scholarly journals A phage display approach to identify highly selective covalent binders

2019 ◽  
Author(s):  
Shiyu Chen ◽  
Matthew Bogyo
Keyword(s):  
Phage Display ◽  
Cyclic Peptides ◽  
Covalent Binding ◽  
Screening Method ◽  
Covalent Bond ◽  
Covalent Modification ◽  
Linear Peptide ◽  
Binding Inhibition ◽  
Covalent Bond Formation ◽  
Activity Of Enzymes

AbstractMolecules that bind macromolecular targets through direct covalent modification have found widespread applications as activity-based probes (ABPs) and as irreversible drugs. Covalent binders can be used to dynamically monitor the activity of enzymes in complex cellular environments, identify targets and induce permanent binding/inhibition of therapeutically important biomolecules. However, the general reactivity of the electrophiles needed for covalent bond formation makes control of selectivity difficult. There is currently no rapid, robust and unbiased screening method to identify new classes of covalent binding ligands from highly diverse pools of candidate molecules. Here we describe the development of a phage display method to screen for highly selective covalent binding ligands. This approach makes use of a reactive linker to form cyclic peptides on the phage surface while simultaneously introducing an electrophilic ‘warhead’ to covalently react with a nucleophile on the target. Using this approach, we identified cyclic peptides that selectively and irreversibly inhibited a cysteine protease with nanomolar potency, exceptional specificity and increased serum stability compared to a linear peptide containing the same electrophile. This approach should enable rapid, unbiased screening to identify new classes of highly selective covalent binding ligands for diverse molecular targets.

scholarly journals Mechanism of Covalent Binding of Ibrutinib to Bruton’s Tyrosine Kinase revealed by QM/MM Calculations

2020 ◽  
Author(s):  
Angus Voice ◽  
Gary Tresadern ◽  
Rebecca Twidale ◽  
Herman Van Vlijmen ◽  
Adrian Mulholland
Keyword(s):  
Tyrosine Kinase ◽  
Covalent Binding ◽  
Bond Formation ◽  
Covalent Bond ◽  
Covalent Modification ◽  
Mechanistic Study ◽  
Bruton’S Tyrosine Kinase ◽  
Bruton's Tyrosine Kinase ◽  
Covalent Inhibitors ◽  
Covalent Bond Formation

<p>Ibrutinib is the first covalent inhibitor of Bruton’s tyrosine kinase (BTK) to be used in the treatment of B-cell cancers. Understanding the mechanism of covalent inhibition is crucial for the design of safer and more selective covalent inhibitors that target BTK. There are questions surrounding the precise mechanism of covalent bond formation in BTK as there is no appropriate active site residue that can act as a base to deprotonate the cysteine thiol prior to covalent bond formation. To address this, we have investigated several mechanistic pathways of covalent modification of C481 in BTK by ibrutinib using QM/MM reaction simulations. The lowest energy pathway we identified involves a direct proton transfer from C481 to the acrylamide warhead in ibrutinib, followed by covalent bond formation to form an enol intermediate. There is a subsequent rate-limiting keto-enol tautomerisation step (DG<sup>‡</sup>=10.5 kcal mol<sup>-1</sup>) to reach the inactivated BTK/ibrutinib complex. Our results represent the first mechanistic study of BTK inactivation by ibrutinib to consider multiple mechanistic pathways. These findings should aid in the design of covalent drugs that target BTK and related proteins. </p>

scholarly journals Mechanism of Covalent Binding of Ibrutinib to Bruton’s Tyrosine Kinase revealed by QM/MM Calculations

2020 ◽  
Author(s):  
Angus Voice ◽  
Gary Tresadern ◽  
Rebecca Twidale ◽  
Herman Van Vlijmen ◽  
Adrian Mulholland
Keyword(s):  
Tyrosine Kinase ◽  
Covalent Binding ◽  
Bond Formation ◽  
Covalent Bond ◽  
Covalent Modification ◽  
Mechanistic Study ◽  
Bruton’S Tyrosine Kinase ◽  
Bruton's Tyrosine Kinase ◽  
Covalent Inhibitors ◽  
Covalent Bond Formation

<p>Ibrutinib is the first covalent inhibitor of Bruton’s tyrosine kinase (BTK) to be used in the treatment of B-cell cancers. Understanding the mechanism of covalent inhibition is crucial for the design of safer and more selective covalent inhibitors that target BTK. There are questions surrounding the precise mechanism of covalent bond formation in BTK as there is no appropriate active site residue that can act as a base to deprotonate the cysteine thiol prior to covalent bond formation. To address this, we have investigated several mechanistic pathways of covalent modification of C481 in BTK by ibrutinib using QM/MM reaction simulations. The lowest energy pathway we identified involves a direct proton transfer from C481 to the acrylamide warhead in ibrutinib, followed by covalent bond formation to form an enol intermediate. There is a subsequent rate-limiting keto-enol tautomerisation step (DG<sup>‡</sup>=10.5 kcal mol<sup>-1</sup>) to reach the inactivated BTK/ibrutinib complex. Our results represent the first mechanistic study of BTK inactivation by ibrutinib to consider multiple mechanistic pathways. These findings should aid in the design of covalent drugs that target BTK and related proteins. </p>

scholarly journals A Perspective on the Kinetics of Covalent and Irreversible Inhibition

2016 ◽  
Vol 22 (1) ◽  
pp. 3-20 ◽  
Author(s):  
John M. Strelow
Keyword(s):  
Covalent Bond ◽  
Pharmacokinetic Profile ◽  
Covalent Modification ◽  
Target Protein ◽  
Biochemical Assays ◽  
Direct Exposure ◽  
Potent Inhibition ◽  
Covalent Inhibitors ◽  
Covalent Bond Formation

The clinical and commercial success of covalent drugs has prompted a renewed and more deliberate pursuit of covalent and irreversible mechanisms within drug discovery. A covalent mechanism can produce potent inhibition in a biochemical, cellular, or in vivo setting. In many cases, teams choose to focus on the consequences of the covalent event, defined by an IC50 value. In a biochemical assay, the IC50 may simply reflect the target protein concentration in the assay. What has received less attention is the importance of the rate of covalent modification, defined by kinact/KI. The kinact/KI is a rate constant describing the efficiency of covalent bond formation resulting from the potency (KI) of the first reversible binding event and the maximum potential rate (kinact) of inactivation. In this perspective, it is proposed that the kinact/KI should be employed as a critical parameter to identify covalent inhibitors, interpret structure-activity relationships (SARs), translate activity from biochemical assays to the cell, and more accurately define selectivity. It is also proposed that a physiologically relevant kinact/KI and an (unbound) AUC generated from a pharmacokinetic profile reflecting direct exposure of the inhibitor to the target protein are two critical determinants of in vivo covalent occupancy. A simple equation is presented to define this relationship and improve the interpretation of covalent and irreversible kinetics.

scholarly journals Modeling covalent-modifier drugs

2017 ◽  
Author(s):  
Ernest Awoonor-Williams ◽  
Andrew G Walsh ◽  
Christopher N Rowley
Keyword(s):  
Computer Modeling ◽  
Covalent Binding ◽  
Covalent Bond ◽  
Bioinformatic Analysis ◽  
Covalent Modification ◽  
Binding Affinities ◽  
Chemical Bonds ◽  
Irreversible Inhibition ◽  
Large Sets ◽  
Covalent Inhibitor

In this review, we present a summary of how computer modeling has been used in the development of covalent modifier drugs. Covalent modifier drugs bind by forming a chemical bond with their target. This covalent binding can improve the selectivity of the drug for a target with complementary reactivity and result in increased binding affinities due to the strength of the covalent bond formed. In some cases, this results in irreversible inhibition of the target, but some targeted covalent inhibitor (TCI) drugs bind covalently but reversibly. Computer modeling is widely used in drug discovery, but different computational methods must be used to model covalent modifiers because of the chemical bonds formed. Structural and bioinformatic analysis has identified sites of modification that could yield selectivity for a chosen target. Docking methods, which are used to rank binding poses of large sets of inhibitors, have been augmented to support the formation of protein--ligand bonds and are now capable of predicting the binding pose of covalent modifiers accurately. The pKa's of amino acids can be calculated in order to assess their reactivity towards electrophiles. QM/MM methods have been used to model the reaction mechanisms of covalent modification. The continued development of these tools will allow computation to aid in the development of new covalent modifier drugs.

scholarly journals Modeling covalent-modifier drugs

2017 ◽  
Author(s):  
Ernest Awoonor-Williams ◽  
Andrew G Walsh ◽  
Christopher N Rowley
Keyword(s):  
Computer Modeling ◽  
Covalent Binding ◽  
Covalent Bond ◽  
Bioinformatic Analysis ◽  
Covalent Modification ◽  
Binding Affinities ◽  
Chemical Bonds ◽  
Irreversible Inhibition ◽  
Large Sets ◽  
Covalent Inhibitor

In this review, we present a summary of how computer modeling has been used in the development of covalent modifier drugs. Covalent modifier drugs bind by forming a chemical bond with their target. This covalent binding can improve the selectivity of the drug for a target with complementary reactivity and result in increased binding affinities due to the strength of the covalent bond formed. In some cases, this results in irreversible inhibition of the target, but some targeted covalent inhibitor (TCI) drugs bind covalently but reversibly. Computer modeling is widely used in drug discovery, but different computational methods must be used to model covalent modifiers because of the chemical bonds formed. Structural and bioinformatic analysis has identified sites of modification that could yield selectivity for a chosen target. Docking methods, which are used to rank binding poses of large sets of inhibitors, have been augmented to support the formation of protein--ligand bonds and are now capable of predicting the binding pose of covalent modifiers accurately. The pKa's of amino acids can be calculated in order to assess their reactivity towards electrophiles. QM/MM methods have been used to model the reaction mechanisms of covalent modification. The continued development of these tools will allow computation to aid in the development of new covalent modifier drugs.

scholarly journals Modeling covalent-modifier drugs

2017 ◽  
Author(s):  
Ernest Awoonor-Williams ◽  
Andrew G Walsh ◽  
Christopher N Rowley
Keyword(s):  
Computer Modeling ◽  
Covalent Binding ◽  
Covalent Bond ◽  
Bioinformatic Analysis ◽  
Covalent Modification ◽  
Binding Affinities ◽  
Chemical Bonds ◽  
Irreversible Inhibition ◽  
Large Sets ◽  
Covalent Inhibitor

In this review, we present a summary of how computer modeling has been used in the development of covalent modifier drugs. Covalent modifier drugs bind by forming a chemical bond with their target. This covalent binding can improve the selectivity of the drug for a target with complementary reactivity and result in increased binding affinities due to the strength of the covalent bond formed. In some cases, this results in irreversible inhibition of the target, but some targeted covalent inhibitor (TCI) drugs bind covalently but reversibly. Computer modeling is widely used in drug discovery, but different computational methods must be used to model covalent modifiers because of the chemical bonds formed. Structural and bioinformatic analysis has identified sites of modification that could yield selectivity for a chosen target. Docking methods, which are used to rank binding poses of large sets of inhibitors, have been augmented to support the formation of protein--ligand bonds and are now capable of predicting the binding pose of covalent modifiers accurately. The pKa's of amino acids can be calculated in order to assess their reactivity towards electrophiles. QM/MM methods have been used to model the reaction mechanisms of covalent modification. The continued development of these tools will allow computation to aid in the development of new covalent modifier drugs.

scholarly journals Covalent binding of proteinases in their reaction with α2-macroglobulin

1980 ◽  
Vol 187 (3) ◽  
pp. 695-701 ◽  
Author(s):  
G S Salvesen ◽  
A J Barrett
Keyword(s):  
Active Site ◽  
Proteinase Inhibitors ◽  
Covalent Binding ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Covalent Bond ◽  
Leucocyte Elastase ◽  
Α2 Macroglobulin ◽  
Covalent Bond Formation ◽  
The Stability

Although it is known that most of the plasma proteinase inhibitors form complexes with proteinases that are not dissociated by SDS (sodium dodecyl sulphate), there has been disagreement as to whether this is true for alpha 2M (alpha 2-macroglobulin). We have examined the stability to SDS with reduction of complexes between alpha 2M and several 125I-labelled proteinases (trypsin, plasmin, leucocyte elastase, pancreatic elastase and papain) by gel electrophoresis. For each enzyme, some molecules were separated from the denatured alpha 2M chains, but amounts ranging from 8.3% (papain) to 61.2% (trypsin) were bound with a stability indicative of a covalent link. Proteolytic activity was essential for the covalent binding to occur, and the proteinase molecules became attached to the larger of the two proteolytic derivatives (apparent mol.wt. 111 000) of the alpha 2M subunit. We take this to mean that cleavage of the proteinase-susceptible site sometimes leads to covalent-bond formation between alpha 2M and proteinase. Whatever the nature of this bond, it does not involve the active site of the proteinase, as bound serine-proteinase molecules retain the ability to react with the active-site-directed reagent [3H]Dip-F (di-isopropyl phosphorofluoridate). Our conclusion is that the ability to form covalent links is not essential for the inhibitory capacity of alpha 2M. It may, however, help to stabilize the complexes against dissociation or proteolysis.

scholarly journals Small-molecule covalent bond formation at tyrosine creates a binding site and inhibits activation of Ral GTPases

2020 ◽  
Vol 117 (13) ◽  
pp. 7131-7139 ◽  
Author(s):  
Khuchtumur Bum-Erdene ◽  
Degang Liu ◽  
Giovanni Gonzalez-Gutierrez ◽  
Mona K. Ghozayel ◽  
David Xu ◽  
...  
Keyword(s):  
High Resolution ◽  
Binding Site ◽  
Bond Formation ◽  
Covalent Bond ◽  
Covalent Modification ◽  
Tyrosine Residue ◽  
Nucleotide Exchange ◽  
Oncogenic Ras ◽  
Ras Gtpases ◽  
Covalent Bond Formation

Ral (Ras-like) GTPases are directly activated by oncogenic Ras GTPases. Mutant K-Ras (G12C) has enabled the development of covalent K-Ras inhibitors currently in clinical trials. However, Ral, and the overwhelming majority of mutant oncogenic K-Ras, are devoid of a druggable pocket and lack an accessible cysteine for the development of a covalent inhibitor. Here, we report that covalent bond formation by an aryl sulfonyl fluoride electrophile at a tyrosine residue (Tyr-82) inhibits guanine exchange factor Rgl2-mediated nucleotide exchange of Ral GTPase. A high-resolution 1.18-Å X-ray cocrystal structure shows that the compound binds to a well-defined binding site in RalA as a result of a switch II loop conformational change. The structure, along with additional high-resolution crystal structures of several analogs in complex with RalA, confirm the importance of key hydrogen bond anchors between compound sulfone oxygen atoms and Ral backbone nitrogen atoms. Our discovery of a pocket with features found on known druggable sites and covalent modification of a bystander tyrosine residue present in Ral and Ras GTPases provide a strategy that could lead to therapeutic agent targeting oncogenic Ras mutants that are devoid of a cysteine nucleophile.

scholarly journals Binding Interactions of Peptide Aptamers

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 6055
Author(s):  
Roger R. C. New ◽  
Tam T. T. Bui ◽  
Michal Bogus
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Cyclic Peptides ◽  
Binding Interactions ◽  
Peptide Aptamers ◽  
Linear Peptide ◽  
Signalling Molecules ◽  
Antibody Binding Site ◽  
Linear Chains ◽  
Protein Receptors

Peptide aptamers are short amino acid chains that are capable of binding specifically to ligands in the same way as their much larger counterparts, antibodies. Ligands of therapeutic interest that can be targeted are other peptide chains or loops located on the surface of protein receptors (e.g., GCPR), which take part in cell-to-cell communications either directly or via the intermediary of hormones or signalling molecules. To confer on aptamers the same sort of conformational rigidity that characterises an antibody binding site, aptamers are often constructed in the form of cyclic peptides, on the assumption that this will encourage stronger binding interactions than would occur if the aptamers were simply linear chains. However, no formal studies have been conducted to confirm the hypothesis that linear peptides will engage in stronger binding interactions with cyclic peptides than with other linear peptides. In this study, the interaction of a model cyclic decamer with a series of linear peptide constructs was compared with that of a linear peptide with the same sequence, showing that the cyclic configuration does confer benefits by increasing the strength of binding.

scholarly journals Phage display demonstrates durable differences in serological profile by route of inoculation in primary infections of non-human primates with Dengue Virus 1

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jayant V. Rajan ◽  
Michael McCracken ◽  
Caleigh Mandel-Brehm ◽  
Greg Gromowski ◽  
Simon Pollett ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Phage Display ◽  
Denv Infection ◽  
Phage Library ◽  
Humoral Immune ◽  
Linear Peptide ◽  
Humoral Immune Responses ◽  
High Resolution Map ◽  
Domain Iii ◽  
Inoculation Route

AbstractNatural dengue virus (DENV) infections occur by mosquito bite but how the inoculation route affects the humoral immune response is unknown. We serologically profiled 20 non-human primates (NHP) from a prior study of DENV1 infection where animals were inoculated by mosquito (N = 10) or subcutaneous injection (N = 10). Using a comprehensive, densely tiled and highly redundant pan-flavivirus programmable phage library containing 91,562 overlapping 62 amino acid peptides, we produced a high-resolution map of linear peptide sequences enriched during DENV seroconversion. Profiles in mosquito-inoculated and subcutaneously-inoculated animals were similar up to 90 days after primary infection, but diverged at 1 year with differences in sero-reactivity in the Envelope (E; residues 215–406; p < 0.08), and Nonstructural-3 (NS3; residues 549–615; p < 0.05) proteins in mosquito-inoculated versus subcutaneously-inoculated animals. Within the E protein, residues 339–384 in domain III accounted for > 99% of the observed sero-reactivity difference. Antibody breadth did not vary by mode of inoculation. The differential reactivity to E domain III seen by phage display validated orthogonally by ELISA, but did not correlate with late neutralization titers. Serological profiling of humoral immune responses to DENV infection in NHP by programmable phage display demonstrated durable differences in sero-reactivity by route of inoculation.

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