Nanopore Enzymology to Study Protein Kinases and Their Inhibition by Small Molecules

Drug discovery using small molecule inhibitors is reaching a stalemate due to low selectivity, adverse off-target effects and inevitable failures in clinical trials. Conventional chemical screening methods may miss potent small molecules because of their use of simple but outdated kits composed of recombinant enzyme proteins. Non-canonical inhibitors targeting a hidden pocket in a protein have received considerable research attention. Kii and colleagues identified an inhibitor targeting a transient pocket in the kinase DYRK1A during its folding process and termed it FINDY. FINDY exhibits a unique inhibitory profile; that is, FINDY does not inhibit the fully folded form of DYRK1A, indicating that the FINDY-binding pocket is hidden in the folded form. This intriguing pocket opens during the folding process and then closes upon completion of folding. In this review, we discuss previously established kinase inhibitors and their inhibitory mechanisms in comparison with FINDY. We also compare the inhibitory mechanisms with the growing concept of “cryptic inhibitor-binding sites.” These sites are buried on the inhibitor-unbound surface but become apparent when the inhibitor is bound. In addition, an alternative method based on cell-free protein synthesis of protein kinases may allow the discovery of small molecules that occupy these mysterious binding sites. Transitional folding intermediates would become alternative targets in drug discovery, enabling the efficient development of potent kinase inhibitors.

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Challenges in design of biochemical assays for the identification of small molecules to target multiple conformations of protein kinases

Drug Discovery Today ◽

10.1016/j.drudis.2008.03.023 ◽

2008 ◽

Vol 13 (11-12) ◽

pp. 522-529 ◽

Cited By ~ 15

Author(s):

P CHENE

Keyword(s):

Small Molecules ◽

Protein Kinases ◽

Biochemical Assays ◽

Multiple Conformations

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Evolution of kinase polypharmacology across HSP90 drug discovery

10.1101/2020.09.09.288936 ◽

2020 ◽

Author(s):

Albert A. Antolin ◽

Paul A. Clarke ◽

Ian Collins ◽

Paul Workman ◽

Bissan Al-Lazikani

Keyword(s):

Drug Discovery ◽

Drug Design ◽

Small Molecules ◽

Protein Kinases ◽

Experimental Methods ◽

Experimental Characterization ◽

Hsp90 Inhibitors ◽

Target Drug ◽

Clinical Candidate

AbstractMost small molecules interact with several target proteins but this polypharmacology is seldom comprehensively investigated or explicitly exploited during drug discovery. Here, we use computational and experimental methods to systematically characterize the kinase cross-pharmacology of representative HSP90 inhibitors. We demonstrate that the resorcinol clinical candidates ganetespib and, to a lesser extent, luminespib, display unique off-target kinase pharmacology as compared to other HSP90 inhibitors. We also demonstrate that polypharmacology evolved during the optimisation to discover luminespib and that the hit, leads and clinical candidate all have different polypharmacological profiles. We conclude that the submicromolar target inhibition of protein kinases by ganetespib may have potential clinical significance and we recommend the computational and experimental characterization of polypharmacology earlier in drug discovery projects to unlock new multi-target drug design opportunities.

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Benzofuran Small Molecules as Potential Inhibitors of Human Protein Kinases. A Review

Current Pharmaceutical Design ◽

10.2174/1381612822666151209152457 ◽

2016 ◽

Vol 22 (7) ◽

pp. 879-894 ◽

Cited By ~ 9

Author(s):

Halina Kwiecie| ◽

Agata Goszczy|ska ◽

Paulina Rokosz

Keyword(s):

Small Molecules ◽

Protein Kinases ◽

Human Protein ◽

Potential Inhibitors

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High-content screening of feeder-free human embryonic stem cells to identify pro-survival small molecules

Biochemical Journal ◽

10.1042/bj20101022 ◽

2010 ◽

Vol 432 (1) ◽

pp. 21-35 ◽

Cited By ~ 31

Author(s):

Paul D. Andrews ◽

Melissa Becroft ◽

Anders Aspegren ◽

Jane Gilmour ◽

Martyn J. James ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Survival ◽

Small Molecules ◽

Protein Kinases ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Basic Research ◽

Rock Inhibitor

The propensity of human embryonic stem cells to die upon enzymatic disaggregation or low-density plating is an obstacle to their isolation and routine use in drug discovery and basic research. Equally, the very low rate of establishment of implanted cells hinders cell therapy. In the present study we have developed a high-content assay for human embryonic stem cell survival and used this to screen a range of libraries of ‘lead-like’ small molecules and known bioactives. From this we identified 18 confirmed hits with four structural classes being represented by multiple compounds: a series of 5-(acyl/alkyl-amino)indazoles, compounds with a 4-(acylamino)pyridine core, simple N6,N6-dialkyladenines and compounds with a 5-(acylamino)indolinone core. In vitro kinase profiling indicated that the ROCK (Rho-associated kinase)/PRK2 (protein kinase C-related kinase 2) protein kinases are of pivotal importance for cell survival and identified previously unreported compound classes that inhibited this important biological activity. An evaluation using an extensive panel of protein kinases showed that six of our hit compounds exhibited better selectivity for ROCK inhibition than the routinely used commercially available ROCK inhibitor Y-27632. In this screen we also identified the K+-ATP channel opener pinacidil and show that it probably promotes cell survival, by ‘off-target’ inhibition of ROCK/PRK2. We have therefore identified novel pro-survival compounds of greater specificity, equivalent potency and reduced toxicity relative to the routinely employed ROCK inhibitor Y-27632.

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Synthetic Strategies of Pyrimidine-Based Scaffolds as Aurora Kinase and Polo-like Kinase Inhibitors

Molecules ◽

10.3390/molecules26175170 ◽

2021 ◽

Vol 26 (17) ◽

pp. 5170

Author(s):

Mrunal Jadhav ◽

Kaksha Sankhe ◽

Richie R. Bhandare ◽

Zehra Edis ◽

Samir Haj Bloukh ◽

...

Keyword(s):

Small Molecules ◽

Protein Kinases ◽

Anticancer Drugs ◽

Anticancer Agents ◽

Kinase Inhibitors ◽

Antitumor Agents ◽

Aurora Kinase ◽

Pyrimidine Ring ◽

Pyrimidine Derivatives ◽

Comprehensive Overview

The past few decades have witnessed significant progress in anticancer drug discovery. Small molecules containing heterocyclic moieties have attracted considerable interest for designing new antitumor agents. Of these, the pyrimidine ring system is found in multitude of drug structures, and being the building unit of DNA and RNA makes it an attractive scaffold for the design and development of anticancer drugs. Currently, 22 pyrimidine-containing entities are approved for clinical use as anticancer drugs by the FDA. An exhaustive literature search indicates several publications and more than 59 patents from the year 2009 onwards on pyrimidine derivatives exhibiting potent antiproliferative activity. These pyrimidine derivatives exert their activity via diverse mechanisms, one of them being inhibition of protein kinases. Aurora kinase (AURK) and polo-like kinase (PLK) are protein kinases involved in the regulation of the cell cycle. Within the numerous pyrimidine-based small molecules developed as anticancer agents, this review focuses on the pyrimidine fused heterocyclic compounds modulating the AURK and PLK proteins in different phases of clinical trials as anticancer agents. This article aims to provide a comprehensive overview of synthetic strategies for the preparation of pyrimidine derivatives and their associated biological activity on AURK/PLK. It will also present an overview of the synthesis of the heterocyclic-2-aminopyrimidine, 4-aminopyrimidine and 2,4-diaminopyrimidine scaffolds, and one of the pharmacophores in AURK/PLK inhibitors is described systematically.

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The Anti-Candida albicans Agent 4-AN Inhibits Multiple Protein Kinases

Molecules ◽

10.3390/molecules24010153 ◽

2019 ◽

Vol 24 (1) ◽

pp. 153 ◽

Cited By ~ 2

Author(s):

Maciej Masłyk ◽

Monika Janeczko ◽

Aleksandra Martyna ◽

Sławomir Czernik ◽

Małgorzata Tokarska-Rodak ◽

...

Keyword(s):

Small Molecules ◽

Protein Kinases ◽

Zebrafish Embryos ◽

Compound I ◽

Force Microscopy ◽

Atomic Force ◽

Multiple Protein ◽

Competitive Inhibitors ◽

Reversible Protein Phosphorylation ◽

Anti Fungal

Small molecules containing quinone and/or oxime moieties have been found as promising anti-fungal agents. One of them is 4-AN, a recently reported potent anti-Candida compound, which inhibits the formation of hyphae, decreases the level of cellular phosphoproteome, and finally shows no toxicity towards human erythrocytes and zebrafish embryos. Here, further research on 4-AN is presented. The results revealed that the compound: (i) Kills Candida clinical isolates, including these with developed antibiotic resistance, (ii) affects mature biofilm, and (iii) moderately disrupts membrane permeability. Atomic force microscopy studies revealed a slight influence of 4-AN on the cell surface architecture. 4-AN was also shown to inhibit multiple various protein kinases, a characteristic shared by most of the ATP-competitive inhibitors. The presented compound can be used in novel strategies in the fight against candidiasis, and reversible protein phosphorylation should be taken into consideration as a target in designing these strategies.

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