Structural Characterization of Proline-rich Tyrosine Kinase 2 (PYK2)
Reveals a Unique (DFG-out) Conformation and Enables Inhibitor Design |
| |
Authors: | Seungil Han Anil Mistry Jeanne S Chang David Cunningham Matt Griffor Peter C Bonnette Hong Wang Boris A Chrunyk Gary E Aspnes Daniel P Walker Arthur D Brosius and Leonard Buckbinder |
| |
Institution: | Pfizer Global Research & Development, Groton, Connecticut 06340 |
| |
Abstract: | Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor
tyrosine kinase implicated in multiple signaling pathways. It is a negative
regulator of osteogenesis and considered a viable drug target for osteoporosis
treatment. The high-resolution structures of the human PYK2 kinase domain with
different inhibitor complexes establish the conventional bilobal kinase
architecture and show the conformational variability of the DFG loop. The
basis for the lack of selectivity for the classical kinase inhibitor,
PF-431396, within the FAK family is explained by our structural analyses.
Importantly, the novel DFG-out conformation with two diarylurea inhibitors
(BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine
kinases identifiable by the gatekeeper Met-502 and the unique hinge loop
conformation of Leu-504. This is the first example of a leucine residue in the
hinge loop that blocks the ATP binding site in the DFG-out conformation. Our
structural, biophysical, and pharmacological studies suggest that the unique
features of the DFG motif, including Leu-504 hinge-loop variability, can be
exploited for the development of selective protein kinase inhibitors.Proline-rich tyrosine kinase 2
(PYK2)2 and focal
adhesion kinase (FAK) comprise the focal adhesion kinase subfamily of
non-receptor tyrosine kinases. PYK2 and FAK are large multidomain proteins
containing an N-terminal FERM domain, a central catalytic domain, and a
C-terminal segment containing dual proline rich (PR) subdomains and a focal
adhesion targeting (FAT) region
(1,
2). While FAK is widely
expressed, PYK2 expression is relatively restricted with highest levels in
brain and the hematopoeitic system. Unlike FAK, optimal PYK2 activation is
dependent on Ca2+ mobilization. PYK2 (-/-) animals have been
described previously, and develop normally
(3,
4). Characterization of the
immune system of PYK2(-/-) animals revealed the absence of marginal zone
B-cells along with abnormal T-cell independent type II responses
(4), and altered macrophage
morphology, migration and signaling in response to cell attachment or
chemokine treatment (3). These
studies strengthen the link between PYK2 and signaling through chemokine and
integrin receptors. In addition, PYK2(-/-) mice were shown to have increased
susceptibility to diet-induced obesity and diabetes
(5).Recently, the characterization of PYK2(-/-) mice showed a high bone mass
phenotype resulting from increased osteogenesis and osteoblast activity. Using
PYK2(-/-) mouse bone marrow cultures and hMSCs expressing a PYK2 shRNA,
elimination or reduction of PYK2 protein levels resulted in significantly
enhanced osteogeogenesis. Importantly, the daily administration of a
pyrimidine-based PYK2 inhibitor, PF-431396, increased bone formation, and
protected against bone loss in ovariectomized rats
(6). PYK2(-/-) mice showed mild
osteopetrosis which was attributed to the impairment in osteoclast function
(7). Therefore, the high bone
mass phenotype may result from both enhanced osteoblast and impaired
osteoclast elements.PYK2 is one member of a family of over 500 evolutionarily conserved enzymes
with high amino acid and structural conservation within the catalytic ATP
binding pocket. Classical kinase inhibitors bind to the ATP site and compete
for substrate binding. Thus, while classical inhibitors based on ATP binding
analogs have been readily identified, the inherent promiscuity of action for
this class has presented significant challenges to drug design
(8). With the exception of
cancer therapeutics, where additional therapeutic benefits may be gained by
the inhibition of multiple kinase targets (e.g. Sutent, Sorafenib),
minimizing off-target activity is most often desired. Therefore, there is
great interest in identifying unique allosteric regulatory domains for
specific kinase targets. Despite intense effort, small molecule inhibitors
exploiting extra-catalytic allosteric sites have been limited to a few
examples including IKK (9) and
MEK (10). Alternatively,
bipartite inhibitors have been developed that stabilize an inactive
conformation of the protein kinase, the prototypical example being BIRB796
binding to p38 and Gleevec binding to Abl. Such compounds make contact with
both the conserved ATP site and less conserved regions of the activation loop,
thus offering the potential for improved selectivity
(11). The N terminus of the
activation loop contains an invariant Asp-Phe-Gly (DFG) motif, and is an
important determinant of enzyme activity. In the active or
“DFG-in” conformation, these amino acids are involved in the
coordination of ATP. Conversely, the “DFG-out” state does not bind
ATP and the kinase is inactive. While a handful of kinases are known to adopt
a DFG-out conformation (e.g. p38, Abl, etc), it remains to be
determined how general this strategy might be in the design of selective
kinase inhibitors.To help elucidate the molecular mechanism of PYK2 and its substrate
specificity, we used biophysical methods and determined multiple x-ray
structures of the PYK2 kinase domain. High-resolution structures of apo and
ATPγS-bound forms were obtained as well as a complex with PF-431396, a
“classical” kinase inhibitor. Empirical screening identified
BIRB796 as a weak PYK2 kinase inhibitor. Surface plasmon resonance (SPR) and
NMR studies indicated that PYK2 could adopt a “DFG-out”
conformation. Despite the low affinity, a 1.75-Å co-crystal structure
was obtained with BIRB796 revealing a novel binding mode. Our biophysical and
structural results provide insight into the enzyme-substrate complex and
allowed us to advance the rational design of a selective DFG-out inhibitor
with improved PYK2 selectivity and potency. The compound, PF-4618433, showed
robust osteogenic activity in hMSC cultures. |
| |
Keywords: | |
|
|