Santosh Pasha

Interaction studies of novel cell selective antimicrobial peptides with model membranes and E. coli ATCC 11775

Cationic antimicrobial peptides (CAMPs) are novel candidates for drug development. Here we describe design of six short and potent CAMPs (SA-1 to SA-6) based on a minimalist template of 12 residues H+HHG+HH+HH+NH2 (where H: hydrophobic amino acid and +: charged hydrophilic amino acid). Designed peptides exhibit good antibacterial activity in micro molar concentration range (1-32 mug/ml) and rapid clearance of Gram-positive and Gram-negative bacterial strains at concentrations higher than MIC. For elucidating mode of action of designed peptides various biophysical studies including CD and Trp fluorescence were performed using model membranes. Further based on activity, selectivity and membrane bound structure; modes of action of Trp rich peptide SA-3 and template based peptide SA-4 were compared. Calcein dye leakage and transmission electron microscopic studies with model membranes exhibited selective membrane active mode of action for peptide SA-3 and SA-4. Extending our work from model membranes to intact E. coli ATCC 11775 in scanning electron micrographs we could visualize different patterns of surface perturbation caused by peptide SA-3 and SA-4. Further at low concentration rapid translocation of FITC-tagged peptide SA-3 into the cytoplasm of E. coli cells without concomitant membrane perturbation indicates involvement of intracellular targeting mechanism as an alternate mode of action as was also evidenced in DNA retardation assay. For peptide SA-4 concentration dependent translocation into the bacterial cytoplasm along with membrane perturbation was observed. Establishment of a non specific membrane lytic mode of action of these peptides makes them suitable candidates for drug development.

Nonprocessive [2 + 2]e- off-loading reductase domains from mycobacterial nonribosomal peptide synthetases.

In mycobacteria, polyketide synthases and nonribosomal peptide synthetases (NRPSs) produce complex lipidic metabolites by using a thio-template mechanism of catalysis. In this study, we demonstrate that off-loading reductase (R) domain of mycobacterial NRPSs performs two consecutive [2 + 2]e- reductions to release thioester-bound lipopeptides as corresponding alcohols, using a nonprocessive mechanism of catalysis. The first crystal structure of an R domain from Mycobacterium tuberculosis NRPS provides strong support to this mechanistic model and suggests that the displacement of intermediate would be required for cofactor recycling. We show that 4e- reductases produce alcohols through a committed aldehyde intermediate, and the reduction of this intermediate is at least 10 times more efficient than the thioester-substrate. Structural and biochemical studies also provide evidence for the conformational changes associated with the reductive cycle. Further, we show that the large substrate-binding pocket with a hydrophobic platform accounts for the remarkable substrate promiscuity of these domains. Our studies present an elegant example of the recruitment of a canonical short-chain dehydrogenase/reductase family member as an off-loading domain in the context of assembly-line enzymology.

Role of histidine interruption in mitigating the pathological effects of long polyglutamine stretches in SCA1: A molecular approach

Polyglutamine expansions, leading to aggregation, have been implicated in various neurodegenerative disorders. The range of repeats observed in normal individuals in most of these diseases is 19–36, whereas mutant proteins carry 40–81 repeats. In one such disorder, spinocerebellar ataxia (SCA1), it has been reported that certain individuals with expanded polyglutamine repeats in the disease range (Q12HQHQ12HQHQ14/15) but with histidine interruptions were found to be phenotypically normal. To establish the role of histidine, a comparative study of conformational properties of model peptide sequences with (Q12HQHQ12HQHQ12) and without (Q42) interruptions is presented here. Q12HQHQ12HQHQ12 displays greater solubility and lesser aggregation propensity compared to uninterrupted Q42 as well as much shorter Q22. The solvent and temperature‐driven conformational transitions (β structure ↔ random coil → α helix) displayed by these model polyQ stretches is also discussed in the present report. The study strengthens our earlier hypothesis of the importance of histidine interruptions in mitigating the pathogenicity of expanded polyglutamine tract at the SCA1 locus. The relatively lower propensity for aggregation observed in case of histidine interrupted stretches even in the disease range suggests that at a very low concentration, the protein aggregation in normal cells, is possibly not initiated at all or the disease onset is significantly delayed. Our present study also reveals that besides histidine interruption, proline interruption in polyglutamine stretches can lower their aggregation propensity.

Chimeric peptide of Met-enkephalin and FMRFa induces antinociception and attenuates development of tolerance to morphine antinociception

A synthetic chimeric peptide of Met-enkephalin and FMRFamide (YGGFMKKKFMRFa), based on MERF was synthesized. This peptide was tested for possible antinociceptive effects using the tail flick test in mice. The effect of the chimeric peptide on morphine antinociception and development of tolerance to the antinociceptive action of morphine was also investigated. The chimeric peptide produced significant, dose-dependent antinociception (40, 60 and 90 mg/kg) in the tail flick test. Pretreatment with naloxone (5 mg/kg, IP) significantly attenuated the antinociceptive effect induced by the chimeric peptide (90 mg/kg, IP), indicating involvement of an opioidergic mechanism. In combination experiments with morphine, the antinociceptive dose of the chimeric peptide (60 mg/kg, IP) potentiated morphine (7 mg/kg, IP) antinociception. A low dose of the chimeric peptide (10 mg/kg, IP), that did not produce significant antinociception on its own, also potentiated morphine antinociception. In the tolerance studies, male albino mice received twice daily injections of morphine (20 mg/kg, IP) followed by either saline (0.1 ml) or chimeric peptide (80 mg/kg, IP) for a period of 4 days. A control group received twice daily injections of saline (0.1 ml) for the same period. When tested on Day 5, tolerance to antinociceptive action of morphine (15 mg/kg, IP) was evidenced by decreased response in chronic morphine plus saline treated mice compared to control group. Concurrent administration of chimeric peptide (80 mg/kg, IP) with morphine significantly attenuated the development of tolerance to the antinociceptive action of morphine. The preliminary results of this study demonstrate that peripherally administered chimeric peptide can produce dose dependent, naloxone reversible, antinociception; potentiate morphine antinociception and attenuate morphine tolerance, indicating a possible role of these type of amphiactive sequences in antinociception and its modulation. These chimeric peptides may also prove to be useful tools for further ascertaining the role of FMRFa family of peptides in mechanisms leading to opiate tolerance and dependence.

Various drug delivery approaches to the central nervous system.

Importance of the field: The presence of the blood–brain barrier (BBB), an insurmountable obstacle, in particular, and other barriers in brain and periphery contribute to hindrance of the successful diagnosis and treatment of a myriad of central nervous system pathologies. This review discusses several strategies adopted to define a rational drug delivery approach to the CNS along with a short description of the strategies implemented by the authors’ group to enhance the analgesic activity, a CNS property, of chimeric peptide of Met-enkephalin and FMRFa (YGGFMKKKFMRFa-YFa). Areas covered in this review: Various approaches for drug delivery to the CNS with their beneficial and non-beneficial aspects, supported by an extensive literature survey published recently, up to August 2009. What the reader will gain: The reader will have the privilege of gaining an understanding of previous as well as recent approaches to breaching the CNS barriers. Take home message: Among the various strategies discussed, the potential for efficacious CNS drug targeting in future lies either with the non-invasively administered multifunctional nanosystems or these nanosystems without characterstics such as long systemic circulating capability and avoiding reticuloendothelial system scavenging system of the body, endogenous transporters and efflux inhibitors administered by convection-enhanced delivery.

YFa, a Chimeric Opioid Peptide, Induces Kappa-Specific Antinociception with No Tolerance Development during 6 Days of Chronic Treatment

Our previous study showed that YGGFMKKKFMRFamide (YFa), a chimeric peptide of Met‐enkephalin, and Phe‐Met‐Arg‐Phe‐NH2 induced naloxone‐reversible antinociception and attenuated the development of tolerance to morphine analgesia. In continuation, the present study investigated which specific opioid receptors—μ, δ or κ—mediate the observed YFa antinociception pharmacologically using specific antagonists and whether chronic administration of YFa at 26.01 μmol/kg per day induces tolerance and its effect on the expression of μ and κ opioid receptors from day 4 to day 6, with endomorphine‐1 (EM‐1) and saline taken as positive and negative controls, respectively. Quantitative differential expression analysis was carried out by real‐time reverse‐transcriptase polymerase chain reaction, and the corresponding changes in protein levels were assessed by Western blot. A pharmacological investigation revealed that nor‐binaltorphimine, a specific κ opioid receptor–1 (KOR1) antagonist, completely antagonized the antinociception induced by 39.01 μmol/kg of YFa. Importantly, its chronic intraperitoneal administration did not result in significant tolerance over 6 days, whereas EM‐1 induced significant tolerance after day 4. Differential expression analysis revealed that EM‐1 caused up‐regulation of μ opioid receptor–1 on day 4, followed by down‐regulation on later days. Interestingly, YFa treatment caused a decrease on day 4, followed by an increase in the expression of KOR1 from day 5 onward. In conclusion, YFa induces kappa‐specific antinociception, with no development of tolerance during 6 days of chronic treatment, which further articulates new directions for improved designing of peptide‐based analgesics that may be devoid of adverse effects like tolerance.

Effect of varying chain length between P1 and P1′ position of tripeptidomimics on activity of angiotensin-converting enzyme inhibitors

One of the efficient modes of treatments of chronic hypertension and cardiovascular disorders has been to restrain the formation of angiotensin-II by inhibiting the action of angiotensin-converting enzyme (ACE) on angiotensin-I. The efforts continue towards achieving superior molecules or drugs with improved affinities, better bioavailability and thus to achieve long duration of action with minimum side effects. Previously, we reported a library of tripeptidomimics of Ornithyl-Proline (Orn-Pro) conjugated with various unnatural amino acids and carboxylic acid derived heterocyclics based on the SAR studies of existing ACE inhibitors. Their synthesis and screening for possible inhibitors of angiotensin-converting enzyme (ACE) revealed that increase in the backbone chain length by one carbon atom results in a sudden decrease in their activity. Therefore, in the present study heterocycles with different chain length were introduced to interact with the hydrophobic S(2) sub-site of ACE and screened for their in vitro ACE inhibition activity. Further, their binding interaction with C-domain of somatic ACE was also determined. Docking and consequent LUDI scores showed good correlation with binding of these molecules in the active site of ACE. Results suggest that heterocycles with C(3) chain length are more appropriate for the effective binding of the tripeptidomimics within the active site of ACE.

Hypotensive effect of novel chimeric peptides of met-enkephalin and FMRFa

Endogenous opioid peptides like endomorphins, met-enkephalin and NPFF/FMRFamide family of neuropeptides, besides playing a role in modulation of antinociception, also affect cardiovascular system. Based on MERF, which consists of overlapping sequences of FMRFa and met-enkephalin, two chimeric peptides YGGFMKKKFMRFamide (YFa) and [D-Ala2] YAGFMKKKFMRFamide ([D-Ala2] YFa) were designed and synthesized. In this study, effect of YFa and [D-Ala2] YFa on arterial blood pressure and heart rate was evaluated in anaesthetized rats. Both YFa and [D-Ala2] YFa showed a dose-dependent fall in mean arterial pressure in dose-range of 13-78 micromol/kg. After naloxone treatment (5 mg/kg), vasodepressor effect of [D-Ala2] YFa and YFa was only partially blocked as compared to met-enkephalin. Partial blockade of vasodepressive effect of YFa and [D-Ala2] YFa by naloxone may be attributed to interaction of these chimeric peptides with receptors other than naloxone-sensitive receptors such as anti-opioid receptors, adrenergic receptors and D-analogue receptors.

Nanoparticles of cationic chimeric peptide and sodium polyacrylate exhibit striking antinociception activity at lower dose

The current study investigates the performance of polyelectrolyte complexes based nanoparticles in improving the antinociceptive activity of cationic chimeric peptide-YFa at lower dose. Size, Zeta potential and morphology of the nanoparticles were determined. Size of the nanoparticles decreases and zeta potential increases with concomitant increase in charge ratio (Z(+/-)). The nanoparticles at Z(+/-)12 are spherical with 70+/-7 nm diameter in AFM and displayed positive surface charge and similar sizes (83+/-8 nm) by Zetasizer. The nanoparticles of Z(+/-) 12 are used in this study. Cytotoxicity by MTT assay on three different mammalian cell lines (liver, neuronal and kidney) revealed lower toxicity of nanoparticles. Hematological parameters were also not affected by nanoparticles compared to normal counts of water treated control group. Nanoparticles containing 10 mg/kg YFa produced increased antinociception, approximately 36%, in tail-flick latency test in mice, whereas the neat peptide at the same concentration did not show any antinociception activity. This enhancement in activity is attributed to the nanoparticle associated protection of peptide from proteolytic degradation. In vitro peptide release study in plasma also supported the antinociception profile of nanoparticles. Thus, our results suggest of a potential nanoparticle delivery system for cationic peptide drug candidates for improving their stability and bioavailability.

Chimeric peptide of met-enkephalin and FMRFa: Effect of chlorination on conformation and analgesia

In our previous study YFa (YGGFMKKKFMRFa), a chimeric peptide of met-enkephalin and FMRFa, not only produced analgesia but also did not let the tolerance develop. In the continuation of the same study, Phe4 is chlorinated so as to assess the effect of chlorination on the conformation, lipophilicity and analgesia of chimeric peptide [p-Cl Phe(4)] YFa. Not only does the chlorination increase the lipophilicity but also enhances the propensity of [p-Cl Phe(4)] YFa to form alpha helix in comparison of YFa in presence of membrane mimicking solvent trifluoroethanol (TFE). This increase in lipophilicity and helix-forming ability results in more bioavailability and naloxone-reversible analgesia by [p-Cl Phe(4)] YFa. Though analgesia produced by [p-Cl Phe(4)] YFa is more than YFa at all doses, there is sudden decrease in analgesia at 45 and 60 min at 60 mg/kg. This sudden decrease of analgesia seems to be due to desensitization of opioid receptors.