Mechanism of Protease Resistance of D-Amino Acid Residue Containing Cationic Antimicrobial Heptapeptides.

Antimicrobial peptides (AMPs) have been an alternate promising class of therapeutics in combating global antibiotic resistance threat. However, the short half-life of AMPs, owing to protease degradability, is one of the major bottlenecks in its commercial success. In this study, we have developed all-D-amino acid containing small cationic peptides P4C and P5C, which are completely protease-resistant, noncytotoxic, nonhemolytic, and potent against the ESKAPE pathogens in comparison to their L analogues. MD simulations suggested marginal improvement in the peptide-binding affinity to the membrane-mimetic SDS micelle (∼ 1 kcal/mol) in response to L → D conversion, corroborating the marginal improvement in the antimicrobial activity. However, L → D chirality conversion severely compromised the peptide:protease (trypsin) binding affinity (≥10 kcal/mol). The relative distance between the scissile peptide carbonyl and the catalytic triad of the protease (H57, D102, and S195) was found to be significantly altered in the D-peptide:protease complex (inactive conformation) relative to the active L-peptide:protease complex. Thus, the poor binding affinity between D-peptides and the protease, resulting in the inactive complex formation, explained their experimentally observed proteolytic stability. This mechanistic insight might be extended to the proteolytic stability of the D-peptides in general and stimulate the rational design of protease-resistant AMPs.

Delineating the Mechanism of Action of a Protease Resistant and Salt Tolerant Synthetic Antimicrobial Peptide against Pseudomonas aeruginosa.

Rapidly growing antimicrobial resistance (AMR) against antibiotics has propelled the development of synthetic antimicrobial peptides (AMPs) as potential antimicrobial agents. An antimicrobial peptide Nle-Dab-Trp-Nle-Dab-Dab-Nle-CONH2 (P36; Nle = norleucine, Dab = diaminobutyric acid, Trp = tryptophan) potent against Pseudomonas aeruginosa (P. aeruginosa) has been developed in the present study. Rational design strategy adopted in this study led to the improvisation of the therapeutic qualities such as activity, salt tolerance, cytotoxicity, and protease resistance of the template peptide P4, which was earlier reported from our group. P36 exhibited salt tolerant antimicrobial potency against P. aeruginosa, along with very low cytotoxicity against mammalian cell lines. P36 was found to be nonhemolytic and resistant toward protease degradation which qualified it as a potent antimicrobial agent. We have investigated the mechanism of action of this molecule in detail using several experimental techniques (spectroscopic, biophysical, and microscopic) and molecular dynamics simulations. P36 was a membrane active AMP with membrane destabilization and deformation abilities, leading to leakage of the intracellular materials and causing eventual cell death. The interaction between P36 and the microbial membrane/membrane mimics was primarily driven by electrostatics. P36 was unstructured in water and upon binding to the microbial membrane mimic SDS, suggesting no influence of secondary structure on its antimicrobial potency. Positive charge, optimum hydrophobic-hydrophilic balance, and chain length remained the most important concerns to be addressed while designing small cationic antimicrobial peptides.

Effect of Differential Geminal Substitution of γ Amino Acid Residues at the (i + 2) Position of αγ Turn Segments on the Conformation of Template ß-Hairpin Peptides.

The effect of insertion of three geminally dimethyl substituted γ amino acid residues [γ2,2 (4-amino-2,2-dimethylbutanoic acid), γ3,3 (4-amino-3,3-dimethylbutanoic acid), and γ4,4 (4-amino-4,4-dimethylbutanoic acid)] at the (i + 2) position of a two-residue αγ C12 turn segment in a model octapeptide sequence Leu-Phe-Val-Aib-Xxx-Leu-Phe-Val (where Xxx = γ amino acid residues) has been investigated in this study. Solution conformational studies (NMR, CD, and IR) and ab initio calculations indicated that γ3,3 and γ4,4 residues were well accommodated in the ß-hairpin nucleating αγ C12 turns, which gave rise to well-registered hairpins, in contrast to γ2,2, which was unable to form a tight C12 ß-hairpin nucleating turn and promote a well-registered ß-hairpin. Geminal disubstitution at the Cα carbon in γ2,2 led to unfavorable steric contacts, disabling its accommodation in the αγ C12 hairpin nucleating turn unlike the γ3,3 and γ4,4 residues. Geminal substitutions at different carbons along the backbone constrained backbone torsion angles for the three γ amino acid residues differently, generating diverse conformational preferences in them. Folded hairpins were energetically more stable (∼8 to 9 kcal/mol) than the unfolded peptides. Conformational preference of the peptides was independent of the N-terminal protecting group. Such fundamental understanding will instrumentalize the future directed design of foldamers.

Effect of monovalent salt concentration and peptide secondary structure in peptide-micelle binding.

Recently, we reported a cationic 14 residue peptide LL-14 (LKWLKKLLKWLKKL) with salt-sensitive broad-spectrum antimicrobial potency. However, the mechanism of its salt (NaCl) sensitivity remained unclear. In this study, we have reported computational (∼14.2 µs of MD) and experimental (CD, fluorescence) investigations to examine the salt-sensitivity and the role of peptide secondary structure on LL-14 binding to simple membrane mimetic (SDS, DPC) systems. LL-14 was shown to adopt a random coil (Pc) conformation in water and α-helical conformation (Ph) in the peptide:SDS micelle complex, accompanied by tryptophan burial, using both simulations and experiments. Simulations successfully deconvoluted the LL-14:micelle binding event in terms of secondary structure (random coil Pc versus helix Ph) and gave atomic insight into the initial and final LL-14:SDS complexes. Electrostatics drove the N-terminus (L1 and K2) of LL-14 (Pc or Ph) to bind the SDS micellar surface, initiating complex formation. LL-14 in amphipathic Ph conformation bound faster and buried deeper into the SDS micelle relative to Pc. Increasing NaCl concentration incrementally delayed LL-14:micelle binding by shielding the overall charges of the interacting partners. LL-14 binding to the SDS micelle was significantly faster relative to that of the zwitterionic DPC micelle due to electrostatic reasons. Cationic α-helical amphipathic peptides (with positively charged N-terminus) with low salt-ion concentration seemed to be ideal for faster SDS binding.

Effect of Secondary Structure and Side Chain Length of Hydrophobic Amino Acid Residues on the Antimicrobial Activity and Toxicity of 14-Residue-Long de novo AMPs.

Herein we report the efficacy and toxicity of three de novo designed cationic antimicrobial peptides (AMPs) LL-14, VV-14 and ßß-14, where side chains of the hydrophobic amino acids were reduced gradually. The AMPs showed broad-spectrum antimicrobial activity against three pathogens from the ESKAPE group and two fungal strains. This study showed that side chains which are either too long or too short increase toxicity and lower antimicrobial activity, respectively. VV-14 was found to be non-cytotoxic and highly potent under physiological salt concentrations against several pathogens, especially Salmonella typhi TY2. These AMPs acted via membrane deformation, depolarization, and lysis. The activity of the AMPs is related to their ability to take on amphipathic helical conformations in the presence of microbial membrane mimics. Among AMPs with the same charge, hydrophobic interactions between the side chains of the residues with cell membrane lipids determine their antimicrobial potency and cytotoxicity. Strikingly, an optimum hydrophobic interaction is the crux of generating highly potent non-cytotoxic AMPs.

Rationally designed antimicrobial peptides: Insight into the mechanism of eleven residue peptides against microbial infections.

The widespread abuse of antibiotics has led to the use of antimicrobial peptides (AMPs) as a replacement for the existing conventional therapeutic agents for combating microbial infections. The broad-spectrum activity and the resilient nature of AMPs has mainly aggrandized their utilization. Here, we report the design of non-toxic, non-hemolytic and salt tolerant undecapeptides (AMP21-24), derived by modification of a peptide P5 (NH2-LRWLRRLCONH2) reported earlier by our group. Our results depict that the designed peptides show potency against several bacterial as well as fungal strains. Circular dichroism (CD) spectroscopy in combination with molecular dynamic (MD) simulations confirm that the peptides are unstructured. Intrinsic tryptophan fluorescence quenching as well as interaction studies using isothermal calorimetry (ITC) of these peptides in the presence of biological microbial membrane mimics establish the strong microbial membrane affinity of these AMPs. Membrane permeabilization assay and cytoplasmic membrane depolarization studies of Pseudomonas aeruginosa and Candida albicans in the presence of AMPs also hint towards the AMP-membrane interactions. Leakage of calcein dye from membrane mimic liposomes, live cell NMR and field emission scanning electron microscopy (FESEM) studies suggest that the AMPs may be primarily involved in membrane perturbation leading to release of intracellular substances resulting in subsequent microbial cell death. Confocal laser scanning microscopy (CLSM) shows localization of the peptides throughout the cell, indicating the possibility of secondary mode of actions. Electrostatic interactions seem to govern the preferential binding of the AMPs to the microbial membranes in comparison to the mammalian membranes as seen from the MD simulations.

Peptide Hydrogels as Platforms for Sustained Release of Antimicrobial and Antitumor Drugs and Proteins.

A charged synthetic peptide-based noncytotoxic hydrogelator was employed in encapsulation, storage, and sustainable release of different kinds of drugs, namely, ciprofloxacin (CP), an antibiotic; 5-fluorouracil (5-FU), an anticancer drug and proteins like lysozyme and bovine serum albumin (BSA). Hydrogelation of the peptide and its coassembly with the drug molecules were studied to obtain mechanistic details. All of the different cargos were capable of sustained and efficient release from the delivery platform. The drugs were found to retain their activity post release, while the proteins showed complete retention of their secondary structure. While about 80% CP was released at physiological pH over a period of 3 days, 5-FU was better released (73%) at an acidic pH (5.5) in comparison to the physiological pH (68%). Lysozyme was better released (82%) than BSA (43%) owing to the smaller size of the former and negative charge on the latter. Such biocompatible multicargo-releasing platforms from simple economically viable biomaterials, capable of sustained and tissue-specific release of cargo, are extremely promising in topical delivery of therapeutics.

Selective detection of fluoride via amplified donor-acceptor interaction of 6H-indolo[2,3-b]quinoline.

In this report, 6H-indolo[2,3-b]quinoline (hereafter 2a) was synthesized and employed as an optical chemosensor for fluoride. The sensitivity of 2a towards fluoride was established from the change in both the absorption and emission signals. The various in-situ1H NMR, UV-Vis, and density functional studies indicate that the 1:2 binding interaction between 2a and fluoride followed by deprotonation to its corresponding di-anion (2a2-), which in turn boosted the donor-acceptor interaction between indole and quinoline moiety in 2a2-via expansion of torsion angle by 10.2° as compared to 2a. Consequently the significant changes in both the absorption and emission signal of 2a allow us to detect and estimate the concentration of fluoride up to 0.2 µM from the mixture of different anions.

Insights into the Mechanism of Antimicrobial Activity of Seven-Residue Peptides.

Antimicrobial peptides have gained widespread attention as an alternative to the conventional antibiotics for combating microbial infections. Here, we report a detailed structure-function correlation of two nontoxic, nonhemolytic, and salt-tolerant de novo designed seven-residue leucine-lysine-based peptides, NH2LKWLKKLCONH2 (P4) and NH2LRWLRRLCONH2 (P5), with strong antimicrobial and antifungal activity. Biological experiments, low- and high-resolution spectroscopic techniques in conjunction with molecular dynamics simulation studies, could establish the structure-function correlation. The peptides are unstructured both in water and in bacterial membrane mimicking environment, suggesting that the secondary structure does not play a major role in their activity. Our studies could justify the probable membranolytic mode of action for killing the pathogens. Attempts to understand the mode of action of these small AMPs is fundamental in the rational design of more potential therapeutic molecules beyond serendipity in the future.

Self-Assembly Mechanism of a Peptide-Based Drug Delivery Vehicle.

We report the mechanism of the concentration-dependent self-assembly of a tetrapeptide. Peptide Boc-Trp-Leu-Trp-Leu-OMe self-assembles to form discrete nanospheres at a low concentration. Tryptophan side chains point outwards of the nanospheres while leucine side chains point towards the core of the nanospheres. The nanospheres fuse together to become microspheres with the increase in the peptide concentration. At higher concentrations of the peptide, the microspheres start clustering. This is stabilized by the aromatic interactions between the side chains of the tryptophan residues that cover the outer surface of the peptide microspheres. In addition to behaving like the conventional hollow sphere-based drug delivery vehicles which entraps the drug and performs stimuli-responsive release, this prototype can interact, stabilize, and intercalate hydrophobic dye carboxyfluorescein and anti-cancer drug curcumin even on the surface through aromatic interactions. The dye/drug can be released in acidic pH and in the presence of physiologically relevant ions such as potassium.

The Study of Coagulation Parameters in Polytrauma Patients and Their Effects on Outcome.

BACKGROUND: Mortality from trauma remains a major public health issue as it is the leading cause of death in persons aged 5 - 44 years. There is a dearth of information on polytrauma from developing countries such as ours. Hence, this topic was studied at our institute. The objective is to study the coagulation parameters in polytrauma patients at our institute and to correlate the findings with the prognosis. METHODS: A prospective study was carried out in the department of pathology in a tertiary care center, during a period of 20 months from December 2012 to July 2014. All the polytrauma patients (injury severity score (ISS) ≥ 15) with injuries to head and neck, face, thorax, abdomen, extremities and external (skin) were included. Sampling was done within 20 min of arrival during primary survey of the patient. Screening tests like bleeding time (BT) and clotting time (CT) were carried out bedside. Other tests carried out were complete blood count (CBC), prothrombin time (PT), activated thromboplastin time (aPTT), thrombin time (TT) and D-dimer assay. Tests were carried out on fresh samples within 2 h of collection. RESULTS: The incidence of coagulopathy was 59.86%. There was significant prolongation of PT, aPTT and TT in those patients who developed coagulopathy. PT was found to be a stronger predictor of mortality among polytrauma patients. CONCLUSION: A significant proportion of polytrauma patients were coagulopathic. Initial coagulation profile is very useful in predicting outcomes for major polytrauma patients. This study emphasizes the importance of early suspicion and basic screening for coagulopathy in polytrauma patients in developing countries.

Congenital pulmonary airway malformation: A report of two cases.

Congenital pulmonary airway malformation (CPAM), previously known as congenital cystic adenomatoid malformation is a congenital disorder of the lung similar to bronchopulmonary sequestration. In CPAM, usually an entire lobe of lung is replaced by a non-working cystic piece of abnormal lung tissue. This abnormal tissue will never function as normal lung tissue. The underlying cause for CPAM is not known. It occurs in approximately 1 in every 30000 pregnancies. The association between CPAM and malignancy has been well documented. There is a small risk (0.7%) of malignant transformation within the cyst. So early diagnosis and surgical resection is important to prevent the grave complications. Herein, we are reporting two interesting cases of CPAM and one belonged to Type II and other belonged to Type III of Stocker's classification.

Unusual histological variant of malignant peripheral nerve sheath tumor with rhabdomyoblastic differentiation.

Malignant peripheral nerve sheath tumor (MPNST) with rhabdomyoblastic differentiation is called as malignant triton tumor (MTT). It is highly aggressive soft tissue tumor with higher local recurrence rate. MTT has poor prognosis than MPNST. MTT seems to be more aggressive in patients with neurofibromatosis (NF-1). We herein, reporting an interesting case of 55 years male with multiple neurofibromas all over the body since 30 years and multiple café-au-lait spots, diagnosed as NF-1. Since 6 years, he had an enlarged mass in left thigh. Wide excision of mass was done. On histopathological examination revealed the diagnosis of MTT and diagnosis of which was confirmed on immunohistochemistry.

Potter's syndrome: a report of 5 cases.

Bilateral renal agenesis or Potter's Syndrome is an extremely rare congenital anomaly associated with oligohydramnios. The infants die off pulmonary hypoplasia. We herewith report five cases of Potter's Syndrome over a span of one year.

Fibrosarcoma of penis--a case report.

Malignant mesenchymal tumours of penis are rare. A rare case of fibrosarcoma of penis in 60 year male is presented herewith.