Inhibitor hijacking of Akt activation.

The kinase Akt plays a central role as a regulator of multiple growth factor input signals, thus making it an attractive anticancer drug target. A-443654 is an ATP-competitive Akt inhibitor. Unexpectedly, treatment of cells with A-443654 causes paradoxical hyperphosphorylation of Akt at its two regulatory sites (Thr308 and Ser473). We explored whether inhibitor-induced hyperphosphorylation of Akt by A-443654 is a consequence of disrupted feedback regulation at a pathway level or whether it is a direct consequence of inhibitor binding to the ATP binding site of Akt. Catalytically inactive mutants of Akt revealed that binding of an inhibitor to the ATP site of Akt is sufficient to directly cause hyperphosphorylation of the kinase in the absence of any pathway feedback effects. We conclude that ATP-competitive Akt inhibitors impart regulatory phosphorylation of their target kinase Akt. These results provide new insights into both natural regulation of Akt activation and Akt inhibitors entering the clinic.

Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases.

The clinical success of multitargeted kinase inhibitors has stimulated efforts to identify promiscuous drugs with optimal selectivity profiles. It remains unclear to what extent such drugs can be rationally designed, particularly for combinations of targets that are structurally divergent. Here we report the systematic discovery of molecules that potently inhibit both tyrosine kinases and phosphatidylinositol-3-OH kinases, two protein families that are among the most intensely pursued cancer drug targets. Through iterative chemical synthesis, X-ray crystallography and kinome-level biochemical profiling, we identified compounds that inhibit a spectrum of new target combinations in these two families. Crystal structures revealed that the dual selectivity of these molecules is controlled by a hydrophobic pocket conserved in both enzyme classes and accessible through a rotatable bond in the drug skeleton. We show that one compound, PP121, blocks the proliferation of tumor cells by direct inhibition of oncogenic tyrosine kinases and phosphatidylinositol-3-OH kinases. These molecules demonstrate the feasibility of accessing a chemical space that intersects two families of oncogenes.

Characterization of cytochrome P450 2D6 alleles using the Invader system.

The cytochrome p450 (CYP) superfamily comprises enzymes that play an essential role in the transformation of medically relevant compounds. Accurate genotyping of polymorphisms in members of this family is drawing increasing interest because certain allelic variants may result in either loss of efficacy or toxic accumulation of therapeutic agents. Debrisoquine 4-hydroxylase, or CYP2D6, is among the most widely studied of the CYPs. The complexity of the CYP2D6 genomic region, including pseudogenes, gene deletions, and gene duplications, has offered numerous challenges to developing a genotyping strategy. We describe a comprehensive CYP2D6 genotyping strategy that employs both a PCR/Invader genotyping assay system and an Invader genomic copy number assay The Invader system is a homogeneous, isothermal, highly specific, and robust signal amplification system. Resultsfrom II CYP2D6 assays in an alle frequency study compare well to published allele frequency values for Caucasians. Further, Invader assays provided unambiguous genotyping determinations for 100% of the 171 samples that yielded a visible PCR product on an agarose gel. A copy number assay yielded only one equivocal result in 205 samples. We identified 17 single-copy individuals and 17 three-copy (or more) individuals.

Synthesis and evaluation of indazole based analog sensitive Akt inhibitors.

The kinase Akt is a key signaling node in regulating cellular growth and survival. It is implicated in cancer by mutation and its role in the downstream transmission of aberrant PI3K signaling. For these reasons, Akt has become an increasingly important target of drug development efforts and several inhibitors are now reaching clinical trials. Paradoxically it has been observed that active site kinase inhibitors of Akt lead to hyperphosphorylation of Akt itself. To investigate this phenomenon we here describe the application of a chemical genetics strategy that replaces native Akt with a mutant version containing an active site substitution that allows for the binding of an engineered inhibitor. This analog sensitive strategy allows for the selective inhibition of a single kinase. In order to create the inhibitor selective for the analog sensitive kinase, a diversity of synthetic approaches was required, finally resulting in the compound PrINZ, a 7-substituted version of the Abbott Labs Akt inhibitor A-443654.

Discovery of dual inhibitors of the immune cell PI3Ks p110delta and p110gamma: a prototype for new anti-inflammatory drugs.

PI3Kdelta and PI3Kgamma regulate immune cell signaling, while the related PI3Kalpha and PI3Kbeta regulate cell survival and metabolism. Selective inhibitors of PI3Kdelta/gamma represent a potential class of anti-inflammatory agents lacking the antiproliferative effects associated with PI3Kalpha/beta inhibition. Here we report the discovery of PI3Kdelta/gamma inhibitors that display up to 1000-fold selectivity over PI3Kalpha/beta and evaluate these compounds in a high-content inflammation assay using mixtures of primary human cells. We find selective inhibition of only PI3Kdelta is weakly anti-inflammatory, but PI3Kdelta/gamma inhibitors show superior inflammatory marker suppression through suppression of lipopolysaccharide-induced TNFalpha production and T cell activation. Moreover, PI3Kdelta/gamma inhibition yields an anti-inflammatory signature distinct from pan-PI3K inhibition and known anti-inflammatory drugs, yet bears striking similarities to glucocorticoid receptor agonists. These results highlight the potential of selectively designing drugs that target kinases with shared biological function.

Noninvasive detection of acute and chronic injuries in human renal transplant by elevation of multiple cytokines/chemokines in urine.

BACKGROUND: Injuries in kidney transplant is currently diagnosed by needle biopsy. A noninvasive test that sensitively detects these injuries would benefit the patients. METHODS: Urine samples were collected from healthy controls and kidney transplant recipients. Urine samples were screened first with an antibody array consisting of 120 chemokines and cytokines and then with a multiplex beads assay. Representative parameters, including macrophage inflammatory protein-1Delta, osteoprotegerin, monokine induced by interferon-gamma (IFN), and IFN-gamma-induced protein of 10 kDa, were simultaneously determined by a quadruplex assay in urine samples from 84 patients with renal allograft injury, 29 patients with stable graft function, and 19 healthy individuals. RESULTS: Twenty-three cytokines/chemokines were found to be elevated in urine samples of patients with acute rejection by the antibody array. The second round of screening confirmed that 11 of the 23 parameters were elevated in the patients but not in the healthy controls. Induced protein of 10 kDa and monokine induced by IFN-gamma were significantly elevated in urine samples of patients with acute renal injury, and macrophage inflammatory protein-1Delta and osteoprotegerin were significantly elevated in patients with both acute and chronic renal injuries. The combination of the four parameters had a high positive detection rate (97.6%) for renal transplant injury and could differentiate between acute and chronic injury. CONCLUSION: These results might indicate that the present multiplex assay provides a basis to establish a noninvasive method for the diagnosis and monitoring of renal transplant injury.

Elevation of CXCR3-binding chemokines in urine indicates acute renal-allograft dysfunction.

A noninvasive urinary test that diagnoses acute renal allograft dysfunction would benefit renal transplant patients. We aimed to develop a rapid urinary diagnostic test by detecting chemokines. Seventy-three patients with renal allograft dysfunction prompting biopsy and 26 patients with stable graft function were recruited. Urinary levels of CXCR3-binding chemokines, monokine induced by IFN-gamma (Mig/CXCL9), IFN-gamma-induced protein of 10 kDa (IP-10/CXCL10), and IFN-inducible T-cell chemoattractant (I-TAC/CXCL11), were determined by a particle-based triplex assay. IP-10, Mig and I-TAC were significantly elevated in renal graft recipients with acute rejection, acute tubular injury and BK virus nephritis. Using 100 pg/mL as the threshold level, both IP-10 and Mig had diagnostic value (sensitivity 86.4%; specificity 91.3%) in differentiating acute graft dysfunction from other clinical conditions. In terms of monitoring the response to antirejection therapy, this urinary test is more sensitive and predictive than serum creatinine. These results indicate that this rapid test is clinically useful.