Design of Protein Kinase inhibitors

• Background

Computer modelling is an important part of rational drug design. Protein kinase inhibitors are often used in medical practice. Our lab is focused on the development of the protein kinase inhibitors. Our kinase targets are: CK2, ASK1, FGFR1, JNK3, ROCK1, LCK, DAPK1.

Initially, we perform receptor-based virtual screening of in-house drug-like collection (about 500,000 compounds). On the base of analysis of ligand-receptor interactions, our qualified scientists select promising compounds for in vitro tests. Results of the tests together with SAR analysis allowed us to indicate a number of promising chemical cores. Using these data, we synthesize and test their derivatives.

• Disclosed results

A number of new and potent classes of human CK2 ATP-competitive inhibitors with in vitro activity in submicromolar range were recently obtained. These classes of inhibitors are not reported in the literature as inhibitors of CK2. Two of the classes were protected by patents.

- 3-carboxy-4(1H)-quinolones:

This class of inhibitors has been selected via receptor-based virtual screening of the Otava compound library. It was revealed that the most active compounds, 5,6,8-trichloro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (7) (IC₅₀=0.3 µM) and 4-oxo-1,4-dihydrobenzo[h]quinoline-3-carboxylic acid (9) (IC₅₀=1 µM), are ATP competitive (K_і values are 0.06 and 0.28 µM, respectively). Evaluation of the inhibitors on seven protein kinases shows considerable selectivity toward CK2. According to theoretical calculations and experimental data, a structural model describing the key features of 3-carboxy-4(1H)-quinolones responsible for tight binding to CK2 active site has been developed.

3-Carboxy-4-(1H)-Quinolones 7 and 9 - inhibitors of human Protein Kinase CK2

Protein kinase	Inhibitor 7	Inhibitor 9
CK2 JNK3 ROCK-I p56 LCK DYRK1a MSK1 GSK3 CDK5	11 74 85 87 35 71 >50 >50	25 81 78 80 85 89 >50 >50

Specificity spectrum of inhibitors 7 and 9. Residual activity determined in the presence of 10 µM inhibitor is expressed as a percentage of the control without inhibitor. Final concentration of ATP in the experiment was 100 µM

Binding mode of 3-Carboxy-4-(1H)-Quinolones 7 (А) and 9 (B) predicted using flexible docking and molecular dynamics simulation

References: Golub A.G., Yakovenko O.Y., Bdzhola V.G., Sapelkin V.M., Zien P., Yarmoluk S.M. Evaluation of 3-carboxy-4(1H)-quinolones as inhibitors of human protein kinase CK2. J Med Chem. 2006 Nov 2; 49(22) : 6443-50.
Pat. UА68984 А, C07D215/00, 2004-08-16. Application of 4-substituted 3-carboxyquinolines as protein kinase CK2 inhibitors. Sapelkin V.M., Lukashov S.S., Golub A.G., Bdzhola V.G., Yakovenko O.Ya., Yarmoluk S.M., Dubinina G.G.

- 4,5,6,7-tetrahalogeno-1H-isoindole-1,3(2H)-diones

Here we present the 4,5,6,7-tetrahalogeno-1H-isoindole-1,3(2H)-diones (TID) as a novel potent class of CK2 inhibitors. We identified this class of inhibitors by high-throughput docking of a compound collection in the ATP binding site of human CK2. The most active compounds are 2-(4,5,6,7-tetraiodo-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propanoic acid and 2-(4,5,6,7-tetraiodo-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)acetic acid with IC₅₀ values 0.15 µM and 0.3 µM, respectively. These inhibitors are ATP-competitive and they only minimally inhibit the activities of protein kinases DYRK1a, MSK1, GSK3 and CDK5. Binding modes for the most active inhibitors are proposed.

Structures and IC₅₀ values of TID 43, 45, 46, 48, 58, 59

Protein kinase	TID43	TID46	TID48	TID58
CK2 DYRK1a MSK1 GSK3 CDK5	11 80 64 >50 >50	7 83 70 >50 >50	15 60 78 >50 >50	18 70 76 >50 >50

Specificity spectrum of TID 43, 46, 48 and 58. Residual activity, determined in the presence of 10 µM inhibitor, is expressed as a percentage of the control without inhibitor. Final concentration of ATP in the experiment was 100 µM.

TID46-CK2 complex obtained by molecular docking. (A) General view. (B) Detailed view. Intermolecular hydrogen bonds are shown as dotted lines, their lengths (in A) are indicated. Key hydrophobic interactions are indicated by arrows

References: Golub A.G., Yakovenko O.Y., Prykhod'ko A.O., Lukashov S.S., Bdzhola V.G., Yarmoluk S.M. Evaluation of 4,5,6,7-tetrahalogeno-1H-isoindole-1,3(2H)-diones as inhibitors of human protein kinase CK2. Biochim Biophys Acta. 2008 Jan; 1784(1) : 143-9.
Pat. UA69165 А, С07D215/00, 2004-08-16. Application of 4,5,6,7-tetrahalogeno-1,3-isoindolinediones as protein kinase CK2 inhibitors. Golub A.G., Yakovenko O.Ya., Yarmoluk S.M., Dubinina G.G., Bdzhola V.G., Prykhod'ko A.O.

- thienopyrimidines

A novel series of substituted (thieno[2,3-d]pyrimidin-4-ylthio)carboxylic acids has been synthesized and tested in vitro towards human protein kinase CK2. It was revealed that the most active compound inhibiting CK2 is 3-{[5-(4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl]thio}propanoic acid (IC₅₀=0.1 µM). Structure-activity relationships of 28 tested thienopyrimidine derivatives have been studied and binding mode of this chemical class has been predicted. Evaluation of the inhibitors on seven protein kinases revealed considerable selectivity towards CK2.

The binding mode of inhibitor in the active site of the CK2 catalytic subunit. Hydrogen bonds are shown by the dotted lines

Reference: Golub A.G., Bdzhola V.G., Briukhovetska N.V., Balanda A.O., Kukharenko O.P., Kotey I.M., Ostrynska O.V., Yarmoluk S.M. Synthesis and biological evaluation of substituted (thieno[2,3-d]pyrimidin-4-ylthio)carboxylic acids as inhibitors of human protein kinase CK2 // Eur. J. Med. Chem. – 2011 Mar; 46(3): 870-6.

We have identified two classes of human ASK1 ATP-competitive inhibitors:

- 3H-naphtho[1,2,3-de]quinoline-2,7-diones

In vitro experiments revealed that ethyl 2,7-dioxo-2,7-dihydro-3H-naphtho[1,2,3-de]quinoline-1-carboxylate (NQDI-1) inhibited ASK1 with a K_і of 500 nM. The competitive character of inhibition is demonstrated in Lineweaver-Burk plots. In our preliminary selectivity study this compound exhibited strong specific inhibitory activity toward ASK1. Structure-activity relationships of 15 tested 3H-naphtho[1,2,3-de]quinoline-2,7-dione derivatives have been studied and binding mode of this chemical class has been predicted.

ASK1	Aurora A	ROCK	HGFR	FGFR1	Tie2	JNK3	CK2
12,5	79	100	62	44	84	100	78

Residual Activity of Protein Kinases (%) in the Presence of NQDI-1 at 25 µM Concentration

The binding mode of NQDI-1 in the active site of the ASK1 catalytic subunit. Hydrogen bonds are shown by the dotted lines.

Reference: Volynets G.P., Chekanov O.M., Synyugin A.R., Golub A.G., Kukharenko O.P., Bdzhola V.G., Yarmoluk S.M. Identification of 3H-naphtho[1,2,3-de]quinoline-2,7-diones as inhibitors of apoptosis signal-regulating kinase 1 (ASK1) http://pubs.acs.org/doi/abs/10.1021/jm200117h

2-thioxo-thiazolidines

In vitro tests revealed that seven derivatives of 2-Thioxo-thiazolidin-4-one exhibited inhibitory activity against ASK1. The most active compound was 5-bromo-3-(4-oxo-2-thioxo-thiazolidin-5-ylidene)-1,3-dihydro-indol-2-one (IC₅₀=2 µM). Binding mode for inhibitors of this class with ASK1 ATP-binding site was proposed.

The binding mode of inhibitor in the active site of the ASK1 catalytic subunit. Hydrogen bond is shown by the dotted line

Reference: Volynets G.P., Bdzhola V.G., Kukharenko O.P., Yarmoluk S.M. Identification of ASK1 small-molecule inhibitors. Ukr. Biochim. J. 2010; 82 (5): 26-34.

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