrichment studies in structure-based virtual screening. In addition, few compounds were screened against homologous human cysteine proteases for selectivity analysis. Further evaluation of structure-activity relationships around these nonpeptidic scaffolds could help in the development of selective leads for antimalarial chemotherapy.

Melanin concentrating hormone (MCH) is a cyclic 19-amino-acid peptide expressed mainly in the hypothalamus. It is involved in the control of feeding behavior, energy homeostasis, and body weight. Administration of MCH-R1 antagonists has been proved to reduce food intake and cause weight loss in animal models. In the present study, a homology model of the human MCH-R1 was constructed using the crystal structure of bovine rhodopsin (PDB: 1u19) as a template. Based on the observation that MCH-R1 can bind ligands of high chemical diversity, the initial model was subjected to an extensive ligand-supported refinement using antagonists of different chemotypes. The refinement process involved stepwise energy minimizations and molecular dynamics simulations. The refined model was inserted into a pre-equilibrated DPPC/TIP3P membrane system and then simulated for 20 ns in complex with structurally diverse antagonists. This protocol was able to explain the SAR of MCH-R1 antagonists with diverse chemical structures. Moreover, it reveals new insights into the critical recognition sites within the receptor. This work represents the first detailed study of molecular dynamics of MCH-R1 inserted into a membrane-aqueous environment.

Design and synthesis of a guaianolide-endoperoxide (thaperoxide) 3 was pursued as a new antimalarial lead which was found to be noncytotoxic as compared to the natural product lead thapsigargin 2. Several analogues of 3 were successfully synthesized and found to be comparable to derivatives of artemisinin 1 in in vitro antimalarial assay. Among the synthesized compounds, 22 showed excellent in vitro potency against the cultured parasites (W2 IC(50) = 13 nM) without apparent cytotoxicity. Furthermore, SAR trends in thaperoxide analogues are presented and explained with the help of docking studies in the homology model of PfSERCA(PfATP6).

Background: Multiple cysteine proteases of malaria parasites are required for maintenance of parasite metabolic homeostasis and egress from the host erythrocyte. In Plasmodium falciparum these proteases appear to mediate the processing of hemoglobin and aspartic proteases (plasmepsins) in the acidic food vacuole and the hydrolysis of erythrocyte structural proteins at neutral pH. Two cysteine proteases, vivapain (VX)-2 and VX-3 have been characterized in P. vivax, but comprehensive studies of P. vivax cysteine proteases remain elusive., Findings: We characterized a novel cysteine protease of P. vivax, VX-4, of which orthologs appears to have evolved differentially in primate plasmodia with strong cladistic affinity toward those of rodent Plasmodium. Recombinant VX-4 demonstrated dual substrate specificity depending on the surrounding micro-environmental pH. Its hydrolyzing activity against benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA was highest at acidic pH (5.5), whereas that against Z-Arg-Arg-MCA was maximal at neutral pH (6.5-7.5). VX-4 preferred positively charged amino acids and Gln at the P1 position, with less strict specificity at P3 and P4. P2 preferences depended on pH (Leu at pH 5.5 and Arg at pH 7.5). Three amino acids that delineate the S2 pocket were substituted in VX-4 compared to VX-2 and VX-3 (Ala90, Gly157 and Glu180). Replacement of Glu180 abolished activity against Z-Arg-Arg-MCA at neutral pH, indicating the importance of this amino acid in the pH-dependent substrate preference. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. VX-4 showed maximal activity against actin at neutral pH, and that against P. vivax plasmepsin 4 and hemoglobin was detected at neutral/acidic and acidic pH, respectively., Conclusion: VX-4 demonstrates pH-dependent substrate switching, which might offer an efficient mechanism for the specific cleavage of different substrates in different intracellular environments. VX-4 might function as a hemoglobinase in the acidic parasite food vacuole, a maturase of P. vivax plasmepsin 4 at neutral or acidic pH, and a cytoskeleton-degrading protease in the neutral erythrocyte cytosol.

The objective of the present research was to evaluate the physicochemical characteristics of berberine chloride and to assess the complexation of drug with 2-hydroxypropyl-β-cyclodextrin (HPβCD), a first step towards solution dosage form development. The parameters such as log P value were determined experimentally and compared with predicted values. The pH-dependent aqueous solubility and stability were investigated following standard protocols at 25°C and 37°C. Drug solubility enhancement was attempted utilizing both surfactants and cyclodextrins (CDs), and the drug/CD complexation was studied employing various techniques such as differential scanning calorimetry, Fourier transform infrared, nuclear magnetic resonance, and scanning electron microscopy. The experimental log P value suggested that the compound is fairly hydrophilic. Berberine chloride was found to be very stable up to 6 months at all pH and temperature conditions tested. Aqueous solubility of the drug was temperature dependent and exhibited highest solubility of 4.05 ± 0.09 mM in phosphate buffer (pH 7.0) at 25°C, demonstrating the effect of buffer salts on drug solubility. Decreased drug solubility was observed with increasing concentrations of ionic surfactants such as sodium lauryl sulfate and cetyl trimethyl ammonium bromide. Phase solubility studies demonstrated the formation of berberine chloride-HPβCD inclusion complex with 1:1 stoichiometry, and the aqueous solubility of the drug improved almost 4.5-fold in the presence of 20% HPβCD. The complexation efficiency values indicated that the drug has at least threefold greater affinity for hydroxypropyl-β-CD compared to randomly methylated-β-CD. The characterization techniques confirmed inclusion complex formation between berberine chloride and HPβCD and demonstrated the feasibility of developing an oral solution dosage form of the drug.

Delta(9)-Tetrahydrocannabinol hemisuccinate (THC-HS), an ester prodrug of Delta(9)-tetrahydrocannabinol (THC) has been investigated for its potential to form inclusion complexes with modified synthetic beta-cyclodextrins (CDs). Phase solubility studies were performed to determine the stoichiometric ratio of complexation of THC-HS with random methylated beta-cyclodextrin (RAMEB) and 2-hydroxypropyl beta-cyclodextrin (HPBCD). THC-HS/RAMEB and THC-HS/HPBCD solid systems were prepared by lyophilization and the lyophilized complexes were characterized by Fourier transform infrared (FT-IR) spectroscopy, proton nuclear magnetic spectroscopy, and molecular modeling techniques. The formation of inclusion complexes of THC-HS/RAMEB and THC-HS/HPBCD was demonstrated by an A(L) type curve with the slopes less than unity by the phase solubility method. The association constants for THC-HS/RAMEB and THC-HS/HPBCD were found to be 562.48 and 238.83 M(-1), respectively. The stoichiometry of both of the complexes was found to be 1:1 as determined from the Job's plot. This was confirmed by (1)H NMR and FT-IR techniques. The results obtained from the molecular modeling studies were in accordance with the data obtained from nuclear magnetic resonance and FT-IR. The docking studies revealed the most probable mode of binding of THC-HS with RAMEB in which the alkyl chain was submerged in the hydrophobic pocket of the CD molecule and hydrogen bonding interactions were observed between the hemisuccinate ester side chain of THC-HS and the rim hydroxy groups of RAMEB. The solubility of THC-HS was significantly higher in RAMEB compared to HPBCD. Solid dispersions of THC-HS with CDs will be further utilized to develop oral formulations of THC-HS with enhanced bioavailability.

Apoptosis, or programmed cell death, forms an important part of the cellular regulation machinery. The Bcl-2 protein family, comprising of proapoptotic and antiapoptotic members, forms an important part of the cells internal apoptotic pathway. Overexpression of the antiapoptotic members of the family in a number of cancer cell lines renders them immune to apoptosis and the ability to survive under conditions of cellular stress. Inhibition of the antiapoptotic members of the Bcl-2 family are, therefore, an interesting target for the development of anticancer therapy. An innovative structure-based virtual screening strategy was developed to identify inhibitors of Bcl-xL, an antiapoptotic member of the Bcl-2 family. Various innovative filters, such as receptor-based pharmacophore, cascade docking approach, cross-docking, and composite scoring with docking pose based descriptors were designed through exhaustive validation studies and implemented in the screening funnel. The 1.8 million 'big-n-greasy' subset from ZINC was screened using the protocol, and 45 compounds were finally selected for biological evaluation against Bcl-xL. The evaluation led to the identification of one low-micromolar and two weaker inhibitors belonging to novel scaffolds. Further evaluation of structure-activity relationships around these scaffolds could help in the development of anticancer leads against Bcl-xL.

The asymmetric total synthesis of (-)-berkeleyamide A (1), a naturally occurring caspase-1 inhibitor, has been achieved by employing Evans' syn-aldol reaction of N-acyl-(4R)-benzyl oxazolidin-2-one 3 as the key step.

Cysteine proteases are implicated in a variety of human physiological processes and also form an essential component of the life cycle of a number of pathogenic protozoa and viruses. The present review highlights the drug design approaches utilized to understand the mechanism of inhibition and discovery of inhibitors against protozoal cysteine protease, falcipain (a cysteine protease of P. falciparum which causes malaria), and viral cysteine protease, SARS-CoV M(pro) (a cysteine protease of severe acute respiratory syndrome corona virus). The article describes rational approaches for the design of inhibitors and focuses on a variety of structure as well as ligand-based modeling strategies adopted for the discovery of the inhibitors. Also, the key features of ligand recognition against these targets are accentuated. Although no apparent similarities exist between viral and protozoal cysteine proteases discussed here, the goal is to provide examples of rational drug design approaches adopted to design inhibitors against these proteases. The current review would be of interest to scientists engaged in the development of drug design strategies to target the cysteine proteases present in mammals and other lower order organisms.

In addition to lowering blood pressure, telmisartan, an angiotensin (AT(1)) receptor blocker, has recently been shown to exert pleiotropic effects as a partial agonist of nuclear peroxisome proliferator-activated receptor gamma (PPAR gamma). On the basis of these findings and docking pose similarity between telmisartan and rosiglitazone in PPAR gamma active site, two classes of benzimidazole derivatives were designed and synthesized as dual PPAR gamma agonist/angiotensin II antagonists for the possible treatment of metabolic syndrome. Compound 4, a bisbenzimidazole derivative showed the best affinity for the AT(1) receptor with a K(i) = 13.4 nM, but it was devoid of PPAR gamma activity. On the other hand 9, a monobenzimidazole derivative, showed the highest activity in PPAR gamma transactivation assay (69% activation) with no affinity for the AT(1) receptor. Docking studies lead to the designing of a molecule with dual activity, 10, with moderate PPARgamma activity (29%) and affinity for the AT(1) receptor (K(i) = 2.5 microM).

The malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our laboratory have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal glycine motif, which forms a characteristic Rossman dinucleotide-binding fold in the co-substrate binding pocket, differentiates PfMDH (GlyXGlyXXGly) from other eukaryotic and prokaryotic malate dehydrogenases (GlyXXGlyXXGly). The amino acids lining the co-substrate binding pocket are completely conserved in MDHs from different species of human, primate and rodent malaria parasites. Based on this knowledge and conserved domains among prokaryotic and eukaryotic MDH, the role of critical amino acids lining the co-substrate binding pocket was analyzed in catalytic functions of PfMDH using site-directed mutagenesis. Insertion of Ala at the 9th or 10th position, which converts the N-terminal GlyXGlyXXGly motif (characteristic of malarial MDH and LDH) to GlyXXGlyXXGly (as in bacterial and eukaryotic MDH), uncoupled regulation of the enzyme through substrate inhibition. The dinucleotide fold GlyXGlyXXGly motif seems not to be responsible for the distinct affinity of PfMDH to 3-acetylpyridine-adenine dinucleotide (APAD, a synthetic analog of NAD), since Ala9 and Ala10 insertion mutants still utilized APADH. The Gln11Met mutation, which converts the signature glycine motif in PfMDH to that of PfLDH, did not change the enzyme function. However, the Gln11Gly mutant showed approximately a 5-fold increase in catalytic activity, and higher susceptibility to inhibition with gossypol. Asn119 and His174 participate in binding of both co-substrate and substrate. The Asn119Gly mutant exhibited approximately a 3-fold decrease in catalytic efficiency, while mutation of His174 to Asn or Ala resulted in an inactive enzyme. These studies provide critical insights into the co-substrate binding pocket of PfMDH, which may be important in design of selective PfMDH/PfLDH inhibitors as potential antimalarials.

Progress made in the past decade pertaining to the development of anti-parasitic agents based on artemisinin is presented. Apart from discussions on important derivatives obtained through functionalization at the C-3, C-9, C-10 and O-11 positions of artemisinin, an outline on its seco analogs and artemisinin bundles are given for a broader perspective on structure-activity relationships. Success with synthetic peroxides, drug-hybrid approaches, broad-spectrum anti-infective properties of peroxide compounds and a survey on peroxide-containing natural products other than artemisinin with available biological data are included to highlight recent trends and avenues for future research. A supplementary material with details on the biological properties of a larger collection of molecules belonging to the above structural classes is also given for reference.

The purpose of the present investigation was to characterize the partitioning of artemisinin and its derivatives into both non-parasitized as well as Plasmodium falciparum parasitized red blood cells (RBCs). Artemisinin and selected derivatives at concentrations of 3.55microM were incubated in RBCs with a hematocrit of 33% for 2h at 37 degrees C, extracted from RBCs by solid phase extraction, and analyzed using liquid chromatography-mass spectrometry in positive electro-spray ionization mode with methanol as mobile phase. The uptake percent of artemisinin and selected derivatives into the non-parasitized RBCs ranged between 35% and 45%, while that into parasitized RBCs was between 51% and 72%. The results suggested that artemisinin and selected derivatives were preferentially distributed in parasitized RBCs. A Multiple Linear Regression model was built to gain insight about the essential structural properties required for the uptake of this class of compounds in parasitized RBCs and will provide instruction for designing of new derivatives of this class of compounds with improved uptake.

This study reports the isolation of nine new terpenoids (2-10), possessing two novel skeletons, from the Red Sea sponge Callyspongia (=Siphonochalina) siphonella. The identity of these novel skeletons was based on X-ray crystallography and extensive spectral analyses. These compounds were evaluated for their ability to reverse P-glycoprotein (P-gp)-mediated multidrug resistance in human epidermoid cancer cells. Sipholenone E (3) was better than sipholenol A (1), a known P-gp modulator from this sponge, in reversing the P-gp-mediated multidrug resistance. Sipholenol L (6) and siphonellinol D (8) were nearly as active as sipholenol A. On the basis of X-ray crystallographic data and the established identity of 3-7, the structure of sipholenol I (11) is revised. A pharmacophore model of three hydrophobic points and two H-bond acceptors was generated for the active sipholane P-gp modulators.

L-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent for the most severe form of malaria, has shown remarkable similarities to L: -lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs characterized in archae and other prokaryotes. Initial sequence analysis and identification of critical amino acid residues involved in inter-subunit salt-bridge interactions predict tetrameric structure for PfMDH. The catalytically active recombinant PfMDH was characterized as a tetramer. The enzyme is localized primarily in the parasites cytosol. To gain molecular insights into PfMDH/PfLDH relationships and to understand the quaternary structure of PfMDH, dimers were generated by mutation to the potential salt-bridge interacting sites. The R183A and R214G mutations, which snapped the salt bridges between the dimers and resulted in lower dimeric state, did not affect catalytic properties of the enzyme. The mutant dimers of PfMDH were active equally as the wild-type PfMDH. The studies reveal structure of PfMDH as a dimer of dimers. The tetrameric state of PfMDH was not essential for catalytic functions of the enzyme but may be an evolutionary adaptation for cytosolic localization to support its role in NAD/NADH coupling, an important metabolic function for survival of the malaria parasite.

A novel ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method has been established for the determination of a newly synthesized epothilone D analog (AV-EPO-106) in human plasma. The plasma samples were prepared by liquid-liquid extraction with cold tert-butyl methyl ether. The chromatographic separation was achieved within 5 min on a C(18) column with water-methanol (10:90, v/v) as mobile phase at a flow-rate of 0.8 mL/min. Mass transition of m/z 568.2 to 386.1 was measured for AV-EPO-106 in positive atmospheric pressure chemical ionization mode. A detailed validation of the method was performed as per the USFDA guidelines. For AV-EPO-106 at the concentrations of 1.0, 5.0 and 10.0 microg/mL in human plasma, the absolute extraction recoveries were 86.17, 85.24 and 85.69%, respectively. The linear quantification range of the method was 0.10-20.0 microg/mL in human plasma with linear correlation coefficients greater than 0.999. The intra-day and inter-day accuracy for AV-EPO-106 at the levels of 1.0, 5.0 and 10.0 microg/mL in human plasma fell in the ranges of 98.25-100.47 and 94.19-97.25%, and the intra- and inter-day precision were in the ranges of 4.75-6.30% and 8.89-10.45%, respectively. The method was successfully applied to quantify AV-EPO-106 in human plasma to determine the half-life of this compound in human plasma., (2008 John Wiley & Sons, Ltd.)

The asymmetric unit of the title compound, C(22)H(26)O(8), contains two crystallographically independent mol-ecules with closely comparable conformations (r.m.s. overlay = 0.54 Å for 30 non-H atoms). All six-membered rings display chair conformations, with a slight distortion for the lactone ring. The mol-ecules are connected by O-H⋯O hydrogen bonds into chains along [010], with the independent mol-ecules segregated into separate chains. The two mol-ecules in the asymmetric unit face each other in a head-to-tail fashion, with the furan ring of one mol-ecule turned towards the carboxylic acid terminal of the other mol-ecule.

The title compound, C(22)H(30)O(7), is a fused five-ring system that is of inter-est for its anti-cancer and anti-malarial activity. The six-membered C(6) and C(5)O rings display chair conformations. The six-membered C(3)O(3) ring containing the ether and per-oxy functionalities has a distorted boat conformation, with a C-O-O-C torsion angle of 42.6 (1)° for the per-oxy group. The seven-membered C(6)O ring has a distorted boat-type conformation, while the seven-membered C(5)O(2) ring has a very distorted chair-type conformation. The structure contains inter-molecular O-H⋯O and O-H⋯(O,O) bonds that link the mol-ecules into sheets parallel to the (100) planes.

On the basis of molecular modeling and QSAR analysis of the known human progesterone receptor (hPR) inhibitor Mifepristone (RU-486) and other hPR ligands, a new class of potential nonsteroidal hPR inhibitors was designed. The parent racemic compound 1 was synthesized through an efficient 13-step synthetic pathway. The key constructive steps are a stereoselective epoxide ring opening and the reductive Heck cyclization to form the main framework of (+/-)-1. The current established flexible synthetic route allows for further chemical diversification.

Severe acute respiratory syndrome (SARS) is an illness caused by a novel corona virus wherein the main proteinase called 3CL(Pro) has been established as a target for drug design. The mechanism of action involves nucleophilic attack by Cys145 present in the active site on the carbonyl carbon of the scissile amide bond of the substrate and the intermediate product is stabilized by hydrogen bonds with the residues of the oxyanion hole. Based on the X-ray structure of 3CL(Pro) co-crystallized with a trans-alpha,beta-unsaturated ethyl ester (Michael acceptor), a set of QM/QM and QM/MM calculations were performed, yielding three models with increasingly higher the number of atoms. A previous validation step was performed using classical theoretical calculation and PROCHECK software. The Michael reaction studies show an exothermic process with -4.5 kcal/mol. During the reaction pathway, an intermediate is formed by hydrogen and water molecule migration from a histidine residue and solvent, respectively. In addition, similar with experimental results, the complex between N3 and 3CL(Pro) is 578 kcal/mol more stable than N1-3CL(Pro) using Own N-layer Integrated molecular Orbital molecular Mechanics (ONIOM) approach. We suggest 3CL(Pro) inhibitors need small polar groups to decrease the energy barrier for alkylation reaction. These results can be useful for the development of new compounds against SARS.

Four new cycloartane glycosides, sutherlandiosides A-D (1-4), were isolated from the South African folk medicine Sutherlandia frutescens and their structures established by spectroscopic methods and X-ray crystallography as 1 S,3 R,24S,25-tetrahydroxy-7S,10S-epoxy-9,10- seco-9,19-cyclolanost-9(11)-ene 25-O-beta-D-glucopyranoside (1), 3R,7S,24S,25-tetrahydroxycycloartan-1-one 25-O-beta-D-glucopyranoside (2), 3R,24S,25-trihydroxycycloartane-1,11-dione 25-O-beta-D-glucopyranoside (3), and 7S,24S,25-trihydroxycycloart-2-en-1-one 25-O-beta-D-glucoyranoside (4). Compound 1 represents the first secocycloartane skeleton possessing a 7,10-oxygen bridge. Compounds 2- 4 are also the first examples of naturally occurring cycloartanes with a C-1 ketone functionality. Biosynthetic considerations and chemical evidence suggest that the presence of the C-1 ketone in 2 may facilitate the ring opening of the strained cyclopropane system.

In the crystal structure of the title compound, C(28)H(36)O(4)S, the p-tolyl ring is inclined at 35.8° to the aromatic ring. The cyclohexene ring adopts a boat conformation and the heterocyclic ring is in a slightly distorted screw boat conformation.

A series of 53 nitro derivatives rationally designed were obtained by parallel synthesis and screened against Leishmania donovani. Six compounds exhibited IC(50) values lower than standard drugs. Brief SAR analysis revealed that substitution is important to the activity. Nitrothiophene analogues were more potent than the nitrofuran ones. This was attributed to the ability of sulfur atoms in accommodating electrons from nitro group, which facilitate its reduction and therefore the formation of free radicals lethal to parasites.

The salvinorin A analog dimethyl (2R,3aR,4R,6aR,7R,9S,9aS,9bS)-2-(3-fur-yl)-9,9a-dihydr-oxy-3a,6a-dimethyl-dodeca-hydro-benzo[de]chromene-4,7-dicarboxyl-ate, C(22)H(30)O(8), has a relatively simple spatial arrangement in which mol-ecules are linked into layers by two pairs of O-H⋯O hydrogen bonds. Each mol-ecule has as the central feature a dodeca-hydro-1H-phenalene ring system. Its three six-membered rings are in the chair conformation, with two axial methyl groups, one axial OH, and one equatorial OH, these OH groups being directly responsible for linking of the mol-ecules in the crystal structure.

Two new cyclic hexapeptides, mollamides B (1) and C (2), were isolated from the Indonesian tunicate Didemnum molle along with the known peptide keenamide A (3). The structures were established using 1D and 2D NMR experiments. The relative configuration of mollamide B at the thiazoline moiety was determined using molecular modeling coupled with NMR-derived restraints. Their absolute configuration was determined using Marfey's method. The new peptides have been evaluated for their antimicrobial, antimalarial, anticancer, anti-HIV-1, anti-Mtb, and anti-inflammatory activities. Keenamide A and mollamide B show cytotoxicity against several cancer cell lines.

The rare indazole-type alkaloid nigellidine (2) is accompanied by its 4-O-sulfite (4) in the seeds of Nigella sativa. Compound 4 may represent the true natural product leading to nigellidine (2) via hydrolysis of the sulfate functionality during the isolation process. The structure of nigellidine-4-O-sulfite (4) is confirmed by NMR, MS, and X-ray crystallographic data. This is the first report of the natural occurrence of sulfated indazole-type alkaloids.

A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to quantify artemisinin in rat serum. The lower limit of quantification (LLOQ) was 4 ng/mL. The calibration curve was linear from 4 ng/mL to 10,000 ng/mL (R=0.998). The assay was based on the selected reaction monitoring (SRM) transitions at m/z 305.4-151.10 for artemisinin and m/z 335.2-163.10 for arteether (internal standard). The artemisinin and internal standard can be separated from endogenous interferences in rat serum. Inter- and intra-day assay variation was less than 15%. The extraction recoveries ranged from 80.0 to 107.3% at the three concentrations (5000, 2000, and 200 ng/mL). This method was successfully applied to pharmacokinetic studies of artemisinin after intravenous and oral administration to rats.

The histone deacetylase (HDAC) enzyme from Plasmodium falciparum has been identified as a novel target for the development of antimalarial therapy. A ligand-refined homology model of PfHDAC-1 was generated from the crystal structures of human HDAC8 and HDLP using a restraint guided optimization procedure involving the OPLS/GBSA potential setup. The model was extensively validated using protein structure checking tools. A predictive docking study was carried out using a set of known human HDAC inhibitors, which were shown to have in vitro antimalarial activity against the chloroquine sensitive D6 and resistant W2 strains of P. falciparum. Pose validation and score-based active/inactive separation studies provided independent validation of the geometric accuracy and the predictive ability of the generated model. Comparative analysis was carried out with the human HDACs to identify differences in the binding site topology and interacting residues, which might be utilized to develop selective PfHDAC-1 inhibitors.

Leishmania donovani and Leishmania major farnesyl pyrophosphate synthase ( LdFPPS and LmFPPS) are potential targets for the development of antileishmanial therapy. The protein sequence for LdFPPS was recently elucidated in our laboratory. Highly refined homology models were generated using the protein sequences of LdFPPS and the closely related LmFPPS enzyme. A ligand-refined model of LmFPPS with a bound bisphosphonate ligand was generated using restraint-guided molecular mechanics followed by quantum mechanics/molecular mechanics refinement. The ligand-refined model of LmFPPS was further validated through extensive pose validation, enrichment, and other docking studies involving known bisphosphonate inhibitors. The model was able to explain the critical binding site interactions and site-directed mutagenesis data obtained from experimental studies on related FPPS enzymes. The ligand-refined model in conjunction with the validated docking protocol could be utilized in the future for structure-based virtual screening and rational drug design studies against these targets.