The identification of novel targets may prove useful in the development of new antimicrobials effective against chlamydiae. Chlamydial genomic studies have identified three Ser/Thr protein kinases, Pkn1, Pkn5, and PknD. Our laboratory has shown previously that C. pneumoniae PknD is a dual-specific protein kinase that autophosphorylates on threonine and tyrosine residues and phosphorylates serine and tyrosine residues of the
FHA-2 domain of Cpn0712, a putative Yersinia YscD ortholog called CdsD . In this report we show that a 3′-pyridyl oxindole compound, a known inhibitor of Janus kinase 3 (JAK3), inhibits C. pneumoniae PknD activity. CP673451 ic50 This compound prevented PknD autophosphorylation and phosphorylation of CdsD, a type III secretion apparatus protein. When added to infected HeLa cells, the compound retarded C. pneumoniae growth and significantly reduced the amount of infectious C. pneumoniae produced suggesting that PknD plays an important role in chlamydial replication.
Results Identification of an inhibitor of C. pneumoniae PknD protein kinase activity We have recently shown that C. pneumoniae contains three Ser/Thr protein kinases  and that one of these, PknD, phosphorylates CdsD, a structural component of the type III secretion SBE-��-CD concentration system (T3SS) . In order to determine whether PknD plays an essential role in Chlamydia development, we screened an existing library Vitamin B12 of 80 small molecule kinase inhibitors, including inhibitors of eukaryotic receptor tyrosine kinases and atypical kinases, for their ability to inhibit PknD autophosphorylation in vitro. Recombinant GST-tagged PknD kinase domain (GST-PknD KD) was pre-incubated with 10 μM of each compound
and reactions initiated with the addition of kinase assay buffer containing Mn2+ and ATP. SDS-PAGE and Western blotting followed by autoradiography was used to visualize the extent of PknD autophosphorylation in the presence of each compound. Nine compounds (EMD designations: D7, E8, F4, F5, F6, F7, G5, H10, and H11) of the 80 Selleck Epacadostat tested exhibited some level of inhibition of PknD autophosphorylation when tested at 10 μM (data not shown). Of these nine compounds only one, compound D7, a 3′-pyridyl oxindole, completely inhibited PknD autophosphorylation. Fig. 1A shows a dose response for PknD inhibition. At 1 μM compound D7 reduced PknD autophosphorylation by greater than 50% (fig. 1A). Similar results were obtained with two different lots of the inhibitor. Compound D4, a pan-specific inhibitor of the Janus kinase (JAK) family, did not significantly inhibit PknD autophosphorylation at concentrations of 0.2 to 10 μM (figs. 1A and 1B). Similarly, two other JAK3 inhibitors, compounds D5 and D6, did not inhibit PknD autophosphorylation at concentrations of 1 or 10 μM (fig. 1B). Figure 1 Inhibition of PknD by compound D7.