Exploiting the Unique ATP-Binding Pocket ofToxoplasmaCalcium-Dependent Protein Kinase 1 To Identify Its Substrates

AbstractUpon Plasmodium falciparum merozoites exposure to low [K+] environment in blood plasma, there is escalation of cytosolic [Ca2+] which activates Ca2+-Dependent Protein Kinase 1 (CDPK1), a signaling hub of intra-erythrocytic proliferative stages of parasite. Given its high abundance and multidimensional attributes in parasite life-cycle, this is a lucrative target for desiging antimalarials. Towards this, we have virtually screened MyriaScreenII diversity collection of 10,000 drug-like molecules, which resulted in 18 compounds complementing ATP-binding pocket of CDPK1. In vitro screening for toxicity in mammalian cells revealed that these compounds are non-toxic in nature. Further, SPR analysis demonstrated differential binding affinity of these compounds towards recombinantly purified CDPK1 protein. Selection of lead compound 1 was performed by evaluating their inhibitory effects on phosphorylation and ATP binding activities of CDPK1. Further, in vitro biophysical evaluations by ITC and FS revealed that binding of compound 1 is driven by formation of energetically favorable non-covalent interactions, with different binding constants in presence and absence of Ca2+, and TSA authenticated stability of compound 1 bound CDPK1 complex. Finally, compound 1 strongly inhibited intra-erythrocytic growth of P. falciparum in vitro. Concievably, we propose a novel CDPK1-selective inhibitor, step towards developing pan-CDPK kinase inhibitors, prerequisite for cross-stage anti-malarial protection.

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Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase

Biochemical Journal ◽

10.1042/bj2200677 ◽

1984 ◽

Vol 220 (3) ◽

pp. 677-683 ◽

Cited By ~ 6

Author(s):

J E Kudlow ◽

Y Leung

Keyword(s):

Protein Kinase ◽

Binding Site ◽

Egf Receptor ◽

Light Exposure ◽

Atp Binding ◽

Receptor Kinase ◽

Dependent Protein Kinase ◽

Atp Binding Site ◽

Dependent Protein ◽

Epidermal Growth

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3′-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5′-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5′-[beta gamma-imido]triphosphate or 20 mM-guanosine 5′-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity and that this analogue is an effective photoaffinity label for the kinase. Furthermore, these studies demonstrate that the EGF receptor, identified by using monoclonal antibodies, contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the Mr 170000 protein.

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Affinity labeling of the ATP-binding site of type II calmodulin-dependent protein kinase by 5′-p-fluorosulfonylbenzoyl adenosine

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(88)90052-5 ◽

1988 ◽

Vol 267 (2) ◽

pp. 467-473 ◽

Cited By ~ 16

Author(s):

Marita M. King ◽

David J. Shell ◽

Ann P. Kwiatkowski

Keyword(s):

Protein Kinase ◽

Binding Site ◽

Affinity Labeling ◽

Atp Binding ◽

Type Ii ◽

Dependent Protein Kinase ◽

Atp Binding Site ◽

Dependent Protein

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CC-115, a Dual Mammalian Target of Rapamycin/DNA-Dependent Protein Kinase Inhibitor in Clinical Trial, Is a Substrate of ATP-Binding Cassette G2, a Risk Factor for CC-115 Resistance

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.119.258392 ◽

2019 ◽

Vol 371 (2) ◽

pp. 320-326 ◽

Cited By ~ 4

Author(s):

Jenny Beebe ◽

Jian-Ting Zhang

Keyword(s):

Clinical Trial ◽

Risk Factor ◽

Protein Kinase ◽

Kinase Inhibitor ◽

Mammalian Target Of Rapamycin ◽

Protein Kinase Inhibitor ◽

Target Of Rapamycin ◽

Atp Binding ◽

Dependent Protein Kinase ◽

Dependent Protein

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Mapping the ATP-Binding Site in the Catalytic Subunit of A denosine-3':5'-monophosphate-Dependent Protein Kinase. Spatial Relationship with the ATP Site of the Undissociated Enzyme

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1978.tb12621.x ◽

1978 ◽

Vol 90 (3) ◽

pp. 427-432 ◽

Cited By ~ 54

Author(s):

Jurgen HOPPE ◽

Wolfgang FREIST ◽

Rainer MARUTZKY ◽

Shmuel SHALTIEL

Keyword(s):

Protein Kinase ◽

Binding Site ◽

Spatial Relationship ◽

Catalytic Subunit ◽

Atp Binding ◽

Dependent Protein Kinase ◽

Atp Binding Site ◽

Dependent Protein

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Structural basis for UCN-01 (7-hydroxystaurosporine) specificity and PDK1 (3-phosphoinositide-dependent protein kinase-1) inhibition

Biochemical Journal ◽

10.1042/bj20031119 ◽

2003 ◽

Vol 375 (2) ◽

pp. 255-262 ◽

Cited By ~ 73

Author(s):

David KOMANDER ◽

Gursant S. KULAR ◽

Jennifer BAIN ◽

Matthew ELLIOTT ◽

Dario R. ALESSI ◽

...

Keyword(s):

Protein Kinase ◽

Hydroxy Group ◽

Active Site ◽

Binding Pocket ◽

Kinase Domain ◽

Structural Basis ◽

Active Site Residues ◽

Dependent Protein Kinase ◽

Growth Factor Signalling ◽

Dependent Protein

PDK1 (3-phosphoinositide-dependent protein kinase-1) is a member of the AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family of protein kinases, and has a key role in insulin and growth-factor signalling through phosphorylation and subsequent activation of a number of other AGC kinase family members, such as protein kinase B. The staurosporine derivative UCN-01 (7-hydroxystaurosporine) has been reported to be a potent inhibitor for PDK1, and is currently undergoing clinical trials for the treatment of cancer. Here, we report the crystal structures of staurosporine and UCN-01 in complex with the kinase domain of PDK1. We show that, although staurosporine and UCN-01 interact with the PDK1 active site in an overall similar manner, the UCN-01 7-hydroxy group, which is not present in staurosporine, generates direct and water-mediated hydrogen bonds with active-site residues. Inhibition data from UCN-01 tested against a panel of 29 different kinases show a different pattern of inhibition compared with staurosporine. We discuss how these differences in inhibition could be attributed to specific interactions with the additional 7-hydroxy group, as well as the size of the 7-hydroxy-group-binding pocket. This information could lead to opportunities for structure-based optimization of PDK1 inhibitors.

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Mapping the ATP-binding domain of DNA-dependent protein kinase (DNA-PK) with coumarin- and isocoumarin-derived inhibitors

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2010.04.102 ◽

2010 ◽

Vol 20 (12) ◽

pp. 3649-3653 ◽

Cited By ~ 14

Author(s):

Sara L. Payne ◽

Sonsoles Rodriguez-Aristegui ◽

Julia Bardos ◽

Céline Cano ◽

Bernard T. Golding ◽

...

Keyword(s):

Protein Kinase ◽

Binding Domain ◽

Atp Binding ◽

Dependent Protein Kinase ◽

Dependent Protein

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Deep insights into the mode of ATP-binding mechanism in Zebrafish cyclin-dependent protein kinase-like 1 (zCDKL1): A molecular dynamics approach

Journal of Molecular Graphics and Modelling ◽

10.1016/j.jmgm.2018.02.002 ◽

2018 ◽

Vol 81 ◽

pp. 175-183 ◽

Cited By ~ 10

Author(s):

Ajaya Kumar Rout ◽

Budheswar Dehury ◽

Jitendra Maharana ◽

Chirasmita Nayak ◽

Vishwamitra Singh Baisvar ◽

...

Keyword(s):

Molecular Dynamics ◽

Protein Kinase ◽

Atp Binding ◽

Binding Mechanism ◽

Dependent Protein Kinase ◽

Dependent Protein

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Influence of a mutation in the ATP-binding region of Ca2+/calmodulin-dependent protein kinase II on its interaction with peptide substrates

Biochemical Journal ◽

10.1042/bj20030741 ◽

2004 ◽

Vol 378 (2) ◽

pp. 391-397 ◽

Cited By ~ 8

Author(s):

Mullasseril PRASEEDA ◽

Kurup K. PRADEEP ◽

Ananth KRUPA ◽

S. Sri KRISHNA ◽

Suseela LEENA ◽

...

Keyword(s):

Protein Kinase ◽

Catalytic Domain ◽

3D Structure ◽

Atp Binding ◽

Binding Region ◽

Dependent Protein Kinase ◽

Catalytic Core ◽

Peptide Substrates ◽

Protein Kinase Ii ◽

Dependent Protein

CaMKII (Ca2+/calmodulin-dependent protein kinase II) is expressed in high concentrations in the brain and is found enriched in the postsynaptic densities. The enzyme is activated by the binding of calmodulin to the autoregulatory domain in the presence of high levels of intracellular Ca2+, which causes removal of auto-inhibition from the N-terminal catalytic domain. Knowledge of the 3D (three-dimensional) structure of this enzyme at atomic resolution is restricted to the association domain, a region at the extreme C-terminus. The catalytic domain of CaMKII shares high sequence similarity with CaMKI. The 3D structure of the catalytic core of CaMKI comprises ATP- and substrate-binding regions in a cleft between two distinct lobes, similar to the structures of all protein kinases solved to date. Mutation of Glu-60, a residue in the ATP-binding region of CaMKII, to glycine exerts different effects on phosphorylation of two peptide substrates, syntide and NR2B (N-methyl-d-aspartate receptor subunit 2B) 17-mer. Although the mutation caused increases in the Km values for phosphorylation for both the peptide substrates, the effect on the kcat values for each was different. The kcat value decreased in the case of syntide, whereas it increased in the case of the NR2B peptide as a result of the mutation. This resulted in a significant decrease in the apparent kcat/Km value for syntide, but the change was minimal for the NR2B peptide. These results indicate that different catalytic mechanisms are employed by the kinase for the two peptides. Molecular modelling suggests structural changes are likely to occur at the peptide-binding pocket in the active state of the enzyme as a consequence of the Glu-60→Gly mutation.

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