Structure-Activity Relationships in the Design of Mitochondria-Targeted Peptide Therapeutics

Mitochondria play a central role in metabolic homeostasis; hence, dysfunction of this organelle underpins the etiology of many heritable and aging-related diseases. Mitochondria-targeted tetrapeptides with alternating cationic and aromatic residues, such as SS-31 (Elamipretide), show promise as therapeutic compounds. In this study, we conducted a quantitative structure-activity analysis of three alternative tetrapeptide analogs that differed with respect to aromatic side chain composition and sequence register, benchmarked against SS-31. Using NMR and molecular dynamics approaches, we obtained the first structural models for this class of compounds, showing that all analogs except for SS-31 form compact reverse turn conformations in the membrane-bound state. All peptide analogs bound cardiolipin-containing membranes, yet they had significant differences in equilibrium binding behavior and membrane interactions. Notably, the analogs had markedly different effects on membrane surface charge, supporting a mechanism in which modulation of membrane electrostatics is a key feature of their mechanism of action. All peptide analogs preserved survival and energy metabolism more effectively than SS-31 in cell stress models. Within our peptide set, the analog containing tryptophan side chains, SPN10, had the strongest impact on most membrane properties and showed greatest efficacy in cell culture studies. Taken together, these results show that side chain composition and register strongly influence the activity of these mitochondria-targeted peptides. Furthermore, this work helps provide a framework for the rational design of next-generation therapeutics with enhanced potency.

Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins

Pore-forming repeats in toxins (RTX) are key virulence factors of many Gram-negative pathogens. We have recently shown that the aromatic side chain of the conserved tyrosine residue 940 within the acylated segment of the RTX adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in target cell membrane interaction of the toxin. Therefore, we used a truncated CyaA-derived RTX719 construct to analyze the impact of Y940 substitutions on functional folding of the acylated segment of CyaA. Size exclusion chromatography combined with CD spectroscopy revealed that replacement of the aromatic side chain of Y940 by the side chains of alanine or proline residues disrupted the calcium-dependent folding of RTX719 and led to self-aggregation of the otherwise soluble and monomeric protein. Intriguingly, corresponding alanine substitutions of the conserved Y642, Y643 and Y639 residues in the homologous RtxA, HlyA and ApxIA hemolysins from Kingella kingae, Escherichia coli and Actinobacillus pleuropneumoniae, affected the membrane insertion, pore-forming (hemolytic) and cytotoxic capacities of these toxins only marginally. Activities of these toxins were impaired only upon replacement of the conserved tyrosines by proline residues. It appears, hence, that the critical role of the aromatic side chain of the Y940 residue is highly specific for the functional folding of the acylated domain of CyaA and determines its capacity to penetrate target cell membrane.

Aromatic side chain at position 412 of SERINC5 exerts restriction activity toward HIV-1 and other retroviruses

The host transmembrane protein SERINC5 is incorporated into viral particles and restricts infection by certain retroviruses. But, what motif of SERINC5 mediates this process remains elusive. By conducting mutagenesis analyses, we found that the substitution of phenylalanine with alanine at position 412 (F412A) resulted in >75-fold reduction in SERINC5’s restriction function. The F412A substitution also resulted in the loss of SERINC5’s function to sensitize HIV-1 neutralization by antibodies recognizing the envelope’s membrane proximal region. A series of biochemical analyses revealed that F412A showed steady-state protein expression, localization at the cellular membrane, and incorporation into secreted virus particles to a greater extent than in the wild type. Furthermore, introduction of several amino acid mutations at this position revealed that the aromatic side chains, including phenylalanine, tyrosine and tryptophan, were required to maintain SERINC5 functions to impair the virus-cell fusion process and virion infectivity. Moreover, the wild-type SERINC5 restricted infection of lentiviruses pseudotyped with envelopes of murine leukemia viruses, simian immunodeficiency virus, and HIV-2; and F412A abrogated this function. Taken together, our results highlight the importance of the aromatic side chain at SERINC5 position 412 to maintain its restriction function against diverse retrovirus envelopes. IMPORTANCE The host protein SERINC5 is incorporated into progeny virions of certain retroviruses and restricts the infectivity of these viruses or sensitizes the envelope glycoprotein toward a class of neutralizing antibodies. However, it remains elusive as to how and which part of SERINC5 engages with the diverse array of retroviral envelopes and exerts its antiretroviral functions. During mutagenesis analyses, we eventually found that the single substitution of phenylalanine with alanine, but not with tyrosine or tryptophan, at position 412 (F412A) resulted in the loss of SERINC5’s functions toward diverse retroviruses; whereas F412A showed steady-state protein expression, localization at the cellular membrane, and incorporation into progeny virions to a greater extent than the wild type. Results suggest that the aromatic side chain at position 412 of SERINC5 plays a critical role in mediating antiviral functions toward various retroviruses, thus providing additional important information regarding host and retrovirus interaction.

Insights Into the Micelle-Induced β-Hairpin-to-α-Helix Transition of a LytA-Derived Peptide by Photo-CIDNP Spectroscopy

A choline-binding module from pneumococcal LytA autolysin, LytA239–252, was reported to have a highly stable nativelike β-hairpin in aqueous solution, which turns into a stable amphipathic α-helix in the presence of micelles. Here, we aim to obtain insights into this DPC-micelle triggered β-hairpin-to-α-helix conformational transition using photo-CIDNP NMR experiments. Our results illustrate the dependency between photo-CIDNP phenomena and the light intensity in the sample volume, showing that the use of smaller-diameter (2.5 mm) NMR tubes instead of the conventional 5 mm ones enables more efficient illumination for our laser-diode light setup. Photo-CIDNP experiments reveal different solvent accessibility for the two tyrosine residues, Y249 and Y250, the latter being less accessible to the solvent. The cross-polarization effects of these two tyrosine residues of LytA239–252 allow for deeper insights and evidence their different behavior, showing that the Y250 aromatic side chain is involved in a stronger interaction with DPC micelles than Y249 is. These results can be interpreted in terms of the DPC micelle disrupting the aromatic stacking between W241 and Y250 present in the nativelike β-hairpin, hence initiating conversion towards the α-helix structure. Our photo-CIDNP methodology represents a powerful tool for observing residue-level information in switch peptides that is difficult to obtain by other spectroscopic techniques.

Synthesis of Substituted -N-(5-((7-Methyl-2-Oxo-2H-Chromen-4-yl)-Methyl)-1,3,4-Thiadiazol-2-yl)-Benzamide Derivatives Using TBTU As Coupling Agent And Their Evaluation For Anti Tubercular Activity

: Tuberculosis remains a highly infectious disease across the world. In the identification of new antitubercular agents, coumarin clubbed thiadiazole amides have been synthesized and evaluated for in vitro antitubercular activity. Due to the growing concern about chemicals and their impact on the environment, greener and faster reaction conditions needed to be incorporated. Therefore, we used TBTU as a coupling reagent for efficient and facile synthesis of substituted-N-(5-((7-methyl-2-oxo-2H-chromes-4-yl)-methyl)-1,3, 4 - thiadiazol-2-yl)-benzamide 4a-j with good yields up to 95% in mild reaction condition. All the synthesized compounds were evaluated in vitro for antitubercular activity against the H37Rv strain of M.Tuberculosis. Compounds 4c, 4f, and 4j were found active at 25 µg/mL against M. tb H37Rv. Electron withdrawing substituents present on aromatic side-chain showed promising anti-tubercular activity.

Below 3 Å structure of apoferritin using a multipurpose TEM with a side entry cryoholder

Recently, the structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single particle analysis (SPA) has had great impact as a biophysical method. Many results of cryo-EM SPA are based on data acquired on state-of-the-art cryo-electron microscopes customized for SPA. These are currently only available in limited locations around the world, where securing machine time is highly competitive. One potential solution for this time-competitive situation is to reuse existing multi-purpose equipment, although this comes with performance limitations. Here, a multi-purpose TEM with a side entry cryo-holder was used to evaluate the potential of high-resolution SPA, resulting in a 3 Å resolution map of apoferritin with local resolution extending to 2.6 Å. This map clearly showed two positions of an aromatic side chain. Further, examination of optimal imaging conditions depending on two different multi-purpose electron microscope and camera combinations was carried out, demonstrating that higher magnifications are not always necessary or desirable. Since automation is effectively a requirement for large-scale data collection, and augmenting the multi-purpose equipment is possible, we expanded testing by acquiring data with SerialEM using a β-galactosidase test sample. This study demonstrates the possibilities of more widely available and established electron microscopes, and their applications for cryo-EM SPA.

nSite-Selective, Chemical Modification of Protein at Aromatic Side Chain and Their Emergent Applications

: Site-selective chemical modification of protein side chain has probed enormous opportunities in the fundamental understanding of cellular biology and therapeutic applications. Primarily, in the field of biopharmaceutical where formulation of bioconjugates is found to be potential medicine than an individual constituent. In this regard, Lysine and Cysteine are the most widely used endogenous amino acid for these purposes. Recently, the aromatic side chain residues (Trp, Tyr, and His) that are low abundant in protein have gained more attention in therapeutic applications due to their advantages of chemical reactivity and specificity. This review discusses the site-selective bioconjugation methods for aromatic side chains (Trp, Tyr and His) and highlights the developed strategies in the last three years, along with their applications. Also, the review highlights the prevalent methods published earlier. We have examined that metal-catalyzed and photocatalytic reactions are gaining more attention for bioconjugation, though their practical operation is under development. The review has been summarized with the future perspective of protein and peptide conjugations contemplating therapeutic applications and challenges.

Synthesis of new cyclopeptide analogues of the miuraenamides

Introduction: Miuraenamides belong to marine natural compounds with interesting biological properties. Materials and Methods: They initiate polymerization of monomeric actin and therefore show high cytotoxicity by influencing the cytoskeleton. New derivatives of the miuraenamides have been synthesized containing a N-methylated amide bond instead of the more easily hydrolysable ester in the natural products. Results: Incorporation of an aromatic side chain onto the C-terminal amino acid of the tripeptide fragment also led to highly active new miuraenamides. Conclusion: We could show that the ester bond of the natural product miuraenamide can be replaced by an N-methyl amide. The yields in the cyclization step are high and generally much better that with the corresponding esters. On the other hand, the biological activity of the new amide analogs are lower compared to the natural products, but the activity can significantly be increased by incorporation of a p-nitrophenyl group at the C-terminus of the peptide fragment.

Aromatic side-chain flips orchestrate the conformational sampling of functional loops in Human Histone Deacetylase 8

Human histone deacetylase 8 (HDAC8) is a key hydrolase in gene regulation and an important drug-target. High-resolution structures of HDAC8 in complex with substrates or inhibitors are available, which have...

The Uniqueness of Tryptophan in Biology: Properties, Metabolism, Interactions and Localization in Proteins

Tryptophan (Trp) holds a unique place in biology for a multitude of reasons. It is the largest of all twenty amino acids in the translational toolbox. Its side chain is indole, which is aromatic with a binuclear ring structure, whereas those of Phe, Tyr, and His are single-ring aromatics. In part due to these elaborate structural features, the biosynthetic pathway of Trp is the most complex and the most energy-consuming among all amino acids. Essential in the animal diet, Trp is also the least abundant amino acid in the cell, and one of the rarest in the proteome. In most eukaryotes, Trp is the only amino acid besides Met, which is coded for by a single codon, namely UGG. Due to the large and hydrophobic π-electron surface area, its aromatic side chain interacts with multiple other side chains in the protein, befitting its strategic locations in the protein structure. Finally, several Trp derivatives, namely tryptophylquinone, oxitriptan, serotonin, melatonin, and tryptophol, have specialized functions. Overall, Trp is a scarce and precious amino acid in the cell, such that nature uses it parsimoniously, for multiple but selective functions. Here, the various aspects of the uniqueness of Trp are presented in molecular terms.