Biomedical subjects

Apurba K Bhattacharjee

Publications and source records attributed to Apurba K Bhattacharjee.

At least 19 recordsLinked to original sources

PubMed2006

Novel semi-automated methodology for developing highly predictive QSAR models: application for development of QSAR models for insect repellent amides.

Conventional 3D-QSAR models are built using global minimum conformations or quantum-mechanics based geometry-optimized conformations as bioactive conformers. QSAR models developed using the global minima as bioactive conformers, employing the GFA, PLS and G/PLS methodologies, gave good non-validated r(2) (0.898, 0.868 and 0.922) and performed well on an internal validation test with leave-one-out correlation q(2) (LOO) (0.902, 0.726 and 0.924), leave-10%-out correlation q(2) (L10O) (0.874, 0.728 and 0.883) and leave-20%-out q(2) (L20O) (0.811, 0.716 and 0.907). However, they showed poor predictive ability on an external data set with best predictive r(2) (Pred-r(2)) of 0.349, 0.139 and 0.204 respectively. A novel methodology to mine bioactive conformers, from clusters of conformations with good 3D-spatial representation around pharmacophoric moiety, furnishes highly predictive 3D-QSAR models. The best QSAR model (model A) showed r(2) of 0.989, q(2) (LOO) of 0.989, q(2) (L10O) of 0.980, q(2) (L20O) of 0.963 and Pred-r(2) on eight test compounds of 0.845. The methodology is based on mimicking the multi-way Partial Least Squares (PLS) technique by performing several automated sequential PLS analyses. The poses/shapes of the mined bioactive conformers provide valuable insight into the mechanism of action of the insect repellents. All of the repetitive tasks were automated using Tcl-based Cerius2 scripts.

Algorithms↗

PubMed2006

Identification of smooth and rough forms in cultures of Brucella melitensis strains by flow cytometry.

Brucella melitensis strains may occur as either smooth or rough variants depending on the expression of O-polysaccharides (OPS) as a component of the bacterial outer membrane lipopolysaccharide (LPS). The wboA gene, which codes for the enzyme glycosyl transferase, is essential for the assembly of O-chain in Brucella. Deletion of wboA in smooth virulent B. melitensis 16M results in a rough mutant designated WRR51. We developed a flow cytometric method to determine the proportion of B. melitensis cells displaying surface O-polysaccharide (OPS) in liquid culture. OPS was detected using polyclonal antibodies from rabbits immunized with smooth (S) or rough (R) Brucella LPS. First, we evaluated the binding of these antibodies to 16M (S), WRR51 (R) and complemented WRR51 expressing the wboA gene (S) as well as to their corresponding GFP-expressing derivative strains 16M/GFP, WRR51/GFP and WRR51/GFP+wboA. The rough mutants did not react with anti-S-LPS nor did the smooth strains react with anti-R-LPS. Second, using different ratios of 16M/GFP and WRR51/GFP, we were able to detect the presence of 1% rough bacteria spiked into a sample of smooth organisms. Third, we evaluated the purity of cultures of B. melitensis strains grown in a fermenter. These flow cytometric methods may be useful for quality control of process development for large-scale vaccine production.

Animals↗

PubMed2006

In vitro and in vivo efficacy and in vitro metabolism of 1-phenyl-3-aryl-2-propen-1-ones against Plasmodium falciparum.

Investigation of a series of 1-phenyl-3-aryl-2-propen-1-ones resulted in the identification of nine inhibitors with submicromolar efficacy against at least one Plasmodium falciparum strain in vitro. These inhibitors were inactive when given orally in a Plasmodium berghei infected mouse model. Significant compound degradation occurred upon their exposure to a liver microsome preparation, suggesting metabolic instability may be responsible for the lack of activity in vivo.

Animals↗

PubMed2006

Activity of and initial mechanistic studies on a novel antileishmanial agent identified through in silico pharmacophore development and database searching.

A 3D pharmacophore was generated to describe the antileishmanial activity of dinitroaniline sulfonamides by CATALYST 3D-QSAR methodology, and this pharmacophore was used to search the Maybridge database. Two compounds identified in this search, BTB 06237 and BTB 06256, were highly active with IC(50) values against L. donovani amastigotes of 0.5 +/- 0.2 and 2.3 +/- 0.8 microM, respectively. BTB 06237 also reduced parasite burdens in L. mexicana-infected J774 macrophages at low micromolar concentrations. Unlike the dinitroaniline sulfonamides, the active compounds did not display antimitotic effects against Leishmania. Transmission electron microscopy showed that the single parasite mitochondrion becomes dilated following incubation with BTB 06237, and fluorescence microscopy demonstrated that this organelle fragments into intensely staining spheres when treated with a mitochondrion-specific dye. The mitochondrial membrane potential was also dissipated in BTB 06237-treated parasites. These results indicate that BTB 06237 is an intriguing antileishmanial lead compound that likely interferes with mitochondrial function.

Aniline Compounds↗

PubMed2006

A high efficiency cloning and expression system for proteomic analysis.

The recent description of the complete genomes of the two most pathogenic species of Brucella opens the way for genome-based analysis of the antigenicity of their proteins. In the present report, we describe a bench-level high-efficiency cloning and expression system (HECES) that allow expression of large numbers of Brucella proteins based on genomic sequence information. Purified proteins are produced with high efficiency in a microarray format conducive to analysis of their sero-reactivity against serum from immunized animals. This method is applicable at either small or large scale of protein processing. While it does not require robotics, the format is amenable to robotic implementation for all aspects of the process and subsequent analysis of protein characteristics. This method will allow selection of new reagents for diagnosis of brucellosis and development of vaccine against Brucella, an important zoonotic disease and biothreat agent.

Automation↗

PubMed2006

Comparison of protective efficacy of subcutaneous versus intranasal immunization of mice with a Brucella melitensis lipopolysaccharide subunit vaccine.

Groups of mice were immunized either subcutaneously or intranasally with purified Brucella melitensis lipopolysaccharide (LPS) or with LPS as a noncovalent complex with Neisseria meningitidis group B outer membrane protein (LPS-GBOMP). Control mice were inoculated with sterile saline. Two doses of vaccine were given 4 weeks apart. Mice were challenged intranasally with virulent B. melitensis strain 16M 4 weeks after the second dose of vaccine. Sera, spleens, lungs, and livers of mice were harvested 8 weeks after challenge. The bacterial loads in the organs were determined by culture on brucella agar plates. Protective efficacy was determined by comparing the clearance of bacteria from organs of immunized mice with the clearance of bacteria from organs of control mice. At 8 weeks postchallenge there was significant protection from disseminated infection of spleens and livers of mice intranasally immunized with either vaccine compared to infection of control mice (P < 0.01). There was no significant difference in clearance of bacteria from the lungs of immunized mice and control mice. However, mice immunized subcutaneously with either LPS or LPS-GBOMP vaccine showed significant protection against infection of the spleen (P < 0.001), liver (P < 0.001), and lungs (P < 0.05). These results show that intranasal immunization of mice with either vaccine provided significant protection against disseminated infection of the spleen and liver but subcutaneous immunization of mice with the vaccines conferred significant protection against infection of the spleen, liver, and lungs.

Administration, Cutaneous↗

PubMed2005

Active immunization with a detoxified endotoxin vaccine protects against lethal polymicrobial sepsis: its use with CpG adjuvant and potential mechanisms.

BACKGROUND: An experimental vaccine for sepsis, composed of detoxified Escherichia coli J5 lipopolysaccharide (LPS) complexed with the outer membrane protein (OMP) of Neisseria meningitidis group B, induces anti-core glycolipid antibody and has been tested in pilot studies in human volunteers. METHODS: Mice were immunized with the LPS-J5/OMP vaccine with or without synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs as a vaccine adjuvant (CpG ODN). The efficacy of the vaccine-induced antibody response was tested in a cecal ligation and puncture model. RESULTS: Immunization resulted in a >20-fold increase in anti-core glycolipid antibody levels, which were further increased 5-fold by the addition of CpG ODN, compared with the levels in mice in the control group. The vaccine provided a survival advantage after a cecal ligation and puncture was performed (P < .01) and significantly decreased the levels of bacteria in organs. Immunoglobulin G (IgG) anti-core glycolipid antibodies were decreased in mice to a significantly greater extent than were levels of total circulating IgG or IgG to the OMP part of the vaccine complex, suggesting specific epitope binding and clearance. CONCLUSIONS: These results indicate that the detoxified LPS-J5/OMP vaccine induces high levels of antibody against the core glycolipid of LPS and functions in vivo to promote clearance of gram-negative bacteria and improve the outcome of experimental polymicrobial intra-abdominal sepsis.

Adjuvants, Immunologic↗

PubMed2005

The medicinal chemistry of botulinum, ricin and anthrax toxins.

The potential use of weapons of mass destruction (nuclear, biological or chemical) by terrorist organizations represents a major threat to world peace and safety. Only a limited number of vaccines are available to protect the general population from the medical consequences of these weapons. In addition there are major health concerns associated with a pre-exposure mass vaccination of the general population. To reduce or eliminate the impact of these terrible threats, new drugs must be developed to safely treat individuals exposed to these agents. A review of all therapeutic agents under development for the treatment of the illnesses and injuries that result from exposure to nuclear, biological or chemical warfare agents is beyond the scope of any single article. The intent here is to provide a focused review for medicinal and organic chemists of three widely discussed and easily deployed biological warfare agents, botulinum neurotoxin and ricin toxins and the bacteria Bacillus anthracis. Anthrax will be addressed because of its similarity in both structure and mechanism of catalytic activity with botulinum toxin. The common feature of these three agents is that they exhibit their biological activity via toxin enzymatic hydrolysis of a specific bond in their respective substrate molecules. A brief introduction to the history of each of the biological warfare agents is presented followed by a discussion on the mechanisms of action of each at the molecular level, and a review of current potential inhibitors under investigation.

Animals↗

PubMed2005

Analysis of molecular stereoelectronic similarity between N,N-diethyl-m-toluamide (DEET) analogs and insect juvenile hormone to develop a model pharmacophore for insect repellent activity.

Similarity analysis on molecular stereoelectronic properties of N,N-diethyl-m-toluamide (DEET), natural insect juvenile hormone (JH), a synthetic insect juvenile hormone mimic (JH-mimic, undecen-2-yl carbamate), and DEET compounds reveals remarkable similarities that lead to a reliable pharmacophore for the design of efficacious insect repellents and provide insights for understanding the mechanism of repellent action. The study involves an AM1 quantum chemical computational procedure enabling a conformational search for the lowest and most abundant energy conformers of JH, JH-mimic, and 15 DEET compounds and complete geometry optimization of the conformers. Similarity analyses of stereoelectronic properties such as structural parameters, atomic charges, dipole moments, molecular electrostatic potentials, and highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies were performed on JH, JH-mimic, and the DEET compounds. Similarity of stereoelectronic attributes of the amide/ester moiety, negative electrostatic potential regions beyond the molecular surface, and a large distribution of hydrophobic regions in the compounds appears to be the 3 important factors leading to a similar interaction with the JH receptor. The similarity of electrostatic profiles beyond the molecular surface is likely to play a crucial role toward molecular recognition interaction with the JH receptor from a distance which suggests a possible electrostatic bioisosterism of the amide group of the DEET compounds and JH-mimic and, thus, a model for molecular recognition at the JH receptor.

Aedes↗

PubMed2004

A three-dimensional in silico pharmacophore model for inhibition of Plasmodium falciparum cyclin-dependent kinases and discovery of different classes of novel Pfmrk specific inhibitors.

The cell division cycle is regulated by a family of cyclin-dependent protein kinases (CDKs) that are functionally conserved among many eukaryotic species. The characterization of plasmodial CDKs has identified them as a leading antimalarial drug target in our laboratory. We have developed a three-dimensional QSAR pharmacophore model for inhibition of a Plasmodium falciparum CDK, known as Pfmrk, from a set of fifteen structurally diverse kinase inhibitors with a wide range of activity. The model was found to contain two hydrogen bond acceptor functions and two hydrophobic sites including one aromatic-ring hydrophobic site. Although the model was not developed from X-ray structural analysis of the known CDK2 structure, it is consistent with the structure-functional requirements for binding of the CDK inhibitors in the ATP binding pocket. Using the model as a template, a search of the in-house three-dimensional multiconformer database resulted in the discovery of sixteen potent Pfmrk inhibitors. The predicted inhibitory activities of some of these Pfmrk inhibitors from the molecular model agree exceptionally well with the experimental inhibitory values from the in vitro CDK assay.

Adenosine Triphosphate↗

PubMed2004

The anthrax protective antigen (PA63) bound conformation of a peptide inhibitor of the binding of lethal factor to PA63: as determined by trNOESY NMR and molecular modeling.

Anthrax protective antigen (PA) is one of the three proteins produced by the gram positive bacteria Bacillus anthracis collectively known as the "anthrax toxin" (Ascenzi, P.; Visca, P.; Ippolito, G.; Spallarossa, A.; Bolognesi, M.; et al. Anthrax toxin: a tripartite lethal combination. FEBS Lett. 2002, 531, 384-388). The role played by PA in anthrax intoxication is to transport the two enzymes lethal factor (LF) and edema factor (EF) into the cell. Collier and co-workers (Mourez, M.; Kane, R. S.; Mogridge, J.; Metallo, S.; Deschatelets, P.; et al. Designing a polyvalent inhibitor of anthrax toxin. Nat. Biotechnol. 2001, 958). reported the isolation of two peptides via phage display that bind to the PA63 heptamer and inhibit its interaction with LF and EF, and thereby prevent the transport of LF and EF into the cell. One of these peptides, His-Thr-Ser-Thr-Try-Trp-Trp-Leu-Asp-Gly-Ala-Pro (P1), was selected for structural investigation on the basis of its ability to prevent the binding of LF to the PA63 heptamer bundle. Two-dimensional trNOESY experiments coupled with NOE restrained simulated annealing calculations were used to determine the PA63-bound conformation of P1. On binding to PA63, P1 adopts a helical conformation involving residues 3-9 while the C- and N-terminal residues exhibit dynamic fraying.

Anti-Bacterial Agents↗

PubMed2004

Nuclear magnetic resonance and molecular modeling analysis of the interaction of the antimalarial drugs artelinic acid and artesunic acid with beta-cyclodextrin.

The artemisinin derivatives artelinic acid and artesunic acid are members of a class of compounds that have shown promise for the treatment of multidrug resistant strains of Plasmodium falciparum. Unfortunately, these compounds exhibit poor solubility and stability in aqueous solution. The research presented herein was conducted to determine whether complexation of artelinic acid or artesunic acid with beta-cyclodextrin would result in complexes with increased aqueous solubility while retaining the potent antimalarial activity of these compounds. Preliminary complexation studies with natural beta-cyclodextrins were conducted as a proof of concept, with a primary focus on understanding the electrostatic interactions that stabilize the resulting complexes. Complex formation was monitored using UV spectroscopy. The structures of the resulting complexes were determined using multidimensional nuclear magnetic resonance spectroscopy (NMR) and molecular modeling. NMR results are most consistent for artelinic acid and beta-cyclodextrin forming complexes in a ratio of 2:1; however, the presence of 1:1, 2:2, and 3:1 complexes in solution cannot be excluded based on the experimental data collected. The NMR data also indicate selective insertion of artelinic acid into the hydrophobic cavity of the beta-cyclodextrin via the primary face. NMR results indicate artesunic acid forms a similar complex with beta-cyclodextrin in a ratio of 1:1; again however, the presence of 1:1, 2:2, and 3:1 complexes in solution cannot be ruled out.

Animals↗

PubMed2004

Structure-activity relationship study of antimalarial indolo [2,1-b]quinazoline-6,12-diones (tryptanthrins). Three dimensional pharmacophore modeling and identification of new antimalarial candidates.

A widely applicable three-dimensional QSAR pharmacophore model for antimalarial activity was developed from a set of 17 substituted antimalarial indolo[2,1-b]quinazoline-6,12-diones (tryptanthrins) that exhibited remarkable in vitro activity (below 100 ng/mL) against sensitive and multidrug-resistant Plasmodium falciparum malaria. The pharmacophore, which contains two hydrogen bond acceptors (lipid) and two hydrophobic (aromatic) features, was found to map well onto many well-known antimalarial drug classes including quinolines, chalcones, rhodamine dyes, Pfmrk cyclin dependent kinase inhibitors, malarial FabH inhibitors, and plasmepsin inhibitors. The phamacophore allowed searches for new antimalarial candidates from multiconformer 3D databases and enabled custom designed synthesis of new potent analogues.

Animals↗

PubMed2004

The antimalarial potential of 4-quinolinecarbinolamines may be limited due to neurotoxicity and cross-resistance in mefloquine-resistant Plasmodium falciparum strains.

The clinical potential of mefloquine has been compromised by reports of adverse neurological effects. A series of 4-quinolinecarbinolamines were compared in terms of neurotoxicity and antimalarial activity in an attempt to identify replacement drugs. Neurotoxicity (MTT [thiazolyl blue reduction] assay) was assessed by exposure of cultured embryonic rat neurons to graded concentrations of the drugs for 20 min. The 50% inhibitory concentration (IC(50)) of mefloquine was 25 microM, while those of the analogs were 19 to 200 microM. The relative (to mefloquine) therapeutic indices of the analogs were determined after using the tritiated hypoxanthine assay for assessment of the antimalarial activity of the analogs against mefloquine-sensitive (W2) and -resistant (D6 and TM91C235) Plasmodium falciparum strains. Five analogs, WR157801, WR073892, WR007930, WR007333, and WR226253, were less neurotoxic than mefloquine and exhibited higher relative therapeutic indices (RTIs) against TM91C235 (2.9 to 12.2). Conventional quinoline antimalarials were generally less neurotoxic (IC(50)s of 400, 600, and 900 for amodiaquine, chloroquine, and quinine) or had higher RTIs (e.g., 30 for halofantrine against TM91C235). The neurotoxicity data for the 4-quinolinecarbinolamines were used to develop a three-dimensional (3D), function-based pharmacophore. The crucial molecular features correlated with neurotoxicity were a hydrogen bond acceptor (lipid) function, an aliphatic hydrophobic function, and a ring aromatic function specifically distributed in the 3D surface of the molecule. Mapping of the 3D structures of a series of structurally diverse quinolines to the pharmacophore allowed accurate qualitative predictions of neurotoxicity (or not) to be made. Extension of this in silico screening approach may aid in the identification of less-neurotoxic quinoline analogs.

Animals↗

PubMed2004

Oral vaccination with Brucella melitensis WR201 protects mice against intranasal challenge with virulent Brucella melitensis 16M.

Human brucellosis can be acquired from infected animal tissues by ingestion, inhalation, or contamination of conjunctiva or traumatized skin by infected animal products. In addition, Brucella is recognized as a biowarfare threat agent. Although a vaccine to protect humans from natural or deliberate infection could be useful, vaccines presently used in animals are unsuitable for human use. We tested orally administered live, attenuated, purine auxotrophic B. melitensis WR201 bacteria for their ability to elicit cellular and humoral immune responses and to protect mice against intranasal challenge with B. melitensis 16M bacteria. Immunized mice made serum antibody to lipopolysaccharide and non-O-polysaccharide antigens. Splenocytes from immunized animals released interleukin-2 and gamma interferon when grown in cultures with Brucella antigens. Immunization led to protection from disseminated infection and enhanced clearance of the challenge inoculum from the lungs. Optimal protection required administration of live bacteria, was related to immunizing dose, and was enhanced by booster immunization. These results establish the usefulness of oral vaccination against respiratory challenge with virulent Brucella and suggest that WR201 should be further investigated as a vaccine to prevent human brucellosis.

Administration, Oral↗

PubMed2003

Phase I study of detoxified Escherichia coli J5 lipopolysaccharide (J5dLPS)/group B meningococcal outer membrane protein (OMP) complex vaccine in human subjects.

We previously observed that a detoxified Escherichia coli O111, Rc chemotype J5 lipopolysaccharide (J5dLPS)/group B meningococcal outer membrane protein (OMP) vaccine protected animals from experimental lethal sepsis when immune antibodies were given passively as treatment at the onset of fever or when vaccine was given actively as prophylaxis. To test the safety and immunogenicity of this vaccine, we administered doses of 5, 10 and 25 microg (based on dLPS) of vaccine at days 0, 28 and 56 to 24 human subjects (8 per group). Temperatures of 100.3, 99.5 and 99.4 degrees F occurred in three subjects. At 24h, pain at the injection site was moderate in 38%, mild in 44% and not present in 18%, while at 48 h, it was 1, 25 and 73%, respectively. No alterations in baseline renal, hepatic or hematologic functions occurred. There were two to three times mean-fold increases in anti-J5dLPS IgG (range: 1.9-5.1) and IgM (range: 1.2-9.2) levels in subjects receiving the 10 and 25 microg doses. At 12-month follow-up, three of the original responders had continued elevation of antibody levels. A 25 microg booster dose of vaccine did not increase antibody levels among those responders and did not elicit antibodies among three subjects with no previous antibody response. The plasma from the six volunteers inhibited LPS-induced cytokine generation in human whole blood ex vivo. We conclude that this J5dLPS/OMP vaccine was safe and well-tolerated with transient, local pain at the injection site. Vaccine formulations with different adjuvants are currently under investigation.

Adolescent↗

PubMed2003

Oxindole-based compounds are selective inhibitors of Plasmodium falciparum cyclin dependent protein kinases.

Cyclin dependent protein kinases (CDKs) have become attractive drug targets in an effort to identify effective inhibitors of the parasite Plasmodium falciparum, the causative agent of the most severe form of human malaria. We tested known CDK inhibitors for their ability to inhibit two malarial CDKs: Pfmrk and PfPK5. Many broad spectrum CDK inhibitors failed to inhibit Pfmrk suggesting that the active site differs from other CDKs in important ways. By screening compounds in the Walter Reed chemical database, we identified oxindole-based compounds as effective inhibitors of Pfmrk (IC(50) = 1.5 microM). These compounds have low cross-reactivity against PfPK5 and human CDK1 demonstrating selectivity for Pfmrk. Amino acid comparison of the active sites of Pfmrk and PfPK5 identified unique amino acid differences that may explain this selectivity and be exploited for further drug development efforts.

Amino Acid Sequence↗

PubMed2003

Inhibition of cholinesterases with cationic phosphonyl oximes highlights distinctive properties of the charged pyridine groups of quaternary oxime reactivators.

Oxime-induced reactivation of phosphonylated cholinesterases (ChEs) produces charged phosphonyl pyridine oxime intermediates (POXs) that are most potent organophosphate (OP) inhibitors of ChEs. To understand the role of cationic pyridine oxime leaving groups in the enhanced anti-ChE activity of POXs, the bimolecular rate constants for the inhibition (k(i)) of acetylcholinesterases (AChE) and butyrylcholinesterases (BChE), and the rate of decomposition (k(d)) of authentic O-alkyl methylphosphonyl pyridine oximes (AlkMeP-POXs) and N,N-dimethylamidophosphoryl pyridine oximes (EDMP-POXs), were studied. Stability ranking order in aqueous solutions correlated well with the electronic features and optimized geometries that were obtained by ab initio calculations at 6-31G(**) basis set level. AlkMeP-POXs of the 2-pyridine oxime series were found to be 4- to 8-fold more stable (t(1/2)=0.7 to 1.5 min) than the homologous O,O-diethylphosphoryl (DEP) oxime. Results suggest that re-inhibition of enzyme activity by POX is less likely during the reactivation of DEP-ChEs (obtained by use of DEP-containing pesticides) by certain oximes, compared to nerve agent-inhibited ChEs. The greatest inhibition was observed for the O-cyclohexyl methylphosphonyl-2PAM derivative (4.0 x 10(9)M(-1)min(-1); mouse AChE) and is 10-fold higher than the k(i) of cyclosarin. Increasing the size of the O-alkyl substituent of AlkMeP-POXs had only a small to moderate effect on the k(i) of ChEs, signifying a major role for the cationic pyridine oxime leaving group in the inhibition reaction. The shape of plots of logk(i) vs. pK(a) of the leaving groups for AlkMeP-PAMs and DEP-PAMs, could be used as a diagnostic tool to highlight and rationalize the unique properties of the cationic moiety of pyridine oxime reactivators.

Animals↗