Exploration of some new secretory proteins to be employed for companion diagnosis of Mycobacterium tuberculosis.

Tuberculosis (TB) is a highly infectious disease and its early and precise diagnosis is essential to reduce morbidity and mortality of patients. Since the routine diagnostic tests (like Monteux, AFB smear microscopy, chest X-Ray) do not give infallible results, additional tests are always recommended. Therefore to address the concerns about non-specificity of the present battery of diagnostic tests, we have attempted to analyze some unique secretory antigens which could be able to identify the stage specific infection of MTB. In this study, we have used recombinant proteins CFP-10, ESAT-6, Ag85 A, Ag85B, Ag85C, PE3, PE4 and Mycp1 to eliminate heterogeneity and cross reactivity in clinical diagnosis. Amplified genes were cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinantly purified proteins were used as antigens against 158 sera samples of TB patients. Secretory proteins showed better response than the PPD control. Among all the used antigens PE3 and PE4 proteins showed better reactivity levels among all the groups of TB patients. The secretions of CFP-10 and ESAT-6 were also higher as compared to other secretory proteins like Ag85 A, Ag85B, Ag85C and MycP1.The clinical use of these newly identified secretory antigens could be of significant value for the confirmatory, rapid, simple and low-cost diagnosis of TB patients.

Substituted carbamothioic amine-1-carbothioic thioanhydrides as novel trichomonicidal fungicides: Design, synthesis, and biology.

Sexually transmitted diseases like trichomoniasis along with opportunistic fungal infections like candidiasis are major global health burden in female reproductive health. In this context a novel non-nitroimidazole class of substituted carbamothioic amine-1-carbothioic thioanhydride series was designed, synthesized, evaluated for trichomonacidal and fungicidal activities, and was found to be more active than the standard drug Metronidazole (MTZ). Compounds were trichomonicidal in the MIC ranges of 4.77-294.1 µM and 32.46-735.20 µM against MTZ-susceptible and -resistant strains, respectively. Further, compounds inhibited the growth of at least two out of ten fungal strains tested at MIC of 7.50-240.38 µM. The most active compound (20) of this series was 3.8 and 9.5 fold more active than the MTZ against the two Trichomonas strains tested. Compound 20 also significantly inhibited the sulfhydryl groups present over Trichomonas vaginalis and was found to be more active than the MTZ in vivo. Further, a docking analysis carried out with cysteine proteases supported their thiol inhibiting ability and preliminary pharmacokinetic study has shown good distribution and systemic clearance.

Role of disulfide linkage in action of bis(dialkylaminethiocarbonyl)disulfides as potent double-Edged microbicidal spermicide: Design, synthesis and biology.

Trichomoniasis and candidiasis are amongst the most common morbidity-causing reproductive tract infections, generally treated by Metronidazole and Fluconazole respectively. Poor vaginal efficacy, drug-resistance and non-spermicidal nature limit their use as topical microbicidal contraceptives. Bis(dialkylaminethiocarbonyl)disulfides (4-38) were designed as dually active, non-surfactant molecules capable of eliminating Trichomonas vaginalis and Candida strains as well as irreversibly immobilizing 100% human sperm instantly, at doses non-cytotoxic to human cervical epithelial cells and vaginal microflora in vitro. Compounds 12, 16, 17 were fifty times more active than nonoxynol-9, OTC vaginal spermicide, and compounds 12 and 17 have shown remarkable in vivo activity in rabbit model. Most promising compound 17 has shown promise for further development as a double-edged vaginal microbicide due to their improved activity and safety along with notable in vivo trichomonicidal activity. Role of disulfide group was established by loss of spermicidal activity on chemical modifications (39-56). These disulfides might be targeting thiol groups present over cell membrane of human sperm and Trichomonas as shown by fluorescence labeling of free thiols.

Innovative Disulfide Esters of Dithiocarbamic Acid as Women-Controlled Contraceptive Microbicides: A Bioisosterism Approach.

In an ongoing effort to discover an effective, topical, dual-function, non-surfactant contraceptive vaginal microbicide, a novel series of 2,2'-disulfanediylbis(3-(substituted-1-yl)propane-2,1-diyl) disubstituted-1-carbodithioates were designed by using a bioisosterism approach. Thirty-three compounds were synthesized, and interestingly, most demonstrated multiple activities: they were found to be spermicidal at a minimal effective concentration of 1-0.001 %, trichomonacidal against drug-susceptible and resistant Trichomonas strains at minimal inhibitory concentration (MIC) ranges of 10.81-377.64 and 10.81-754.14 µM, respectively, and fungicidal at MIC 7.93-86.50 µM. These compounds were also found to be non-cytotoxic to human cervical (HeLa) epithelial cells and vaginal microflora (Lactobacilli) in vitro. The most promising compound, 2,2'-disulfanediylbis(3-(pyrrolidin-1-yl)propane-2,1-diyl)dipyrrolidine-1-carbodithioate (5), exhibited spermicidal activity 15-fold higher than that of the marketed spermicide Nonoxynol-9 (N-9) and also demonstrated microbicidal potency. To identify common structural features required for spermicidal activity, a 3D-QSAR analysis was carried out, as well as in vivo efficacy studies and fluorescent labeling studies to determine the biological targets of compound 5.

N-Alkyl/aryl-4-(3-substituted-3-phenylpropyl)piperazine-1-carbothioamide as dual-action vaginal microbicides with reverse transcriptase inhibition.

The growing population and health-care burden (due to STIs and HIV) imposes a particular economic crisis over resource-poor countries. Thus a novel approach as vaginal microbicides emerges as integrated tool to control both population and anti-STIs/HIV. Our continued efforts in this field led to the synthesis of fifteen N-alkyl/aryl-4-(3-substituted-3-phenylpropyl) piperazine-1-carbothioamide (12-26) derivatives as topical vaginal microbicides which were evaluated for anti-Trichomonas, spermicidal, antifungal and reverse transcriptase (RT) inhibitory activities. All compounds were also tested for preliminary safety through cytotoxicity assays against human cervical cell line (HeLa) and the vaginal flora, Lactobacillus. Docking studies were performed to gain an insight into the binding mode and interactions of the most promising compound 12 [oxo derivative], comprising of reverse transcriptase (RT) inhibitory (72.30%), spermicidal (MEC 0.01%), anti-Trichomonas (MIC 46.72 µM) and antifungal (MIC 9.34-74.8 µM) activities, along with its hydroxyl (17) and O-alkylated 4-trifluoromethylphenoxy (22) derivative, with similar activities. The stability of compound 12 in simulated vaginal fluid (SVF) and its preliminary in vivo pharmacokinetics performed in female NZ-rabbits signifies its clinical safety in comparison to marketed spermicide Nonoxynol-9.

Design and synthesis of new fluconazole analogues.

We have synthesized new fluconazole analogues containing two different 1,2,3-triazole units in the side chain. The synthesis of new amide analogues using a variety of acids is also described. All the compounds showed very good antifungal activity. A hemolysis study of the most active compounds 6e and 13j showed that both compounds did not cause any hemolysis at the dilutions tested. These compounds did not exhibit any toxicity to L929 cells at MIC and lower concentrations. In the docking study, the overall binding mode of 6e and 13j appeared to be reasonable and provided a good insight into the structural basis of inhibition of Candida albicans Cyp51 by these compounds.

Novel Glycoconjugate of 8-Fluoro Norfloxacin Derivatives as Gentamicin-resistant Staphylococcus aureus Inhibitors: Synthesis and Molecular Modelling Studies.

Antibiotic resistance has been the subject of interest in clinical practice due to high prevalence of antibiotic-resistant pathogenic organisms. In view of the prevalence of lesser resistance in antibiotics belonging to aminoglycoside class of compounds viz. Food and Drug Administration-approved gentamicin for the treatment of Staphylococcus infections, which also has instances of resistance in the clinical isolates of Staphylococcus aureus, a series of novel glycoconjugates of 8-fluoro norfloxacin analogues with high regio-selectivity by employing copper (I)-catalyzed 1, 3-dipolar cycloaddition of 1-O-propargyl monosaccharides has been synthesized and evaluated for the antibacterial activity against gentamicin resistance Staphylococcus aureus. Among these compounds, the compound 10g showed better antibacterial activity (MIC = 3.12 µg/ml) than gentamicin (Escherichia coli (12.5 µg/ml), Staphylococcus aureus (6.25 µg/ml) and Klebsiella pneumonia (6.25 µg/ml), including gentamicin resistant (>50 µg/ml) strain in vitro). The docking studies suggest DNA gyrase of Staphylococcus aureus as a probable target for the antibacterial action of compound 10g.

A monoclonal antibody against 47.2 kDa cell surface antigen prevents adherence and affects biofilm formation of Candida albicans.

Candida albicans is an opportunistic dimorphic pathogen that exists in both planktonic and biofilm phases causing deep-rooted infections in mainly immunocompromised patients. Antibodies are believed to play anti-Candida activity by different mechanisms, like inhibition of adhesion and neutralization of virulence-related antigens. Inhibition of adhesion is one of the important strategies to prevent Candida infections and biofilm formation. In this study, monoclonal antibody (MAb 7D7) against C. albicans biofilm cell surface antigen (47.2 kDa) was generated to determine the changes in adherence and viability of C. albicans. In this regard XTT assay was carried out in 30, 60, 90 min and 48 h (maturation time) time points using MAb 7D7 and it (MAb 7D7) was found to be effective against adhesion and the formation of C. albicans biofilm on polystyrene as well as monolayer of human epithelial cells (HeLa). This result may also prove to be a valuable addition to the reagents available to study C. albicans cell surface dynamics and interaction of the fungus with host cells.

Amphotericin-B entrapped lecithin/chitosan nanoparticles for prolonged ocular application.

Fungal keratitis is the major cause of vision loss worldwide. Amphotericin-B is considered as the drug of choice for fungal infections. However, its use in ophthalmic drug delivery is limited by the low precorneal residence at ocular surface as a result of blinking reflex, tear turnover and nasopharyngeal drainage. We report Amphotericin-B loaded lecithin/chitosan nanoparticles for prolonged ocular application. The prepared nanoparticles were in the size range of 161.9-230.5 nm, entrapment efficiency of 70-75%, theoretical drug loading of 5.71% with positive zeta potential of 26.6-38.3 mV. As demonstrated by antifungal susceptibility against Candida albicans and Aspergillus fumigatus, nanoparticles were more effective than marketed formulation. They exhibited pronounced mucoadhesive properties. In-vivo pharmacokinetic studies in New Zealand albino rabbit eyes indicated improved bioavailablity (∼ 2.04 fold) and precorneal residence time (∼ 3.36 fold) by nanoparticles prepared from low molecular weight chitosan as compared with marketed formulation.

Design and synthesis of substituted morpholin/piperidin-1-yl-carbamodithioates as promising vaginal microbicides with spermicidal potential.

A series of seventeen morpholin/piperidin-1-yl-carbamodithioate (3-19) were synthesized as topical vaginal microbicidal spermicides. The synthesized compounds were evaluated for their anti-Trichomonas activity against MTZ susceptible and resistant strains along with their spermicidal and antifungal potential. All the synthesized compounds were assessed for their safety through cytotoxic assay against human cervical cell line (HeLa) and compatibility with vaginal flora, Lactobacillus. The study identified eleven dually active compounds with apparent safety. The plausible mode of action of these compounds was through sulfhydryl binding, confirmed via reduction in available free thiols on human sperm. The most promising compound 9 significantly inhibited (P<0.001) thiol-sensitive sperm hexokinase. The stability of compound 9 in simulated vaginal fluid (SVF) was performed via HPLC-PDA method, which supported its utility for vaginal administration.

Corneal targeted nanoparticles for sustained natamycin delivery and their PK/PD indices: an approach to reduce dose and dosing frequency.

Natamycin is the only approved medication for the treatment of mycotic keratitis. Current dosage regimen include one drop of natamycin suspension (5% w/v) instilled in the conjunctival sac at hourly or two hourly intervals for several days which has poor patient compliance. The purpose of the present study was to design a corneal targeted nanoformulation in order to reduce dose and dosing frequency of natamycin and evaluate its pharmacokinetic/pharmacodynamic indices in comparison with clinical marketed preparation. The nanoparticles prepared by nanoprecipitation method were in nanometer size range with high entrapment efficiency and positive surface charge. In-vitro release studies indicated prolonged release of natamycin up to 8h. In-vitro antifungal activity was comparable with marketed preparation. The performance of nanoformulations was evaluated in rabbit eyes. The concentration of natamycin in tear fluid was determined by using LC-MS/MS. The pharmacokinetic parameters such as area under the curve, t½ and mean residence time were significantly higher and clearance was significantly lower for nanoformulations with that of marketed preparation. The optimized dosing schedule to maintain natamycin concentration above tenfold of MIC90 was one instillation in every 5h. Moreover, 1/5th dose reduction of nanoformulation was also effective.

Synthesis of 2,3,6-trideoxy sugar triazole hybrids as potential new broad spectrum antimicrobial agents.

Here, we describe a molecular hybridization inspired design and synthesis of novel 6-triazolyl 2,3,6-trideoxy sugars as promising new broad-spectrum antimicrobial agents using click chemistry in key step. These compounds showed MIC between 0.39 and 50 µg/mL against different native and resistant bacteria and fungi with no toxicity. Among them, compound 29 was the most active molecule with MIC 0.78 µg/mL against Staphylococcus aureus and Klebsiella pneumoniae and 3.12 µg/mL against methicillin- and vancomycin-resistant S. aureus. Compound 26 was the most potent anti-fungal candidate with MIC 0.39 µg/mL against Trichophyton mentagrophytes. Compound 46 was found to be promising with broad-spectrum activity against both bacterial and fungal strains. The bioinformatic studies involving bacteria's protein co-crystals prompted penicillin binding protein-2 as the most likely target of these compounds.

Design and characterization of short antimicrobial peptides using leucine zipper templates with selectivity towards microorganisms.

Design of antimicrobial peptides with selective activity towards microorganisms is an important step towards the development of new antimicrobial agents. Leucine zipper sequence has been implicated in cytotoxic activity of naturally occurring antimicrobial peptides; moreover, this motif has been utilized for the design of novel antimicrobial peptides with modulated cytotoxicity. To understand further the impact of substitution of amino acids at 'a' and/or 'd' position of a leucine zipper sequence of an antimicrobial peptides on its antimicrobial and cytotoxic properties four short peptides (14-residue) were designed on the basis of a leucine zipper sequence without or with replacement of leucine residues in its 'a' and 'd' positions with D-leucine or alanine or proline residue. The original short leucine zipper peptide (SLZP) and its D-leucine substituted analog, DLSA showed comparable activity against the tested Gram-positive and negative bacteria and the fungal strains. The alanine substituted analog (ASA) though showed appreciable activity against the tested bacteria, it showed to some extent lower activity against the tested fungi. However, the proline substituted analog (PSA) showed lower activity against the tested bacterial or fungal strains. Interestingly, DLSA, ASA and PSA showed significantly lower cytotoxicity than SLZP against both human red blood cells (hRBCs) and murine 3T3 cells. Cytotoxic and bactericidal properties of these peptides matched with peptide-induced damage/permeabilization of mammalian cells and bacteria or their mimetic lipid vesicles suggesting cell membrane could be the target of these peptides. As evidenced by tryptophan fluorescence and acrylamide quenching studies the peptides showed similarities either in interaction or in their localization within the bacterial membrane mimetic negatively charged lipid vesicles. Only SLZP showed localization inside the mammalian membrane mimetic zwitterionic lipid vesicles. The results show significant scope for designing antimicrobial agents with selectivity towards microorganisms by substituting leucine residues at 'a' and/or 'd' positions of a leucine zipper sequence of an antimicrobial peptide with different amino acids.

Arachidonic acid affects biofilm formation and PGE2 level in Candida albicans and non-albicans species in presence of subinhibitory concentration of fluconazole and terbinafine

Candida albicans utilizes arachidonic acid (AA) released during the course of infection (Candidiasis) from phospholipids of infected host cell membranes and synthesizes extracellular prostaglandin(s) which play an important role in hyphae formation and host cell damage. C. albicans biofilms secrete significantly more prostaglandin(s) and evidence suggests that Candida biofilms have dramatically reduced susceptibility to majority of antifungal drugs. AA influences the saturation level of lipids and fluidity of yeast cell membranes. Therefore the aim of this study was to evaluate the effect of AA alone or in combination with antifungal agents on biofilm formation and production of prostaglandin (PGE2) in C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, and C. albicans amphotericin B resistant strain (AmBR). Maximum biofilm formation was found to be in the case of C. albicans compared to C. non-albicans species. However, among the non-albicans species C. tropicalis exhibited highest biofilm formation. Treatment with AA in combination with subinhibitory concentrations of fluconazole and terbinafine separately exhibited significant (p < 0.05) reduction in biofilm formation against C. glabrata, C. parapsilosis, C. tropicalis and AmBR as compared to their individual effect. Further, these two antifungal agents in combination with AA caused an increase in production of prostaglandin from fungal cell itself which was significant (p < 0.05) in case of all the strains tested.

Investigation of Ugi-4CC derived 1H-tetrazol-5-yl-(aryl) methyl piperazinyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid: synthesis, biology and 3D-QSAR analysis.

Novel series of 7-piperazinylquinolones with tetrazole derivatives were synthesized and evaluated for their antibacterial activity against various strains of Staphylococcus aureus. All the synthesized compounds showed significant in vitro antibacterial activity against Gram-positive bacteria whereas some compounds displayed moderate activity against Gram-negative bacteria. Among all the synthesized compounds, compounds (6a-c, 6e-g, 6i-k, 6m, 6'f and 6'm) were found to be more effective with MIC ranging from (0.78-3.12 µg/mL) against S. aureus (ATCC-29213) than the control; ciprofloxacin (MIC = 25 µg/mL). Moreover, these analogues displayed no toxicity up to MIC = 0.39 µg/mL against mammalian cell line L-929. Furthermore, to correlate the biological activities of synthesized compounds with their 3D conformation, we attempted 3D-QSAR study.

Arachidonic acid affects biofilm formation and PGE2 level in Candida albicans and non-albicans species in presence of subinhibitory concentration of fluconazole and terbinafine.

Candida albicans utilizes arachidonic acid (AA) released during the course of infection (Candidiasis) from phospholipids of infected host cell membranes and synthesizes extracellular prostaglandin(s) which play an important role in hyphae formation and host cell damage. C. albicans biofilms secrete significantly more prostaglandin(s) and evidence suggests that Candida biofilms have dramatically reduced susceptibility to majority of antifungal drugs. AA influences the saturation level of lipids and fluidity of yeast cell membranes. Therefore the aim of this study was to evaluate the effect of AA alone or in combination with antifungal agents on biofilm formation and production of prostaglandin (PGE2) in C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, and C. albicans amphotericin B resistant strain (AmBR). Maximum biofilm formation was found to be in the case of C. albicans compared to C. non-albicans species. However, among the non-albicans species C. tropicalis exhibited highest biofilm formation. Treatment with AA in combination with subinhibitory concentrations of fluconazole and terbinafine separately exhibited significant (p<0.05) reduction in biofilm formation against C. glabrata, C. parapsilosis, C. tropicalis and AmBR as compared to their individual effect. Further, these two antifungal agents in combination with AA caused an increase in production of prostaglandin from fungal cell itself which was significant (p<0.05) in case of all the strains tested.

Azole-carbodithioate hybrids as vaginal anti-Candida contraceptive agents: design, synthesis and docking studies.

Azole and carbodithioate hybrids were synthesized as alkyl 1H-azole-1-carbodithioates (7-27) and evaluated for spermicidal/microbicidal activities against human sperm, Trichomonas vaginalis and Candida species. Seventeen compounds (7-14, 16-18 and 20-25) showed spermicidal activity at MEC 1.0% (w/v) and permanently immobilized 100% normal human spermatozoa within ∼30 s. Seventeen compounds (7-11, 13-18 and 20-25) exhibited anti-Candida activity (IC50 1.26-47.69 µg/mL). All compounds were devoid of bactericidal activity against four bacterial strains (50.00 µg/mL) and antiprotozoal activity against Trichomonas vaginalis (200.00 µg/mL). Four promising compounds (10, 17, 20 and 22) have better safety profile as compared to Nonoxynol-9 (N-9). Docking study was done to visualize the possible interaction of designed scaffold with prospective receptor (Cyp51) of Candida albicans.

Characterization of antimicrobial, cytotoxic, and antiendotoxin properties of short peptides with different hydrophobic amino acids at "a" and "d" positions of a heptad repeat sequence.

To understand the influence of different hydrophobic amino acids at "a" and "d" positions of a heptad repeat sequence on antimicrobial, cytotoxic, and antiendotoxin properties, four 15-residue peptides with leucine (LRP), phenylalanine (FRP), valine (VRP), and alanine (ARP) residues at these positions were designed, synthesized, and characterized. Although valine is similarly hydrophobic to leucine and phenylalanine, VRP showed significantly lesser cytotoxicity than LRP and FRP; further, the replacement of leucines with valines at "a" and "d" positions of melittin-heptads drastically reduced its cytotoxicity. However, all four peptides exhibited significant antimicrobial activities that correlate well with their interactions with mammalian and bacterial cell membranes and the corresponding lipid vesicles. LRP most efficiently neutralized the LPS-induced pro-inflammatory mediators like NO, TNF-α, and IL-6 in macrophages followed by FRP, VRP, and ARP. The results could be useful for designing short antimicrobial and antiendotoxin peptides with understanding the basis of their activity.

Identification of 1-[4-Benzyloxyphenyl)-but-3-enyl]-1H-azoles as New Class of Antitubercular and Antimicrobial Agents.

A series of 1-[(4-benzyloxyphenyl)-but-3-enyl]-1H-azoles has been identified as potent antitubercular agents against Mycobacterium tuberculosis. Synthesis of compounds involved acid catalyzed ring-opening of cyclopropyl ring of phenyl cyclopropyl methanols followed by nucleophilic attack of the azoles on the carbocation intermediates. Several of the compounds 26, 34, and 36 exhibited significant antitubercular activities with MIC value as low as 1.56, 1.56, and 0.61 µg/mL, respectively, comparable to many standard drugs. These compounds were also screened against other strains of bacteria and fungi, and few of them showed good antifungal activity against A. fumigatus, responsible for lung infection.

Micelles catalyzed chemo- and regio-selective one pot and one step synthesis of 2,3,5,6-tetrakis(alkyl and arylsulfanyl)-1,4-benzoquinones and 2,5-diaminosubstituted-1,4-benzoquinones "In-Water" and their biological evaluation as antibacterial and antifungal agents.

Chemo- and regio-selective one pot and one step synthesis of novel 2,3,5,6-tetrakis (substituted thio)cyclohexa-2,5-diene-1,4-diones (4d-14), 2,5-dichloro-3,6-diaminocyclohexa-2,5-diene-1,4-diones and 2,5-diaminocyclohexa-2,5-diene-1,4-diones (16) by economical green methodology approach using LD (Laundry detergent) as a catalyst "In-Water" by nucleophilic addition and substitution reactions of 1,4-benzoquinone and chloranil with sulfur and nitrogen nucleophiles in high yields has been demonstrated. The antifungal profile of 4 and 16 indicates that compounds 4d and 16f had better antifungal activity compared to clinically prevalent antifungal drugs Fluconazole, 5-Fluorocytosine and Clotrimazole against Sporothrix schenckii and Trichophyton mentagraphytes. 16f had also been found to possess better antibacterial activity compared to Ampicillin in vitro against Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae. Compound 16f did not exhibit any toxicity towards mammalian cells L929.