PDBsum entry 4mx9

Transferase/transferase inhibitor

PDB id: 4mx9

Contents

Protein chain

459 a.a.

Ligands

2E8

Waters ×24

PDB id:

4mx9

Name:

Transferase/transferase inhibitor

Title:

Cdpk1 from neospora caninum in complex with inhibitor uw1294

Structure:

Calmodulin-like domain protein kinase isoenzyme gamma, related. Chain: a. Fragment: residues 22-506. Engineered: yes

Source:

Neospora caninum. Organism_taxid: 572307. Strain: liverpool. Gene: cdpk1, ncliv_011980, xp_003880764. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

3.10Å R-factor: 0.250 R-free: 0.274

Authors:

E.A.Merritt

Key ref:

K.K.Ojo et al. (2014). Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy. Plos One, 9, e92929. PubMed id: 24681759 DOI: 10.1371/journal.pone.0092929

Date:

26-Sep-13 Release date: 09-Oct-13

Protein chain

F0V9W9  (F0V9W9_NEOCL) -  Calmodulin-like domain protein kinase isoenzyme gamma, related from Neospora caninum (strain Liverpool)

Seq: 506 a.a.

Struc: 459 a.a.

Enzyme reactions

• Enzyme class: E.C.2.7.11.17 - calcium/calmodulin-dependent protein kinase.
• Reaction:
  1. L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] + ADP + H⁺
  2. L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] + ADP + H⁺
• Cofactor: Ca(2+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1371/journal.pone.0092929

Plos One 9:e92929 (2014)

PubMed id: 24681759

Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy.

K.K.Ojo, M.C.Reid, L.Kallur Siddaramaiah, J.Müller, P.Winzer, Z.Zhang, K.R.Keyloun, R.S.Vidadala, E.A.Merritt, W.G.Hol, D.J.Maly, E.Fan, W.C.Van Voorhis, A.Hemphill.

ABSTRACT

Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.