Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome

2019 ◽  
Author(s):  
Patrick R. A. Zanon ◽  
Lisa Lewald ◽  
Stephan M. Hacker
Keyword(s):  
Binding Sites ◽  
Bacterial Resistance ◽  
Mevalonate Pathway ◽  
Rapid Development ◽  
High Reactivity ◽  
Functional Importance ◽  
Essential Proteins ◽  
Covalent Inhibitors ◽  
Druggable Targets ◽  
Covalent Ligands

Rapid development of bacterial resistance has led to an urgent need to find new druggable targets for antibiotics. In this context, residue-specific chemoproteomic approaches enable proteome-wide identification of binding sites for covalent inhibitors. Here, we describe isotopically labeled desthiobiotin azide (isoDTB) tags that are easily synthesized, shorten the chemoproteomic workflow and allow an increased coverage of cysteines in bacterial systems. We quantify 59% of all cysteines in essential proteins in <i>Staphylococcus aureus</i> and discover 88 cysteines with high reactivity, which correlates with functional importance. Furthermore, we identify 268 cysteines that are engaged by covalent ligands. We verify inhibition of HMG-CoA synthase, which will allow addressing the bacterial mevalonate pathway through a new target. Overall, a comprehensive map of the bacterial cysteinome is obtained, which will facilitate the development of antibiotics with novel modes-of-action.

Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome

2019 ◽  
Author(s):  
Patrick R. A. Zanon ◽  
Lisa Lewald ◽  
Stephan M. Hacker
Keyword(s):  
Binding Sites ◽  
Bacterial Resistance ◽  
Mevalonate Pathway ◽  
Rapid Development ◽  
High Reactivity ◽  
Functional Importance ◽  
Essential Proteins ◽  
Covalent Inhibitors ◽  
Druggable Targets ◽  
Covalent Ligands

Rapid development of bacterial resistance has led to an urgent need to find new druggable targets for antibiotics. In this context, residue-specific chemoproteomic approaches enable proteome-wide identification of binding sites for covalent inhibitors. Here, we describe isotopically labeled desthiobiotin azide (isoDTB) tags that are easily synthesized, shorten the chemoproteomic workflow and allow an increased coverage of cysteines in bacterial systems. We quantify 59% of all cysteines in essential proteins in <i>Staphylococcus aureus</i> and discover 88 cysteines with high reactivity, which correlates with functional importance. Furthermore, we identify 268 cysteines that are engaged by covalent ligands. We verify inhibition of HMG-CoA synthase, which will allow addressing the bacterial mevalonate pathway through a new target. Overall, a comprehensive map of the bacterial cysteinome is obtained, which will facilitate the development of antibiotics with novel modes-of-action.

Covalent-Fragment Screening of Brd4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites

2019 ◽  
Author(s):  
Michael Olp ◽  
Daniel Sprague ◽  
Stefan Kathman ◽  
Ziyang Xu ◽  
Alexandar Statsyuk ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Site ◽  
Binding Sites ◽  
Computational Prediction ◽  
Chemical Probes ◽  
Computational Docking ◽  
Fragment Screening ◽  
Covalent Inhibitors ◽  
Bet Protein ◽  
Proof Of Principle

<p>Brd4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as a promising epigenetic target in cancer and inflammatory disorders. All reported BET family ligands bind within the bromodomain acetyl-lysine binding sites and competitively inhibit BET protein interaction with acetylated chromatin. Alternative chemical probes that act orthogonally to the highly-conserved acetyl-lysine binding sites may exhibit selectivity within the BET family and avoid recently reported toxicity in clinical trials of BET bromodomain inhibitors. Here, we report the first identification of a ligandable site on a bromodomain outside the acetyl-lysine binding site. Inspired by our computational prediction of hotspots adjacent to non-homologous cysteine residues within the <i>C</i>-terminal Brd4 bromodomain (Brd4-BD2), we performed a mid-throughput mass spectrometry screen to identify cysteine-reactive fragments that covalently and selectively modify Brd4. Subsequent mass spectrometry, NMR and computational docking analyses of electrophilic fragment hits revealed a novel ligandable site near Cys356 that is unique to Brd4 among all human bromodomains. This site is orthogonal to the Brd4-BD2 acetyl-lysine binding site as Cys356 modification did not impact binding of the pan-BET bromodomain inhibitor JQ1 in fluorescence polarization assays. Finally, we tethered covalent fragments to JQ1 and performed NanoBRET assays to provide proof of principle that this orthogonal site can be covalently targeted in intact human cells. Overall, we demonstrate the potential of targeting sites orthogonal to bromodomain acetyl-lysine binding sites to develop bivalent and covalent inhibitors that displace Brd4 from chromatin.</p>

scholarly journals Identification of pyrogallol as a warhead in design of covalent inhibitors for the SARS-CoV-2 3CL protease

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Haixia Su ◽  
Sheng Yao ◽  
Wenfeng Zhao ◽  
Yumin Zhang ◽  
Jia Liu ◽  
...  
Keyword(s):  
Theoretical Calculations ◽  
Cysteine Proteinase ◽  
Covalent Binding ◽  
Food Sources ◽  
Binding Mechanism ◽  
Covalent Inhibitors ◽  
Catalytic Cysteine ◽  
3Cl Protease ◽  
Covalent Ligands ◽  
Covalent Inhibitor

AbstractThe ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urgently needs an effective cure. 3CL protease (3CLpro) is a highly conserved cysteine proteinase that is indispensable for coronavirus replication, providing an attractive target for developing broad-spectrum antiviral drugs. Here we describe the discovery of myricetin, a flavonoid found in many food sources, as a non-peptidomimetic and covalent inhibitor of the SARS-CoV-2 3CLpro. Crystal structures of the protease bound with myricetin and its derivatives unexpectedly revealed that the pyrogallol group worked as an electrophile to covalently modify the catalytic cysteine. Kinetic and selectivity characterization together with theoretical calculations comprehensively illustrated the covalent binding mechanism of myricetin with the protease and demonstrated that the pyrogallol can serve as an electrophile warhead. Structure-based optimization of myricetin led to the discovery of derivatives with good antiviral activity and the potential of oral administration. These results provide detailed mechanistic insights into the covalent mode of action by pyrogallol-containing natural products and a template for design of non-peptidomimetic covalent inhibitors against 3CLpros, highlighting the potential of pyrogallol as an alternative warhead in design of targeted covalent ligands.

scholarly journals Analysis of functional importance of binding sites in the Drosophila gap gene network model

BMC Genomics ◽  
2015 ◽  
Vol 16 (Suppl 13) ◽  
pp. S7 ◽  
Author(s):  
Konstantin Kozlov ◽  
Vitaly V Gursky ◽  
Ivan V Kulakovskiy ◽  
Arina Dymova ◽  
Maria Samsonova
Keyword(s):  
Network Model ◽  
Binding Sites ◽  
Gene Network ◽  
Functional Importance ◽  
Gap Gene

scholarly journals Multi-modal adaptor-clathrin contacts drive coated vesicle assembly

2021 ◽  
Author(s):  
Sarah M Smith ◽  
Gabrielle Larocque ◽  
Katherine M Wood ◽  
Kyle L Morris ◽  
Alan M Roseman ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Binding Sites ◽  
Potential Role ◽  
Fundamental Property ◽  
Structural Knowledge ◽  
Coated Vesicle ◽  
Functional Importance ◽  
Coated Pits ◽  
Interaction Sites ◽  
Cross Links

Clathrin-coated pits are formed by the recognition of membrane and cargo by the heterotetrameric AP2 complex and the subsequent recruitment of clathrin triskelia. A potential role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the b2 subunit of AP2, and several binding sites on b2 and on the clathrin heavy chain have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of b2 hinge and appendage (b2HA) domains. We find that the b2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, b2-appendage cross-links two adjacent terminal domains from different triskelia below the vertex. Functional analysis of b2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the ''sandwich site'' on the appendage, with the appendage ''platform site'' having less importance. From these studies and the work of others, we propose that b2-appendage binding to more than one clathrin triskelion is a key feature of the system and likely explains why clathrin assembly is driven by AP2.

scholarly journals Ginger and Its Derivatives as Promising Alternatives to Antibiotics in Poultry Feed

Animals ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 452 ◽  
Author(s):  
Mohamed E. Abd El-Hack ◽  
Mahmoud Alagawany ◽  
Hazem Shaheen ◽  
Dalia Samak ◽  
Sarah I. Othman ◽  
...  
Keyword(s):  
Bacterial Resistance ◽  
Rapid Development ◽  
Feed Additives ◽  
Enteric Pathogens ◽  
Poultry Feed ◽  
Poultry Production ◽  
Quality Health ◽  
Immune Stimulants ◽  
Growth Promoting ◽  
And Control

Poultry enterprises have sustained rapid development through the last three decennaries. For which reason, higher utilization of antibacterial, either as therapeutic or growth promoting agents, has been accepted. Owing to the concern of developing bacterial resistance among populations towards antibiotic generations, accumulation of antibacterial remaining’s in chicken products and elevating shopper request for outcomes without antibacterial remaining’s, looking for unconventional solutions that could exchange antibacterial without influencing productiveness or product characters. Using natural alternatives including ginger, garlic prebiotics, organic acids, plant extracts, etheric oils and immune stimulants have been applied to advance the performance, hold poultry productiveness, prevent and control the enteric pathogens and minimize the antibacterial utilization in the poultry production in recent years. The use of a single replacement or ideal assemblage of different choices besides good supervision and livestock welfare may play a basic role in maximizing benefits and preserving poultry productiveness. The object of this review was to support an outline of the recent knowledge on the use of the natural replacements (ginger and its derivatives) in poultry feed as feed additives and their effects on poultry performance, egg and meat quality, health as well as the economic efficiency.

Cysteine Mapping in Conformationally Distinct Kinase Nucleotide Binding Sites: Application to the Design of Selective Covalent Inhibitors

2011 ◽  
Vol 54 (5) ◽  
pp. 1347-1355 ◽  
Author(s):  
Emeline Leproult ◽  
Sofia Barluenga ◽  
Dino Moras ◽  
Jean-Marie Wurtz ◽  
Nicolas Winssinger
Keyword(s):  
Binding Sites ◽  
Nucleotide Binding ◽  
Nucleotide Binding Sites ◽  
Covalent Inhibitors

scholarly journals Over-activation of a nonessential bacterial protease DegP as an antibiotic strategy

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Hyunjin Cho ◽  
Yuri Choi ◽  
Kyungjin Min ◽  
Jung Bae Son ◽  
Hyojin Park ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Binding Sites ◽  
Antibiotic Development ◽  
Essential Proteins ◽  
Bacterial Protease ◽  
Novel Targets ◽  
Essential Enzyme ◽  
Substrate Binding Sites ◽  
Antibiotic Discovery

AbstractRising antibiotic resistance urgently begs for novel targets and strategies for antibiotic discovery. Here, we report that over-activation of the periplasmic DegP protease, a member of the highly conserved HtrA family, can be a viable strategy for antibiotic development. We demonstrate that tripodal peptidyl compounds that mimic DegP-activating lipoprotein variants allosterically activate DegP and inhibit the growth of an Escherichia coli strain with a permeable outer membrane in a DegP-dependent fashion. Interestingly, these compounds inhibit bacterial growth at a temperature at which DegP is not essential for cell viability, mainly by over-proteolysis of newly synthesized proteins. Co-crystal structures show that the peptidyl arms of the compounds bind to the substrate-binding sites of DegP. Overall, our results represent an intriguing example of killing bacteria by activating a non-essential enzyme, and thus expand the scope of antibiotic targets beyond the traditional essential proteins or pathways.

Covalent-Fragment Screening of Brd4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites

2019 ◽  
Author(s):  
Michael Olp ◽  
Daniel Sprague ◽  
Stefan Kathman ◽  
Ziyang Xu ◽  
Alexandar Statsyuk ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Site ◽  
Binding Sites ◽  
Computational Prediction ◽  
Chemical Probes ◽  
Computational Docking ◽  
Fragment Screening ◽  
Covalent Inhibitors ◽  
Bet Protein ◽  
Proof Of Principle

<p>Brd4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as a promising epigenetic target in cancer and inflammatory disorders. All reported BET family ligands bind within the bromodomain acetyl-lysine binding sites and competitively inhibit BET protein interaction with acetylated chromatin. Alternative chemical probes that act orthogonally to the highly-conserved acetyl-lysine binding sites may exhibit selectivity within the BET family and avoid recently reported toxicity in clinical trials of BET bromodomain inhibitors. Here, we report the first identification of a ligandable site on a bromodomain outside the acetyl-lysine binding site. Inspired by our computational prediction of hotspots adjacent to non-homologous cysteine residues within the <i>C</i>-terminal Brd4 bromodomain (Brd4-BD2), we performed a mid-throughput mass spectrometry screen to identify cysteine-reactive fragments that covalently and selectively modify Brd4. Subsequent mass spectrometry, NMR and computational docking analyses of electrophilic fragment hits revealed a novel ligandable site near Cys356 that is unique to Brd4 among all human bromodomains. This site is orthogonal to the Brd4-BD2 acetyl-lysine binding site as Cys356 modification did not impact binding of the pan-BET bromodomain inhibitor JQ1 in fluorescence polarization assays. Finally, we tethered covalent fragments to JQ1 and performed NanoBRET assays to provide proof of principle that this orthogonal site can be covalently targeted in intact human cells. Overall, we demonstrate the potential of targeting sites orthogonal to bromodomain acetyl-lysine binding sites to develop bivalent and covalent inhibitors that displace Brd4 from chromatin.</p>

Sign in / Sign up

Export Citation Format

Share Document