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Design, Synthesis, and Bioactivity of Thioamidated Macrocyclic Peptides
No full textThe work presented in this thesis describes the comprehensive study related to thioamidated macrocyclic peptides. We study the underexplored class of peptide bond isostere; thioamides in the context of small macrocyclic peptides The thesis is divided into six chapters. First chapter introduces the literature available for macrocyclic peptides along with the various peptide backbone modifications, and challenges associated with the study of thioamidated peptides. In the second chapter, we have focussed on the optimization of synthetic protocol for the incorporation of a thioamidated residue in site-selective manner in linear and cyclic peptides. Subsequently, in chapter 3 efforts have been put to understand the conformational impact of thioamide incorporation on the backbone of macrocyclic peptides. Understanding the detailed role of bioactivity in structurally characterised thioamidated peptides is the main focus of chapters 4 and 5. Detailed experimental and computational analysis lead us to propose thioamide as a bioisosteric modification that would greatly aid in the development of leads in peptide-based drug discovery efforts. Chapter 6 deals with the summary and conclusion of the work. The later part of thesis has Appendices, each appendix describes the materials and methods used in preceding chapters and characterization of various synthesised thioamidated amino acid precursors and peptides used in this study, via HRMS, HPLC and NMR
Exploring Nature's Inventory: Investigating the Role of Amide to Thioamide Substitution on Protein Stability
No full textThe peptide backbone holds a protein together and plays a crucial role in guiding its three-dimentional structure. The tertiary structure of proteins regulates several biological processes. Therefore, peptide bond modification has gained significant attention to influence protein folding, stability, and functions. However, it is a challenging task to mimic the peptide (amide) bond due to its unique properties, such as planar structure, hydrogen bond donor, and acceptor properties. Thioamide, a single O to S substitution in an amide bond, is the closest isostere and has shown promising results on small peptides. The recent discovery of thioamide in natural protein, methyl-coenzyme M reductase (MCR), raises an important fundamental question of its role in protein conformation and stability. However, the synthesis of thio modified peptides/proteins is challenging. Therefore, we first focused on the synthetic procedure for the synthesis of thioamidated peptides and proteins. We have shown the compatibility of our synthetic method in incorporating the thionated derivative of all the 20 naturally occurring amino acids onto a growing peptide chain. We also report the use of a 2% DBU + 5% piperazine cocktail for fast Fmoc-deprotection that allowed us to synthesize thioamidated Pin1 WW domain and GB1 directly on a solid support. Next, we demonstrated the role of a single n→π* interaction on protein stability by engineering n→π* interaction at the β-turn. Our experimental results at the i+2 residue of type-II’ β-turn in GB1 variants suggest that amino acid side-chain identity and the rotamer conformation can modulate the strength of an n→π* interaction. The altered rotamer conformation as a result of local structural changes within a protein can amplify/weaken an n→π* interaction affecting the backbone torsion angles (phi, psi), and thereby influencing its stability. Further, we amplified the strength of n→π* interaction by replacing i+1 donor carbonyl with thiocarbonyl and validated that the n→π* interaction can indeed influence the structural stability of proteins. We also highlighted the potential impact of a single atom substitution in stabilizing the β-sheet protein. It broadens the scope of this backbone mutation approach in designing/stabilizing the protein scaffold
Insights into the conformational stability & pharmacology of peptides through side chain and backbone modification
No full textThe first part of the work presented in the thesis deals with CH-pi interaction, which is a lesser-known non-covalent interaction in proteins. We identified several CH-pi donor-acceptor pairs involving amino acids side chains and less polarized C-H bonds at a solvent-exposed site between the strands of a model beta-hairpin peptide. Therein, we observe a distance-dependent induction of CH-pi interaction within the aliphatic-aromatic amino acid pair. Our results also suggest an interplay of hydrophobicity and CH-pi interaction in dictating the stability of beta-hairpin. In the second part, we have attempted to establish thioamidation, which is a single atom ‘O’ to ‘S’ substitution, as a chemical strategy for amide bond desolvation. Thioamide substitution on a model and a bioactive cyclic peptide has led to an enhancement in membrane permeability and metabolic stability which are the two major deterrents of peptide bioavailability. We observed improved plasma exposure of the thioamidated peptides upon oral intake. Thioamidation could successfully improve the pharmacokinetic properties of the peptides and subsequently, their oral bioavailability. Additionally, thioamidated somatostatin agonists displayed prolonged inhibition of growth hormone release in vivo.
Altogether, the work done in this thesis highlights the roles of both side chains and amide backbone in modulating conformations and other functional properties of peptides and proteins. Our strategies underscore how these local components can be fine-tuned to achieve the desired biophysical properties and activities to design next generation peptide-based therapeutics.Mo
Engineering Reverse Turns: Implications in Conformation, Stability and Bioactivity of Peptides and Proteins
No full textThe work presented in this thesis provides an overview of the potential role of N-methylation in inducing reverse turns. Backbone N-methylation is one of the common modifications designed by nature and exploited by humans to enhance the oral bioavailability of macrocyclic peptides. However, their predisposition in turns in cyclic peptides are undermined by the backbone cyclization restraint, where conformations are driven mainly by steric interactions. Therefore, the work sheds light on the intrinsic property of N-methylation in inducing turns and its influence in modulating the conformational stability of beta-hairpin containing peptides and proteins.
The thesis is divided into five chapters. In the first chapter, we provide a brief introduction on a stable class of scaffolds known as peptidomimetics and the literature associated in designing of major secondary elements: alpha-helix and beta-sheet. Additionally, we highlight the strategies employed in engineering beta-turn mimetics and the challenges associated with the available repertoire of turn mimetics. In the second chapter, we largely focus on utilization of N-methylated heterochiral amino acids as beta-turn surrogate and their role in inducing beta-hairpin formation in both polar and apolar conditions, with inspiration derived from the conformations of naturally-occurring and designed cyclic peptides. Subsequently in the third chapter, we demonstrate the differential behavior of N-methylation at i+1 and i+2 site of a beta-turn and thereby, tune the steric restraints at the turn through non- covalent interaction. We also show the bio-compatibility of our engineered motif with different functional groups, present on the ribosomal-encoded amino acids. Finally, we validate our design in a protein model system and modulate the global conformational stability of the protein. In the fourth chapter, we venture into the applicability of our designed ‘chemical toolkit’, where we exploit our design in generating highly potent, low-cytotoxic and metabolically stable b-hairpin antimicrobial peptides that has the ability to kill bacteria in a very short period of time. Finally, in the last chapter, we summarize the overall work. The later part of the thesis has appendices, which consists of the materials and methods employed in the aforementioned chapters, characterization of all the compounds synthesized in the individual chapters using MALDI, HPLC and NMR and few supplementary data
Understanding the role of arginine clusters in β-hairpin antimicrobial peptides
No full textThe work described in this thesis describes the role of arginine clusters and the
importance of their position in B-hairpin antimicrobial peptides. Here, we have
tried to explore the role of guanidinium groups in the studied model system
Polyphemeusin-1. The thesis has been divided into four chapters. The first
chapter includes the challenges associated with the antimicrobial resistance, the
literature available for antimicrobial peptides and a broad view of their history,
diversity, physiochemical properties and the mechanism of actions of
antimicrobial peptides. From this chapter we can infer the fact the antimicrobial
peptides are an important part of innate immune system and show broad
spectrum activity proving them as a potential candidate for developing into
therapeutics. Later, in the second chapter we have described the background
study which was done to understand the arginine cluster, how we chose the
model system for our study and the literature available for the model peptide
Polyphemusin-1. In this chapter, we observed that all -hairpin antimicrobial
peptides contain cysteine disulfide bridge. Also, all the peptides analysed
showed a pattern of arginine clusters in terminal region, strand region and turn
region from which we delineated our objective. In the third Chapter we have
detailed all the experiments, which were performed to carry out this project. The
last chapter includes the results and discussion section wherein, we have
described all the detailed analysis of the experiments performed and results
obtained while trying to understand the role of arginine and its position for
overall potency and activity of the peptide. From this chapter we could conclude
the fact that arginine in the terminal clusters plays a key role in antimicrobial
activity of Polyphemusin-1 and removal of guanidinium group leads to
compromise in its antimicrobial activity without compromising its interaction
with the membrane. However, the peptides showed structural difference at
different pH and increased structural rigidity in hydrophobic niche for which
further experiments needs to be done.
Summary and Outlook
The arginine clusters in β-hairpin peptides play an important role for the poteny
of peptides. The mutation of arginine to ornithine in which the guanidine group
was lost led to compromise upon the antimicrobial activity of the Polyphemusin
specially the removal of guanidinium group left the peptide with no
antimicrobial activity. However, the loss of guanidinium group did not showed
any major effect upon the membrane damage and membrane depolarization
concluding the fact that the first interaction of the peptides with membrane is
not getting affected possibly because the Polyphemusin has some internal
targets which still needs to be explored
Development of a Selective Cell-Permeable Protein Phosphatase 1 Inhibitor
Get PDFSelective ‘super-specific’ inhibitors of Protein Phosphatase 1 (PP1) are not available. Several natural product toxins possessing marginal selectivity between PP1 and the closely related Protein Serine/Threonine Phosphatase (PSTP), Protein Phosphatase 2A (PP2A) have been used to study the role of PP1 and PP2A in cellular signaling processes, such as the cyclic peptide inhibitors (microcystins and nodularins); terpenoid (cantharidin); polyketides (okadaic acid, calyculin, and tautomycin). The organic molecule tautomycetin is a natural product which has the highest selectivity for PP1 compared to the closely related PSTP PP2A, albeit slightly so (about 39 times more selective). Calyculin A is equally selective to PP1 and PP2A. On the other hand, okadaic acid is about 100 times more selective towards PP2A compared to PP1.
Specific protein inhibitors are not suitable for cell-based assay due to low, intrinsic cellular permeability of proteins. A si-RNA mediated knockdown approach though feasible, is not ‘fast-acting’. The knockdown often lasts for an extended time period and cannot be modulated (turned on or off) as desired. Also, analysis of knockdown data is complex as the system can regulate itself in complex ways, making any effort to interpret the data liable to misinterpretation.
The ultimate goal of this project is to develop a cell-permeable, potent, and selective inhibitor for PP1 (which does not target the related protein phosphatases PP2A, PP2B and PP5) whose activity inside cells can be modulated as desired so that spatiotemporal control over the activity of PP1 can be achieved. Development of such an inhibitor can be used as a chemical tool to study the cellular signaling of PP1 and not by the related PSTP PP2A.
To address the problem of a lack of inhibitor targeting Protein Phosphatase 1 selectively over the closely related PSTP, PP2A; design of a peptide based inhibitor has been envisioned which targets the acidic groove and hydrophobic groove of Protein Phosphatase 1 in addition to targeting the active site (triple approach combination). The parent peptide (V6.2.10) of this study has been designed using a co-crystal structure of rat PP1cγ complexed with mouse inhibitor-2 (PDB ID: 2O8A).
The parent peptide V6.2.10 has an IC50 value of 4.2 µM, which has been confirmed in the present study. A combination of single site mutations has been made using N-terminus arginine scanning, C-terminus arginine scanning, active site mutations, cyclohexylalanine scanning, and miscellaneous site-specific mutations. A hydrophobic pocket present in Protein Phosphatase 1 has been probed using ortho and meta fluorophenyalanine residue to increase potency and metabolic stability of the peptide. The rationale for such mutations was based upon a combination of approaches: mutagenesis in PyMOL, calculation of binding energies in FoldX, suitability of parent residues to be mutated, and how important are parent and substituent residues for cellular permeability and metabolic stability.
Several peptides were identified from single-site mutations which had lower (improved) IC50 compared to the parent peptide of the study, V6.2.10. Several double mutations combining potent single-mutant peptides identified from this study has lower (improved) IC50 values than either of the single mutant peptides. #30 (combination of #15 and #4.2) has an IC50 value of about 334 nM and #36 (combination of #15 and 4-Fluoro Phenylalanine at the F5 position) has an IC50 value of 531 nM. #30 is the optimized peptide inhibitor from this study which is currently being utilized for crystallization trails in the laboratory.
Far UV Circular dichroism study of #4.2 peptide shows mostly random coil conformation along with contributions from other secondary structures. Moreover, #4.2 is capable of adopting an alpha helical conformation in the presence of the well-known helix inducer chemical trifluoroethanol.
Purification of PP1α protein using affinity chromatography has been optimized in order to increase the yield of pure protein phosphatase 1. Attempts to express and purify PP1α protein in BL21 (DE3) bacterial cells gave low yield. Thus, expression and purification of PP1α protein derived from human genomic sequence has been attempted in BL21 (RIL) codon-optimized cells which resulted in increased production of pure protein
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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