Screening ubiquitin specific protease activities using chemically synthesized ubiquitin and ubiquitinated peptides

Bacchi, Marine; Fould, Benjamin; Jullian, Magali; Kreiter, Aude; Maurras, Amélie; Nosjean, Olivier; Coursindel, Thibault; Puget, Karine; Ferry, Gilles; Boutin, Jean A.

doi:10.1016/j.ab.2016.12.014

Cited by 5 publications

(8 citation statements)

References 43 publications

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“…Roughly half a decade ago, we decided to enter a series of programs aimed at synthesizing a series of proteins of growing length from ubiquitin and calstabin to quinone reductase (which contains 226 amino acids and with which we failed). The difficulties encountered in developing a universal methodology to obtain such proteins, most of which were enzymes, led us to limit ourselves to ~200 amino acids.…”

Section: Peptides? Which Structure Which Refolding?mentioning

confidence: 99%

“…However, in contrast to recombinant expression, in peptide synthesis, no chaperone is present to assist in the refolding of the nascent peptide chain. Thus, refolding is either spontaneous (e.g., ubiquitin) or a long and partial process in which the end product must be thoroughly characterized. As a particularly interesting example, the recent paper by Kuroha et al on a new lasso peptide essentially aimed at describing the structure of the material.…”

Section: Peptides? Which Structure Which Refolding?mentioning

confidence: 99%

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General lack of structural characterization of chemically synthesized long peptides

Boutin

Tartar²,

Dorsselaer

et al. 2019

Protein Science

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Many peptide chemistry scientists have been reporting extremely interesting work on the basis of chemical peptides for which the only characterization was their purity, mass, and biological activity. It seems slightly overenthusiastic, as many of these structures should be thoroughly characterized first to demonstrate the uniqueness of the structure, as opposed to the uniqueness of the sequence. Among the peptides of identical sequences in the final chemical preparation, what amount of well-folded peptide supports the measured activity? The activity of a peptide preparation cannot prove the purity of the desired peptide. Therefore, greater care should be taken in characterizing peptides, particularly those coming from chemical synthesis. At a time when the pharmaceutical industry is changing its paradigm by moving substantially from small molecules to biologics to better serve patients' needs, it is important to understand the limitations of the descriptions of these products and to start to apply the same "good laboratory practices" to our peptide research. Here, we attempt to delineate how synthetic peptides are described and characterized and what will be needed to describe them in regards to how they are well-folded and homogeneous in their tertiary structure. Older studies were done when the tools were not yet discovered, but more recent publications are still lacking proper descriptions of these peptides. Modern tools of analysis are capable of segregating folded and unfolded peptides, even if the preparation is biologically active.

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Section: Peptides? Which Structure Which Refolding?mentioning

confidence: 99%

Section: Peptides? Which Structure Which Refolding?mentioning

confidence: 99%

General lack of structural characterization of chemically synthesized long peptides

Boutin

Tartar²,

Dorsselaer

et al. 2019

Protein Science

Self Cite

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“…Our first step is towards an orthogonal confirmation of the activity of the compound. Obviously, whenever an enzyme is concerned, it is 'quite easy' to invent or copy an alternative assay that engages different physical parameters, going from the old, global, radioactive phosphocellulose paper-based detection assay [112] to a more specific radioactivity-based assay [113] for a kinase, for instance; trying to go from a protein-based phosphorylation assay [114] to a more specific peptide-based assay [66] or lastly, going from an antibody-based assay to a more specific ubiquitinated peptide-based assay [115]. There are multiple examples of such orthogonal assays, such as the use of HPLC, fluorescence polarization, mass spectrometry, and fluorescence resonance energy transfer (FRET) technology.…”

Section: Confirming the Hit Activitymentioning

confidence: 99%

“…In doing so, we not only identified and incorporated strong scientists but also could assign new technologies to categories such as 'dream', 'usable soon', 'needs more time', etc. Among the discoveries that we have published are the ligase use for in cellulo transformation of proteins [163], including antibodies; cellular imaging using large synchrotron instruments; synthetic proteins that can be as active as the recombinants [141]; and synthesis of proteins and peptides modified by ubiquitination the way it happens in the cell (as opposed to what was commercially available at that time) [115]. In addition, we started our first trials in thermodynamics, structural biology, new crystallization techniques, native mass spectrometry for structural biology approaches [152] and for target-molecule relationships, and the use of human stem cells as an alternative to rat cardiomyocytes in first culture, among other initiatives.…”

Section: Outside the Boxmentioning

confidence: 99%

On the Organization of a Drug Discovery Platform

Nosjean¹,

Ferry²

2018

Drug Discovery - Concepts to Market

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Some of the most exciting parts of work in the pharmaceutical industry are the steps leading up to drug discovery. This process can be oversimplified by describing it as a screening campaign involving the systematic testing of many compounds in a test relevant to a given pathology. This naïve description takes place without taking into consideration the numerous key steps that led up to the screening or the steps that might follow. The present chapter describes this whole process as it was conducted in our company during our early drug discovery activities. First, the purpose of the procedures is described and rationalized. Next follows a series of mostly published examples from our own work illustrating the various steps of the process from cloning to biophysics, including expression systems and membrane-bound protein purifications. We believe that what is described here presents an example of how pharmaceutical industry research can organize its platform(s) when the goal is to find and qualify a new preclinical drug candidate using cutting-edge technologies and a lot of hard work.

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“…We saw in this area the possibility to develop a strategy by which the scaffold would be synthesized chemically, permitting the introduction in the sequence of one or several exotic amino acids. We applied those approaches to several proteins (ubiquitin or an enzyme: calstabin) and were interested to extend this strategy to VHHs. Indeed, the size of those proteins permitted to anticipate two things: (a) the feasibility of their production by recombinant host(s) and (b) the possibility to reach those structures by a total chemical synthesis, even at an industrial scale.…”

Section: Introductionmentioning

confidence: 99%

VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

Hartmann

Botzanowski

Galibert³

et al. 2019

Protein Science

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In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody‐like protein, using an anti‐HER2 VHH as a model. The total chemical synthesis of the 125 amino‐acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its “antigen.” Using this combination of orthogonal techniques, it was possible to show that the three VHHs—whether synthetic or recombinant ones—were properly and similarly folded and recognized the “antigen” HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH‐drug conjugates.

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Screening ubiquitin specific protease activities using chemically synthesized ubiquitin and ubiquitinated peptides

Cited by 5 publications

References 43 publications

General lack of structural characterization of chemically synthesized long peptides

General lack of structural characterization of chemically synthesized long peptides

On the Organization of a Drug Discovery Platform

VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

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