A research team led by scientists at proteomics firm Cellzome has developed a selective inhibitor to the PI3 kinase isoform PI3Kγ.
The work, which was detailed in a paper published this week in Nature Chemical Biology, provides a potential new research tool for the study of PI3Kγ, and demonstrates the utility of Cellzome's chemoproteomics platform for developing new inhibitors.
As one of the four class I PI3Ks, PI3Kγ is involved in a number of processes including cell differentiation, survival, migration, and proliferation. From a clinical perspective, it "is an interesting target because of its role in inflammation as well as cancer, and metabolic and cardiovascular diseases," Gitte Neubauer, Cellzome's vice president of research operations and senior author on the paper, told ProteoMonitor this week via e-mail.
"There's a lot of interest in the [drug] industry in the entire PI3K class I family of enzyme," she said.
Study of PI3Kγ, however, has been hampered by a lack of selective inhibitors for the protein, particularly with regard to its use as a therapeutic target for non-fatal diseases. As the Nature Chemical Biology authors note, "lack of highly selective inhibitors … has hitherto precluded human clinical studies in inflammation because adverse effects caused by the simultaneous inhibition of off-target kinases … are intolerable for the treatment of chronic, non-life threatening diseases."
To identify sufficiently selective inhibitors of PI3Kγ, Cellzome and collaborators at the University of Torino and San Francisco-based biotech firm BioSeek used an adapted version of Cellzome's Kinobeads platform, a mass spec-based system for performing competition-binding assays to measure kinase-inhibitor interactions.
The Kinobeads matrix consists of a mix of kinase inhibitors that are incubated with aliquots of cell extracts that have been spiked with varying concentrations of the inhibitor being investigated. The binding patterns of the target kinases to the Kinobead matrix can then be measured by mass spec to quantify the effectiveness and selectivity of a given inhibitor.
The PI3Kγ work involved two changes to the platform, Neubauer said. First, the researchers designed a new matrix to optimize the capture of lipid kinases. Secondly, to enable high-throughput screening, the researchers used antibody detection to screen for potent, selective inhibitors.
These "potent molecules showing some degree of selectivity were then subjected to in-depth off-target profiling using mass spectrometry as a readout," Neubauer said. For their mass spec analyses the researchers used Thermo Fisher Scientific LTQ-Orbitrap XL and Orbitrap Velos instruments.
In recent work employing similar chemoproteomic platforms, researchers have explored the use of MALDI-MS to obtain higher throughput for their systems (PM 4/20/2012). Neubauer said, however, that Cellzome had no plans to pursue a MALDI approach given the high level of throughput the researchers achieved with antibody-based screening.
"We believe that antibody detection is much more scalable for high-throughput applications, and that the employed ESI-MS-based approach offers excellent sensitivity and coverage for in-depth characterization of interesting compounds," she said.
Using the platform, the researchers screened 16,146 compounds against PI3Kγ as well as closely related kinases including PI3Kδ, mTOR, and DNA-PK. Via immunodetection, they identified potential leads, ultimately arriving at a single most promising compound – CZC19091.
Structural changes to improve this compound's selectivity and in vivo activity led to development of the compound CZC24832, which the researchers put through mass-spec based kinome profiling screens in which it demonstrated reactivity with only two off-targets – PI3Kβ and PIP4K2C – out of 154 kinases plus 922 additional proteins.
"The off-target profiling was very comprehensive in two ways," Neubauer said. "First, using the antibody screening assay we measured the potency toward the five closest off-targets for each of the compounds we synthesized. This directed the design of new compounds immediately towards target potency and off-target selectivity."
"Secondly, the profiling is comprehensive with regards to the kinome of the cell or tissue type we use," she added. "Our standard Kinobead [matrix] typically [consists of] 150 to 180 protein kinases and hundreds of potential non-kinase off targets."
The platform is also "tunable toward particular target families," Neubauer said, noting that the new matrix used in the Nature Chemical Biology work "enabled the focused analysis of PI3K isoforms and a large panel of lipid and atypical kinases that are poorly captured on more protein kinase-directed beads."
The researchers also looked at potential off-targets for the compound by using it for pull-down experiments in cell extracts, finding, Neubauer said, that "the obtained selectivity information [from this work] was fully in line with the results from the Kinobeads."
With the compound, the researchers used BioSeek's BioMAP cellular activity mapping system to investigate PI3Kγ's role in inflammation, finding that use of the compound inhibited TH17 cell differentiation, providing evidence of a role for PI3Kγ in the regulation of TH17 cells.
The authors wrote that they expect the inhibitor "will be of use for further pharmacological validation of PI3Kγ in autoimmune and inflammatory disorders as well as in cancer and in metabolic and cardiovascular diseases." However, Neubauer said that Cellzome, which owns IP rights to the compound, currently has no plans to commercialize it as either a research tool or drug.