RESUMEN
We report a new reversible lysine conjugation that features a novel diazaborine product and much slowed dissociation kinetics in comparison to the previously known iminoboronate chemistry. Incorporating the diazaborine-forming warhead RMR1 to a peptide ligand gives potent and long-acting reversible covalent inhibitors of the staphylococcal sortase. The efficacy of sortase inhibition is demonstrated via biochemical and cell-based assays. A comparative study of RMR1 and an iminoboronate-forming warhead highlights the significance and potential of modulating bond dissociation kinetics in achieving long-acting reversible covalent inhibitors.
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LisinaRESUMEN
Binding via reversible covalent bond formation presents a novel and powerful mechanism to enhance the potency of synthetic inhibitors for therapeutically important proteins. Work on this front has yielded the anticancer drug bortezomib as well as the antisickling drug voxelotor. However, the rational design of reversible covalent inhibitors remains difficult even when noncovalent inhibitors are available as a scaffold. Herein, we report chemically modified phage libraries, both linear and cyclic, that incorporate 2-acetylphenylboronic acid (APBA) as a warhead to bind lysines via reversible iminoboronate formation. To demonstrate their utility, these APBA-presenting phage libraries were screened against sortase A of Staphylococcus aureus, as well as the spike protein of SARS-CoV-2. For both protein targets, peptide ligands were readily identified with single-digit micromolar potency and excellent specificity, enabling live-cell sortase inhibition and highly sensitive spike protein detection, respectively. Furthermore, our structure-activity studies unambiguously demonstrate the benefit of the APBA warhead for protein binding. Overall, this contribution shows for the first time that reversible covalent inhibitors can be developed via phage display for a protein of interest. The phage display platform should be widely applicable to proteins including those involved in protein-protein interactions.
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Bacteriófagos , COVID-19 , Bacteriófagos/metabolismo , Humanos , Ligandos , Lisina/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Due to their equivalent lengths, δ-amino acids can serve as surrogates of α-dipeptides. However, δ-amino acids with proteinogenic side chains have not been well studied because of synthetic difficulties and because of their insolubility in organic solvents. Recently we reported the spontaneous supramolecular gelation of δ-peptides composed of ß(O)-δ5 -amino acids. Here, we report the incorporation of ß(O)-δ5 -amino acids as guests into the host α-helix, α,γ-hybrid peptide 12-helix and their single-crystal conformations. In addition, we studied the solution conformations of hybrid peptides composed of 1:1 alternating α and ß(O)-δ5 -amino acids. In contrast to the control α-helix structures, the crystal structure of peptides with ß(O)-δ5 -amino acids exhibit α-helical conformations consisting of both 13- and 10-membered H-bonds. The α,δ-hybrid peptide adopted mixed 13/11-helix conformation in solution with alternating H-bond directionality. Crystal-structure analysis revealed that the α,γ4 -hybrid peptide accommodated the guest ß(O)-δ5 -amino acid without significant deviation to the overall helix folding. The results reported here emphasize that ß(O)-δ5 -amino acids with proteinogenic side chains can be accommodated into regular α-helix or 12-helix as guests without much deviation of the overall helix folding of the peptides.
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Aminoácidos/química , Dipéptidos/química , Péptidos/química , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos MolecularesRESUMEN
The ordered supramolecular assemblies of short peptides have been recently gaining momentum due to their widespread applications in biology and materials sciences. In contrast to the α-peptides, limited success has been achieved from the backbone modified peptides. The proteolytic stability and conformational flexibility of the backbone modified peptides composed of ß-, γ-, and δ-amino acids can be explored to design ordered supramolecular gels and self-assembled materials. In this article, we are reporting the divergent supramolecular gels from a new class of short hybrid dipeptides composed of conformationally flexible new ß(O)-δ5-amino acids. The hybrid dipeptide composed of ß3- and ß(O)-δ5-Phe showed the formation of transparent gels from the aromatic solvents, while the dipeptide composed of ß(O)-δ5-Phe showed the thixotropic gel in phosphate buffered saline (PBS). In contrast, no organic or hydrogels were observed from the dipeptides composed of alternating α- and ß(O)-δ5-Phe as well as γ4 and ß(O)-δ5-Phe. The organogelation property displayed by the ß3,ß(O)-δ5-Phe dipeptide was further explored to recover the oil spills from the oil-water mixture. The thixotropic hydrogels displayed by the ß(O)-δ5, ß(O)-δ5-Phe dipeptide was further utilized as matrix along with cell culture medium to grow the cells in 2D-cell culture. Replacing the backbone -CH2- with "O" in the δ-Phe leads to the drastic change in the supramolecular behavior of δ-peptides. Overall, the short dipeptides from different backbone modified amino acids showed the divergent gelation properties and these properties can be further explored to design new functional biomaterials.
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Hidrogeles/química , Péptidos/química , Conformación Molecular , ProteolisisRESUMEN
Double helices are not common in polypeptides and proteins except in the peptide antibiotic gramicidinâ A and analogous l,d-peptides. In contrast to natural polypeptides, remarkable ß-double-helical structures from achiral γ-peptides built from α,ß-unsaturated γ-amino acids have been observed. The crystal structures suggest that they adopted parallel ß-double helical structures and these structures are stabilized by the interstrand backbone amide H-bonds. Furthermore, both NMR spectroscopy and fluorescence studies support the existence of double-helical conformations in solution. Although a variety of folded architectures featuring distinct H-bonds have been discovered from the ß- and γ-peptide foldamers, this is the first report to show that achiral γ-peptides can spontaneously intertwine into ß-double helical structures.
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Péptidos/química , Cristalografía por Rayos X , Gramicidina/química , Enlace de Hidrógeno , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica en Hélice alfa , Espectrometría de FluorescenciaRESUMEN
Widespread adoption of mirror-image biological systems presents difficulties in accessing the requisite D-protein substrates. In particular, mirror-image phage display has the potential for high-throughput generation of biologically stable macrocyclic D-peptide binders with potentially unique recognition modes but is hindered by the individualized optimization required for D-protein chemical synthesis. We demonstrate a general mirror-image phage display pipeline that utilizes automated flow peptide synthesis to prepare D-proteins in a single run. With this approach, we prepare and characterize 12 D-proteins - almost one third of all reported D-proteins to date. With access to mirror-image protein targets, we describe the successful discovery of six macrocyclic D-peptide binders: three to the oncoprotein MDM2, and three to the E3 ubiquitin ligase CHIP. Reliable production of mirror-image proteins can unlock the full potential of D-peptide drug discovery and streamline the study of mirror-image biology more broadly.
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Péptidos , Proteínas , Ligandos , Descubrimiento de DrogasRESUMEN
Multicyclic peptides are appealing candidates for peptide-based drug discovery. While various methods are developed for peptide cyclization, few allow multicyclization of native peptides. Herein we report a novel cross-linker DCA-RMR1, which elicits facile bicyclization of native peptides via N-terminus Cys-Cys cross-linking. The bicyclization is fast, affords quantitative conversion, and tolerates various side chain functionalities. Importantly, the resulting diazaborine linkage, while stable at a neutral pH, can readily reverse upon mild acidification to give pH-responsive peptides.
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Péptidos Cíclicos , Péptidos , Péptidos/química , Concentración de Iones de Hidrógeno , Ciclización , Péptidos Cíclicos/químicaRESUMEN
Directing Aß42 to adopt a conformation that is free from aggregation and cell toxicity is an attractive and viable strategy to design therapeutics for Alzheimer's disease. Over the years, extensive efforts have been made to disrupt the aggregation of Aß42 using various types of inhibitors but with limited success. Herein, we report the inhibition of aggregation of Aß42 and disintegration of matured fibrils of Aß42 into smaller assemblies by a 15-mer cationic amphiphilic peptide. The biophysical analysis comprising thioflavin T (ThT) mediated amyloid aggregation kinetic analysis, dynamic light scattering, ELISA, AFM, and TEM suggested that the peptide effectively disrupts Aß42 aggregation. The circular dichroism (CD) and 2D-NMR HSQC analysis reveal that upon interaction, the peptide induces a conformational change in Aß42 that is free from aggregation. Further, the cell assay experiments revealed that this peptide is non-toxic to cells and also rescues the cells from the toxicity of Aß42. Peptides with a shorter length displayed either weak or no inhibitory effect on Aß42 aggregation and cytotoxicity. These results suggest that the 15-residue cationic amphiphilic peptide reported here may serve as a potential therapeutic candidate for Alzheimer's disease.
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Unique ε-helical organizations (11-helices) from ß,γ-hybrid peptides composed of chiral ß3-amino acids along with achiral 3,3- or 4,4-dimethyl substituted γ-amino acids are disclosed.
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Synthesis and incorporation of a new amino acid with a nitroalkane side chain into peptides, in situ transformation of a nitroalkane side chain into nitrile oxide, and chemoselective 1,3-dipolar cycloaddition reactions between in situ generated nitrile oxide and different alkynes are reported. The nitroalkane-mediated nitrile oxide-alkyne cycloaddition was found to be orthogonal to the copper(I)-catalyzed azide-alkyne cycloaddition reaction. The combination of orthogonal nitrile oxide-alkyne and azide-alkyne cycloaddition reactions can be explored to tailor different 1,2,3-triazole and 3,5-isoxazoles, respectively, on the peptide backbone.
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Alquinos/química , Azidas , Catálisis , Cobre , Reacción de Cicloadición , Estructura Molecular , Nitrilos , Óxidos , PéptidosRESUMEN
Stimuli responsive controlled release from liposome based vesicles is a promising strategy for the site specific delivery of drugs. Herein, we report the design of pH sensitive coiled coils and their incorporation into the liposome as triggers for the controlled release of encapsulated drugs. The designed coiled coil peptides with the incorporation of environment sensitive fluorescent amino acids were found to be stable at physiological pH and unstructured while changing the pH of the environment to either acidic or basic. This pH dependent conformational switch of the coiled-coil polypeptides was exploited as triggers for the enhanced release of the encapsulated drug molecules from liposomes. The SEM, DLS and TEM analysis revealed the uniform morphology of the peptide liposome hybrid vesicles. Further, the drug encapsulated liposome internalization experiments with cancer cells revealed the enhanced release and accumulation of drugs in the acidic lysosomal compartments in comparison with liposomes without coiled coils.
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Péptidos , Animales , Línea Celular , Concentración de Iones de Hidrógeno , Liposomas , Ratones , Péptidos/química , Péptidos/farmacocinética , Péptidos/farmacologíaRESUMEN
We are reporting the influence of foldamer structures on their self-assembled architectures. In a sharp contrast to the ordered α,γ-hybrid 12-helix obtained from 1 : 1 alternating Aib and γ-Phe, the α,γ-hybrid peptides constituted with α-Phe and 4,4-dimethyl γ-amino acid (Aic) displayed the extended sheet type of conformations in solution and spontaneously self-assembled into thermally and proteolytically stable capsules. In contrast, the conformationally ordered 12-helix self-assembled into a three-dimensional supramolecular polyhedron.
RESUMEN
The stability and compatibility of designed coiled coil peptides towards the selective incorporation of γ(4)-amino acids at the hydrophobic positions of the heptad repeat are studied. Investigations reveal that the low thermal denaturation temperature of γ(4)-residue mutated coiled coils can be utilized as a mild hyperthermia trigger in liposomes.