Current and prospective applications of non-proteinogenic amino acids in profiling of proteases substrate specificity

2012 ◽  
Vol 393 (9) ◽  
pp. 843-851 ◽  
Author(s):  
Paulina Kasperkiewicz ◽  
Anna D. Gajda ◽  
Marcin Drąg
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Posttranslational Modifications ◽  
Cleavage Site ◽  
Point Of View ◽  
Biological Functions ◽  
Diverse Families ◽  
Protease Substrate ◽  
Endogenous Substrates

Abstract Proteases recognize their endogenous substrates based largely on a sequence of proteinogenic amino acids that surrounds the cleavage site. Currently, several methods are available to determine protease substrate specificity based on approaches employing proteinogenic amino acids. The knowledge about the specificity of proteases can be significantly extended by application of structurally diverse families of non-proteinogenic amino acids. From a chemical point of view, this information may be used to design specific substrates, inhibitors, or activity-based probes, while biological functions of proteases, such as posttranslational modifications can also be investigated. In this review, we discuss current and prospective technologies for application of non-proteinogenic amino acids in protease substrate specificity profiling.

scholarly journals Specificity of human rhinovirus 2Apro is determined by combined spatial properties of four cleavage site residues

2013 ◽  
Vol 94 (7) ◽  
pp. 1535-1546 ◽  
Author(s):  
David Neubauer ◽  
Martina Aumayr ◽  
Irene Gösler ◽  
Tim Skern
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Cleavage Site ◽  
Antiviral Agents ◽  
Genetic Group ◽  
Human Rhinovirus ◽  
C Terminus ◽  
Group B

The 2A proteinase (2Apro) of human rhinoviruses cleaves the virally encoded polyprotein between the C terminus of VP1 and its own N terminus. Poor understanding of the 2Apro substrate specificity of this enzyme has hampered progress in developing inhibitors that may serve as antiviral agents. We show here that the 2Apro of human rhinovirus (HRV) 1A and 2 (rhinoviruses from genetic group A) cannot self-process at the HRV14 (a genetic group B rhinovirus) cleavage site. When the amino acids in the cleavage site of HRV2 2Apro (Ile-Ile-Thr-Thr-Ala*Gly-Pro-Ser-Asp) were singly or doubly replaced with the corresponding HRV14 residues (Asp-Ile-Lys-Ser-Tyr*Gly-Leu-Gly-Pro) at positions from P3 to P2′, HRV1A and HRV2 2Apro cleavage took place at WT levels. However, when three or more positions of the HRV1A or 2 2Apro were substituted (e.g. at P2, P1 and P2′), cleavage in vitro was essentially eliminated. Introduction of the full HRV14 cleavage site into a full-length clone of the HRV1A and transfection of HeLa cells with a transcribed RNA did not give rise to viable virus. In contrast, revertant viruses bearing cysteine at the P1 position or proline at P2′ were obtained when an RNA bearing the three inhibitory amino acids was transfected. Reversions in the enzyme affecting substrate specificity were not found in any of the in vivo experiments. Modelling of oligopeptide substrates onto the structure of HRV2 2Apro revealed no appreciable differences in residues of HRV2 and HRV14 in the respective substrate binding sites, suggesting that the overall shape of the substrate is important in determining binding efficiency.

Local and spatial factors determining HIV-1 protease substrate recognition

2001 ◽  
Vol 358 (2) ◽  
pp. 505-510 ◽  
Author(s):  
Stephane HAZEBROUCK ◽  
Valerie MACHTELINCKX-DELMAS ◽  
Jean-Jacques KUPIEC ◽  
Pierre SONIGO
Keyword(s):  
Amino Acids ◽  
Cleavage Site ◽  
Insertional Mutagenesis ◽  
Substrate Recognition ◽  
Enzymic Activity ◽  
Local Sequence ◽  
Protease Substrate ◽  
The Stability ◽  
Potential Cleavage Site ◽  
Hiv 1

Insertional mutagenesis of the Escherichia coli thymidylate synthase (TS) was used to address substrate recognition of HIV-1 protease in a well characterized structural context. By modifying the TS conformation while maintaining its enzymic activity, we investigated the influence of protein folding on protease–substrate recognition. A slight destabilization of the TS structure permitted the cleavage of a target site, which was resistant in the native TS. This result supports a dynamic interpretation of HIV-1 protease specificity. Exposure time of the potential cleavage site, which depends on the stability of the global conformation, must be compatible with the cleavage kinetics, which are determined by the local sequence. Cleavage specificity has been described as the consequence of cumulative interactions, globally favourable, between at least six amino acids around the cleavage site. To investigate influence of local sequence, we introduced insertions of variable lengths in two exposed loops of the TS. In both environments, insertion of only two amino acids could determine specific cleavage. We then inserted libraries of dipeptides naturally cleaved by the HIV-1 protease in order to assess the limitations of established classifications of substrates in different conformational contexts.

In the Pursuit of (Ald)Imine Surrogates for the Direct Asymmetric Synthesis of Non-Proteinogenic α-Amino Acids

Synthesis ◽  
2021 ◽  
Author(s):  
Stéphane P. Roche
Keyword(s):  
Amino Acids ◽  
Asymmetric Synthesis ◽  
Nucleophilic Substitution ◽  
Posttranslational Modifications ◽  
Kinetic Studies ◽  
Structural Diversity ◽  
Building Blocks ◽  
Bioactive Molecules ◽  
Biological Functions ◽  
Imino Esters

Nature remarkably employs posttranslational modifications of the 20 canonical α-amino acids to devise a far larger structural, conformational, and functional diversity found in non-proteinogenic amino acids (NPAAs) which ultimately translates into a plethora of complex biological functions. Synthetic chemists are continuously trying to reproduce and even extrapolate the repertoire of NPAA building blocks to build structural diversity into bioactive molecules and materials. The direct asymmetric functionalization of α-imino esters represents one of the most robust and attractive routes to NPAAs. This review summarizes the most prominent examples of bench-stable (ald)imine surrogates exploited for the synthesis of NPAAs including our most recent results in the nucleophilic substitution of α-haloglycines and other α-haloaminals. A synopsis of kinetic studies, reaction optimizations, and enantioselective catalytic methods is also presented.

scholarly journals On the Use of the Discrete Constant pH Molecular Dynamics to Describe the Conformational Space of Peptides

Polymers ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 99
Author(s):  
Cristian Privat ◽  
Sergio Madurga ◽  
Francesc Mas ◽  
Jaime Rubio-Martínez
Keyword(s):  
Molecular Dynamics ◽  
Amino Acids ◽  
Md Simulations ◽  
Point Of View ◽  
Conformational Space ◽  
Conformational Sampling ◽  
Protonation State ◽  
Constant Ph ◽  
Biochemical Systems ◽  
Constant Ph Molecular Dynamics

Solvent pH is an important property that defines the protonation state of the amino acids and, therefore, modulates the interactions and the conformational space of the biochemical systems. Generally, this thermodynamic variable is poorly considered in Molecular Dynamics (MD) simulations. Fortunately, this lack has been overcome by means of the Constant pH Molecular Dynamics (CPHMD) methods in the recent decades. Several studies have reported promising results from these approaches that include pH in simulations but focus on the prediction of the effective pKa of the amino acids. In this work, we want to shed some light on the CPHMD method and its implementation in the AMBER suitcase from a conformational point of view. To achieve this goal, we performed CPHMD and conventional MD (CMD) simulations of six protonatable amino acids in a blocked tripeptide structure to compare the conformational sampling and energy distributions of both methods. The results reveal strengths and weaknesses of the CPHMD method in the implementation of AMBER18 version. The change of the protonation state according to the chemical environment is presumably an improvement in the accuracy of the simulations. However, the simulations of the deprotonated forms are not consistent, which is related to an inaccurate assignment of the partial charges of the backbone atoms in the CPHMD residues. Therefore, we recommend the CPHMD methods of AMBER program but pointing out the need to compare structural properties with experimental data to bring reliability to the conformational sampling of the simulations.

scholarly journals Recuperative Amino Acids Separation through Cellulose Derivative Membranes with Microporous Polypropylene Fiber Matrix

Membranes ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 429
Author(s):  
Aurelia Cristina Nechifor ◽  
Andreia Pîrțac ◽  
Paul Constantin Albu ◽  
Alexandra Raluca Grosu ◽  
Florina Dumitru ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cellulose Acetate ◽  
Polypropylene Fiber ◽  
Point Of View ◽  
Hydroxyethyl Cellulose ◽  
Separation Performance ◽  
Thermal Gravimetric Analyzer ◽  
Microfiltration Membranes ◽  
Ft Ir ◽  
Structural Point

The separation, concentration and transport of the amino acids through membranes have been continuously developed due to the multitude of interest amino acids of interest and the sources from which they must be recovered. At the same time, the types of membranes used in the sepa-ration of the amino acids are the most diverse: liquids, ion exchangers, inorganic, polymeric or composites. This paper addresses the recuperative separation of three amino acids (alanine, phe-nylalanine, and methionine) using membranes from cellulosic derivatives in polypropylene ma-trix. The microfiltration membranes (polypropylene hollow fibers) were impregnated with solu-tions of some cellulosic derivatives: cellulose acetate, 2-hydroxyethyl-cellulose, methyl 2-hydroxyethyl-celluloseand sodium carboxymethyl-cellulose. The obtained membranes were characterized in terms of the separation performance of the amino acids considered (retention, flux, and selectivity) and from a morphological and structural point of view: scanning electron microscopy (SEM), high resolution SEM (HR-SEM), Fourier transform infrared spectroscopy (FT-IR), energy dispersive spectroscopy (EDS) and thermal gravimetric analyzer (TGA). The re-sults obtained show that phenylalanine has the highest fluxes through all four types of mem-branes, followed by methionine and alanine. Of the four kinds of membrane, the most suitable for recuperative separation of the considered amino acids are those based on cellulose acetate and methyl 2-hydroxyethyl-cellulose.

Designed Synthesis of Diversely Substituted Hydantoins and Hydantoin-Based Hybrid Molecules: A Personal Account

Synlett ◽  
2021 ◽  
Author(s):  
Vinod Kumar
Keyword(s):  
Clinical Applications ◽  
Point Of View ◽  
Biological Functions ◽  
Personal Account ◽  
Pharmacological Activities ◽  
Structural Modifications ◽  
Hybrid Molecules ◽  
Biological Target ◽  
Designed Synthesis ◽  
Mixed Mechanism

Hydantoin and its analogs such as thiohydantoin and iminohydantoin have received substantial attention both from a chemical and biological point of view. Several compounds of this class have shown useful pharmacological activities such as anticonvulsant, antitumor, antiarrhythmic, herbicidal, and others that lead in some cases to clinical applications. Because of broad-spectrum activities, intensive research efforts have been dedicated in industry and academia to the synthesis and structural modifications of hydantoin and its derivatives. Realizing the importance of hydantoin in organic and medicinal chemistry, we also initiated a research program to successfully design and develop the new routes/methods resulting in the formation of hydantoin, thiohydantoin, and iminohydantoin substituted at different positions particularly at the N-1 position without following protection-deprotection strategy. Given the fact that the combination of two or more pharmacophoric groups may lead to hybrid molecules which result in a mixed mechanism of action on the biological target. We, therefore, further extended the developed strategy for the synthesis of new types of hydantoin-based hybrid molecules by combining hydantoin with a triazole, isoxazoline, and phosphate scaffolds as another pharmacophoric group to exploit diverse biological functions.

Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays

PROTEOMICS ◽  
2005 ◽  
Vol 5 (5) ◽  
pp. 1292-1298 ◽  
Author(s):  
Dhaval N. Gosalia ◽  
Cleo M. Salisbury ◽  
Dustin J. Maly ◽  
Jonathan A. Ellman ◽  
Scott L. Diamond
Keyword(s):  
Substrate Specificity ◽  
Serine Protease ◽  
Solution Phase ◽  
Peptide Microarrays ◽  
Protease Substrate ◽  
Fluorogenic Peptide
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