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1.
Adv Healthc Mater ; 11(10): e2102640, 2022 05.
Article in English | MEDLINE | ID: mdl-35038229

ABSTRACT

Tuberculosis (TB), including extrapulmonary TB, is responsible for more than one million deaths in a year worldwide. Existing methods of mycobacteria detection have poor sensitivity, selectivity, and specificity, especially in human tissues. Herein, the synthesis of a cholic acid-derived fluorescent probe (P4) that can specifically stain the mycobacterium species is presented. It is shown that P4 probe specifically binds with mycobacterial lipids, trehalose monomycolate, and phosphatidylinositol mannoside 6. P4 probe can detect mycobacteria in polymicrobial planktonic cultures and biofilms with high specificity, selectivity, and sensitivity. Moreover, it can detect a single mycobacterium in the presence of 10 000 other bacilli. Unlike the probes that depend on active mycobacterial enzymes, the membrane-specific P4 probe can detect mycobacteria even in formalin-fixed paraffin-embedded mice and human tissue sections. Therefore, the ability of the P4 probe to detect mycobacteria in different biological milieu makes it a potential candidate for diagnostic and prognostic applications in clinical settings.


Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Animals , Fluorescent Dyes , Humans , Mice , Paraffin Embedding , Sensitivity and Specificity , Tuberculosis/diagnosis
2.
J Phys Chem Lett ; 10(4): 754-760, 2019 Feb 21.
Article in English | MEDLINE | ID: mdl-30694679

ABSTRACT

As mechanisms underpinning the molecular interactions between membrane-targeting antimicrobials and Gram-negative bacterial membranes at atomistic scale remain elusive, we used cholic acid (CA)-derived amphiphiles with different hydrophobicities as model antimicrobials and assessed the effect of their conformational flexibility on antimicrobial activity. Relative to other hydrophobic counterparts, a compound with a hexyl chain (6) showed the strongest binding with the lipopolysaccharide (LPS) of Gram-negative bacterial membranes and acted as an effective antimicrobial. Biomolecular simulations, validated by complementary approaches, revealed that specific intramolecular hydrogen bonding imparts conformationally rigid character to compound 6. This conformational stability of compound 6 allows minimum but specific interactions of the amphiphile with LPS that are a sum of exothermic processes like electrostatic interactions, membrane insertion, and endothermic contributions from disaggregation of LPS. Therefore, our study reveals that a membrane-targeting mechanism with the help of conformationally selective molecules offers a roadmap for developing future therapeutics against bacterial infections.


Subject(s)
Anti-Bacterial Agents/chemistry , Cholic Acids/chemistry , Lipid Bilayers/chemistry , Surface-Active Agents/chemistry , Anti-Bacterial Agents/pharmacology , Cholic Acids/pharmacology , Escherichia coli/drug effects , Hydrogen Bonding , Lipopolysaccharides/chemistry , Molecular Conformation , Static Electricity , Surface-Active Agents/pharmacology
3.
J Med Chem ; 62(4): 1875-1886, 2019 02 28.
Article in English | MEDLINE | ID: mdl-30688460

ABSTRACT

The presence of lipopolysaccharide and emergence of drug resistance make the treatment of Gram-negative bacterial infections highly challenging. Herein, we present the synthesis and antibacterial activities of cholic acid-peptide conjugates (CAPs), demonstrating that valine-glycine dipeptide-derived CAP 3 is the most effective antimicrobial. Molecular dynamics simulations and structural analysis revealed that a precise intramolecular network of CAP 3 is maintained in the form of evolving edges, suggesting intramolecular connectivity. Further, we found high conformational rigidity in CAP 3 that confers maximum perturbations in bacterial membranes relative to other small molecules. Interestingly, CAP 3-coated catheters did not allow the formation of biofilms in mice, and treatment of wound infections with CAP 3 was able to clear the bacterial infection. Our results demonstrate that molecular conformation and internal connectivity are critical parameters to describe the antimicrobial nature of compounds, and the analysis presented here may serve as a general principle for the design of future antimicrobials.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Cholic Acids/therapeutic use , Gram-Negative Bacteria/drug effects , Gram-Negative Bacterial Infections/drug therapy , Lipopolysaccharides/metabolism , Peptides/therapeutic use , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/metabolism , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Cholic Acids/chemical synthesis , Cholic Acids/pharmacology , Escherichia coli/drug effects , Escherichia coli/metabolism , Escherichia coli/physiology , Gram-Negative Bacteria/metabolism , Gram-Negative Bacteria/physiology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/metabolism , Mice, Inbred BALB C , Microbial Sensitivity Tests , Molecular Conformation , Molecular Dynamics Simulation , Peptides/chemical synthesis , Peptides/pharmacology , Structure-Activity Relationship
4.
Bioconjug Chem ; 30(3): 721-732, 2019 03 20.
Article in English | MEDLINE | ID: mdl-30669829

ABSTRACT

A major impediment to developing effective antimicrobials against Gram-negative bacteria like Salmonella is the ability of the bacteria to develop resistance against existing antibiotics and the inability of the antimicrobials to clear the intracellular bacteria residing in the gastrointestinal tract. As the critical balance of charge and hydrophobicity is required for effective membrane-targeting antimicrobials without causing any toxicity to mammalian cells, herein we report the synthesis and antibacterial properties of cholic acid-derived amphiphiles conjugated with alkyl chains of varied hydrophobicity. Relative to other hydrophobic counterparts, a compound with hexyl chain (6) acted as an effective antimicrobial against different Gram-negative bacteria. Apart from its ability to permeate the outer and inner membranes of bacteria; compound 6 can cross the cellular and lysosomal barriers of epithelial cells and macrophages and kill the facultative intracellular bacteria without disrupting the mammalian cell membranes. Oral delivery of compound 6 was able to clear the Salmonella-mediated gut infection and inflammation, and was able to combat persistent, stationary, and multi-drug-resistant clinical strains. Therefore, our study reveals the ability of cholic acid-derived amphiphiles to clear intracellular bacteria and Salmonella-mediated gut infection and inflammation.


Subject(s)
Anti-Bacterial Agents/administration & dosage , Cholic Acid/administration & dosage , Inflammation/prevention & control , Intestinal Diseases/prevention & control , Salmonella Infections/prevention & control , Administration, Oral , Animals , Drug Resistance, Multiple, Bacterial , Intestinal Diseases/microbiology , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests , Salmonella/isolation & purification , Salmonella/pathogenicity
5.
ACS Biomater Sci Eng ; 5(9): 4764-4775, 2019 Sep 09.
Article in English | MEDLINE | ID: mdl-33448819

ABSTRACT

Inappropriate and uncontrolled use of antibiotics results in the emergence of antibiotic resistance, thereby threatening the present clinical regimens to treat infectious diseases. Therefore, new antimicrobial agents that can prevent bacteria from developing drug resistance are urgently needed. Selective disruption of bacterial membranes is the most effective strategy for combating microbial infections as accumulation of genetic mutations will not allow for the emergence of drug resistance against these antimicrobials. In this work, we tested cholic acid (CA) derived amphiphiles tethered with different alkyl chains for their ability to combat Gram-positive bacterial infections. In-depth biophysical and biomolecular simulation studies suggested that the amphiphile with a hexyl chain (6) executes more effective interactions with Gram-positive bacterial membranes as compared to other hydrophobic counterparts. Amphiphile 6 is effective against multidrug resistant Gram-positive bacterial strains as well and does not allow the adherence of S. aureus on amphiphile 6 coated catheters implanted in mice. Further, treatment of wound infections with amphiphile 6 clears the bacterial infections. Therefore, the current study presents strategic guidelines in design and development of CA-derived membrane-targeting antimicrobials for Gram-positive bacterial infections.

6.
Eur Biophys J ; 44(8): 635-45, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26184723

ABSTRACT

The ability of the membrane skeletal protein spectrin to interact with phospholipids, and aminophospholipids in particular, in both natural and model membranes, is well documented. The present study involves phospholipid-induced quenching of tryptophan fluorescence to probe spectrin-membrane interactions in the presence and absence of cholesterol. We performed the experiments on small unilamellar vesicles of phospholipids made of DMPC and DMPC/DMPE and of DOPC and DOPC/DOPE with and without cholesterol at two different temperatures, one below at 15 °C and another above, at 50 °C, the main phase transition temperature (T m) of the bulk phospholipid. Results indicate that erythroid and brain spectrin binds DMPC/DMPE membranes by tenfold and 40-fold stronger, respectively, in the presence of 20 % cholesterol, up to which both gel (Lß) and liquid crystalline (Lα) phases coexists, at 15 °C particularly in DMPC-based membranes containing saturated fatty acyl chains and not in DOPC-based membranes with appreciably lower T m. Time-resolved fluorescence and circular dichroism spectroscopic studies indicated no significant change in the mean lifetime of the tryptophan residues in spectrin and in the secondary structures of the proteins upon binding to the phospholipid SUVs.


Subject(s)
Cholesterol/chemistry , Phosphatidylethanolamines/chemistry , Spectrin/chemistry , Dimyristoylphosphatidylcholine/chemistry , Fluorescence , Gels/chemistry , Liquid Crystals/chemistry , Phosphatidylcholines/chemistry , Protein Binding , Unilamellar Liposomes/chemistry
7.
J Fluoresc ; 25(3): 707-17, 2015 May.
Article in English | MEDLINE | ID: mdl-25835748

ABSTRACT

Brain spectrin enjoys overall structural and sequence similarity with erythroid spectrin, but less is known about its function. We utilized the fluorescence properties of tryptophan residues to monitor their organization and dynamics in brain spectrin. Keeping in mind the functional relevance of hydrophobic binding sites in brain spectrin, we monitored the organization and dynamics of brain spectrin bound to PRODAN. Results from red edge excitation shift (REES) indicate that the organization of tryptophans in brain spectrin is maintained to a considerable extent even after denaturation. These results are supported by acrylamide quenching experiments. To the best of our knowledge, these results constitute the first report of the presence of residual structure in urea-denatured brain spectrin. We further show from REES and time-resolved emission spectra that PRODAN bound to brain spectrin is characterized by motional restriction. These results provide useful information on the differences between erythroid spectrin and brain spectrin.


Subject(s)
2-Naphthylamine/analogs & derivatives , Brain/metabolism , Protein Conformation , Spectrin/chemistry , Tryptophan/chemistry , 2-Naphthylamine/chemistry , 2-Naphthylamine/metabolism , Animals , Binding Sites , Circular Dichroism , Fluorescence , Hydrophobic and Hydrophilic Interactions , Protein Denaturation , Sheep , Spectrin/metabolism , Spectrometry, Fluorescence/methods , Urea/chemistry
8.
J Biomol Struct Dyn ; 32(6): 852-65, 2014.
Article in English | MEDLINE | ID: mdl-24404769

ABSTRACT

We have used three polarity-sensitive fluorescence probes, 6-propionyl 2-(N,N-dimethyl-amino) naphthalene (Prodan), pyrene and 8-anilino 1-naphthalene sulphonic acid, to study their binding with erythroid and nonerythroid spectrin, using fluorescence spectroscopy. We have found that both bind to prodan and pyrene with high affinities with apparent dissociation constants (Kd) of .50 and .17 µM, for prodan, and .04 and .02 µM, for pyrene, respectively. The most striking aspect of these bindings have been that the binding stoichiometry have been equal to 1 in erythroid spectrin, both in dimeric and tetrameric form, and in tetrameric nonerythroid spectrin. From an estimate of apparent dielectric constants, the polarity of the binding site in both erythroid and nonerythroid forms have been found to be extremely hydrophobic. Thermodynamic parameters associated with such binding revealed that the binding is favored by positive change in entropy. Molecular docking studies alone indicate that both prodan and pyrene bind to the four major structural domains, following the order in the strength of binding to the Ankyrin binding domain > SH3 domain > Self-association domain > N-terminal domain of α-spectrin of both forms of spectrin. The binding experiments, particularly with the tetrameric nonerythroid spectrin, however, indicate more toward the self association domain in offering the unique binding site, since the binding stoichiometry have been 1 in all forms of dimeric and tetrameric spectrin, so far studied by us. Further studies are needed to characterize the hydrophobic binding sites in both forms of spectrin.


Subject(s)
Carrier Proteins/chemistry , Erythrocytes/chemistry , Microfilament Proteins/chemistry , Spectrin/chemistry , 2-Naphthylamine/analogs & derivatives , 2-Naphthylamine/chemistry , Anilino Naphthalenesulfonates/chemistry , Animals , Fluorescent Dyes , Goats , Hydrophobic and Hydrophilic Interactions , Molecular Docking Simulation , Protein Interaction Domains and Motifs , Protein Multimerization , Pyrenes/chemistry , Sheep , Spectrometry, Fluorescence , Thermodynamics
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