Irreversible inhibition of BoNT/A protease: proximity-driven reactivity contingent upon a bifunctional approach

2021 ◽  
Author(s):  
Lewis D. Turner ◽  
Alexander L. Nielsen ◽  
Lucy Lin ◽  
Sabine Pellett ◽  
Takashi Sugane ◽  
...  
Keyword(s):  
Covalent Bond ◽  
Covalent Modification ◽  
Irreversible Inhibition ◽  
Metal Chelating

A proximity-driven covalent bond with intrinsically less reactive warheads has been made possible by using a metal-chelating anchor for directed targeted covalent modification of Cys165 within the BoNT/A protease.

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 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 Irreversible Inhibition of BoNT/A Protease: Unique Warhead Reactivity and Function Contingent upon a Bifunctional Approach

2021 ◽  
Author(s):  
Lewis Turner ◽  
Alexander Lund Nielsen ◽  
Lucy Lin ◽  
Sabine Pellett ◽  
Takashi Sugane ◽  
...  
Keyword(s):  
Enzyme Kinetics ◽  
Therapeutic Potential ◽  
Covalent Bond ◽  
Pharmacological Properties ◽  
Irreversible Inhibition ◽  
And Function ◽  
Chelating Compounds

We describe a comprehensive screening campaign of warheads, linked to a hydroxamate chelating anchor, for the modification of Cys165 within the BoNT/A protease. <div>Engaging thorough enzyme kinetics, we detail a remarkable proximity-driven covalent bond with an epoxide warhead, a weak electrophile; yet, one that possessed superior irreversible inhibition, and pharmacological properties, when compared to intrinsically higher reactive warheads. This directed, selective covalent bond was contingent upon the crucial hydroxamate-Zn<sup>2+ </sup>chelating interaction as exemplified by examining non-chelating compounds. </div><div>We discuss previous approaches using non-target selective cysteine-reactive warheads to modify the BoNT/A protease of which none present any therapeutic potential – our bifunctional strategy allows the use of intrinsically less reactive warheads to intercept the cysteine, which will allow for less off-target modifications of such inhibitors. Moreover, we also broach that this bifunctional approach is not a one-off strategy that we believe can be broadly translated to other metalloproteases that possess non-catalytic, yet, nucleophilic residues within the enzymes catalytic sphere. </div>

scholarly journals The plasma membrane Ca2+ pump mutant lysine591 → arginine retains some activity, but is still inactivated by fluorescein isothiocyanate

1996 ◽  
Vol 317 (1) ◽  
pp. 41-44 ◽  
Author(s):  
Hugo P. ADAMO ◽  
Adelaida G. FILOTEO ◽  
John T. PENNISTON
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Human Plasma ◽  
Fluorescein Isothiocyanate ◽  
Covalent Modification ◽  
Transport Activity ◽  
Wild Type ◽  
Pump Function ◽  
Irreversible Inhibition ◽  
Lysine Residues

Inactivation of the wild-type human plasma membrane Ca2+ pump (isoform 4b) by fluorescein isothiocyanate is accompanied by covalent modification of Lys591. The mutation of Lys591 to arginine reduced the Ca2+ transport activity to 35% of the wild-type, and diminished the amount of acylphosphate formed from ATP by a corresponding amount. When this mutant was treated with fluorescein isothiocyanate, the enzyme was still irreversibly inactivated, even though no reactive residue was available at position 591. The results show that, although Ca2+ pump function is sensitive to the residue at position 591, Lys591 is not essential for enzyme activity. They also demonstrate that irreversible inhibition of the plasma membrane Ca2+ pump by fluorescein isothiocyanate does not require the covalent modification of Lys591. This indicates that fluorescein isothiocyanate reacts with lysine residues at other positions in addition to Lys591.

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 Irreversible inhibition of sodium current and batrachotoxin binding by a photoaffinity-derivatized local anesthetic.

1995 ◽  
Vol 105 (2) ◽  
pp. 267-287 ◽  
Author(s):  
J McHugh ◽  
W M Mok ◽  
G K Wang ◽  
G Strichartz
Keyword(s):  
Local Anesthetic ◽  
Uv Light ◽  
Sodium Current ◽  
Covalent Bond ◽  
Cell Voltage ◽  
Scorpion Venom ◽  
Gh3 Cells ◽  
Post Exposure ◽  
Irreversible Inhibition ◽  
Uv Illumination

We have synthesized a model local anesthetic (LA), N-(2-di-N-butyl-aminoethyl)-4-azidobenzamide (DNB-AB), containing the photoactivatable aryl azido moiety, which is known to form a covalent bond to adjacent molecules when exposed to UV light (Fleet, G.W., J.R. Knowles, and R.R. Porter. 1972. Biochemical Journal. 128:499-508. Ji, T.H. 1979. Biochimica et Biophysica Acta. 559:39-69). We studied the effects of DNB-AB on the sodium current (INa) under whole-cell voltage clamp in clonal mammalian GH3 cells and on 3[H]-BTX-B binding to sheep brain synaptoneurosomes. In the absence of UV illumination, DNB-AB behaved similarly to known LAs, producing both reversible block of peak INa (IC50 = 26 microM, 20 degrees C) and reversible inhibition of 3[H]-BTX-B (50 nM in the presence of 0.12 microgram/liter Leiurus quinquestriatus scorpion venom) binding (IC50 = 3.3 microM, 37 degrees C), implying a noncovalent association between DNB-AB and its receptor(s). After exposure to UV light, both block of INa and inhibition of 3[H]-BTX-B binding were only partially reversible (INa = 42% of control; 3[H]-BTX-B binding = 23% of control) showing evidence of a light-dependent, covalent association between DNB-AB and its receptor(s). In the absence of drug, UV light had less effect on INa (post exposure INa = 96% of control) or on 3[H]-BTX-B binding (post exposure binding = 70% of control). The irreversible block of INa was partially protected by coincubation of DNB-AB with 1 mM bupivacaine (IC50 = 45 microM, for INa inhibition at 20 degrees C, Wang, G.K., and S.Y. Wang. 1992. Journal of General Physiology. 100:1003-1020), (post exposure INa = 73% of control). The irreversible inhibition of 3[H]-BTX-B binding also was partially protected by coincubation with bupivacaine (500 microM, 37 degrees C) (post exposure binding = 51% of control), suggesting that the site of irreversible inhibition of both INa and 3[H]-BTX-B binding is shared with the clinical LA bupivacaine.

Sign in / Sign up

Export Citation Format

Share Document