Small Molecule Therapeutics
Scheduled to speak at the GTC Bio event on kinase inhibitors as therapeutics for chronic inflammatory diseases, Maria Webb, Ph.D., vp, preclinical research, biological and pharmacological sciences at Pharmacopeia (www.pharmacopeia.com), notes that, up to now, most kinase-based drugs have been targeted for oncology, where therapeutics have “targeted more than a single kinase, thus are not selective.” But because of different mechanism of action and risk-benefit issues, kinase drugs for chronic inflammatory diseases are likely to be “selective for the kinase targeted in the disease process.”
Pharmacopeia is focused on small molecule therapeutics and recently entered into an agreement with Wyeth under which the latter will market drugs for immunological and inflammatory diseases. Pharmacopeia retains full commercialization rights for topical treatment of skin and eye diseases that result from the collaboration.
Dr. Webb notes that her group runs both cell-free assays that measure the enzymatic activity of kinases as well as numerous cell-based assays that are carefully developed for the kinase of interest—Jak 3, for example.
“To be certain that we are as thorough as possible in assessing kinase selectivity,” Dr. Webb says, “when we run a Jak 3 assay we also look at Jak 2, Jak 1, and Tyk 2. An enzyme assay alone doesn’t tell you that you’re inhibiting activity in the cell. One must set up functional measures at related kinases such as other members of the Jak family and look downstream for inhibition of cell signaling, stat5 phosphorylation for example, and read-outs of T-cell activity.” Additionally, activity at unrelated kinases is also assessed in broad or specifically chosen kinase panels. To accomplish this selectivity assessment, Pharmacopeia maintains an in-house kinase panel that includes near and distant members of the kinome and uses several outside services to run broad kinase panels.
Reinforcing the need for selectivity, Mark McCoy, research fellow at the Schering-Plough Research Institute (www.schering-plough.com), will report on NMR-guided design of protein kinase inhibitors at the GTC Bio conference. His group uses NMR methods to search for low molecular weight hits that can either be assembled into leads using a fragment-based approach or serve as scaffolds for chemical exploration that is guided by SAR from NMR data.
While the reliable evaluation of weak hits poses a formidable challenge for bioassay-based screening, NMR methods are well-suited to provide accurate data on low affinity binders.
“When we’re looking at the binding of low molecular weight compounds, they generally have low affinities, but generate strong NMR signals even at micro-molar levels,” Dr. McCoy states.
Ligand-detected NMR methods such as ATP-STD NMR are used to rapidly and accurately determine µM–mM Ki’s of ATP-competitive inhibitors. Binding of non-ATP competitive compounds can also be detected, Dr. McCoy notes. Both active and inactive kinases can be screened.
Concepts such as ligand binding efficiency are used to compare the affinities of scaffolds with different molecular weights and to monitor the effectiveness of substitutions to the scaffold. The high reliability NMR data for weak hits can also impact the evaluation of existing kinase screening libraries. In one example, the Schering-Plough group used SAR from NMR data to identify the correct binding mode of a series of low-affinity screening hits that, through structure-guided optimization, ultimately lead to a potent series of Akt inhibitors.
“Although Akt is implicated in tumor development,” Dr. McCoy states, “it might not be desirable to inhibit all three members of the Akt family.”
STD NMR is also useful for looking at the NMR signal of ATP after it has bound to kinase. Dr. McCoy notes that NMR detects atomic properties of the whole molecule, can reliably distinguish ATP from ADP, and determines that the kinase is whole and functional.