Dual kinase-bromodomain inhibitors for rationally designed polypharmacology.

Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multitarget profile has, however, necessitated the application of combination therapies, which can pose major clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as new targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase-bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348, which are clinical PLK1 and JAK2-FLT3 kinase inhibitors, respectively, is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a new strategy for rational single-agent polypharmacological targeting. Furthermore, structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase-bromodomain inhibitors.

A small molecule-kinase interaction map for clinical kinase inhibitors.

Kinase inhibitors show great promise as a new class of therapeutics. Here we describe an efficient way to determine kinase inhibitor specificity by measuring binding of small molecules to the ATP site of kinases. We have profiled 20 kinase inhibitors, including 16 that are approved drugs or in clinical development, against a panel of 119 protein kinases. We find that specificity varies widely and is not strongly correlated with chemical structure or the identity of the intended target. Many novel interactions were identified, including tight binding of the p38 inhibitor BIRB-796 to an imatinib-resistant variant of the ABL kinase, and binding of imatinib to the SRC-family kinase LCK. We also show that mutations in the epidermal growth factor receptor (EGFR) found in gefitinib-responsive patients do not affect the binding affinity of gefitinib or erlotinib. Our results represent a systematic small molecule-protein interaction map for clinical compounds across a large number of related proteins.

Comprehensive analysis of kinase inhibitor selectivity.

We tested the interaction of 72 kinase inhibitors with 442 kinases covering >80% of the human catalytic protein kinome. Our data show that, as a class, type II inhibitors are more selective than type I inhibitors, but that there are important exceptions to this trend. The data further illustrate that selective inhibitors have been developed against the majority of kinases targeted by the compounds tested. Analysis of the interaction patterns reveals a class of 'group-selective' inhibitors broadly active against a single subfamily of kinases, but selective outside that subfamily. The data set suggests compounds to use as tools to study kinases for which no dedicated inhibitors exist. It also provides a foundation for further exploring kinase inhibitor biology and toxicity, as well as for studying the structural basis of the observed interaction patterns. Our findings will help to realize the direct enabling potential of genomics for drug development and basic research about cellular signaling.

Activation state-dependent binding of small molecule kinase inhibitors: structural insights from biochemistry.

Interactions between kinases and small molecule inhibitors can be activation state dependent. A detailed understanding of inhibitor binding therefore requires characterizing interactions across multiple activation states. We have systematically explored the effects of ABL1 activation loop phosphorylation and PDGFR family autoinhibitory juxtamembrane domain docking on inhibitor binding affinity. For a diverse compound set, the affinity patterns correctly classify inhibitors as having type I or type II binding modes, and we show that juxtamembrane domain docking can have dramatic negative effects on inhibitor affinity. The results have allowed us to associate ligand-induced conformational changes observed in cocrystal structures with specific energetic costs. The approach we describe enables investigation of the complex relationship between kinase activation state and compound binding affinity and should facilitate strategic inhibitor design.

A quantitative analysis of kinase inhibitor selectivity.

Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biological consequences of multi-kinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. We present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global analysis of the results, we introduce the concept of a selectivity score as a general tool to quantify and differentiate the observed interaction patterns. We further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.

Wild-type opaque2 and defective opaque2 polypeptides form complexes in maize endosperm cells and bind the opaque2-zein target site.

The Opaque2 (O2) basic leucine (Leu)-zipper transcriptional activator controls the expression of several genes in maize (Zea mays). We investigated the phosphorylation extent of wild-type O2 and mutant-defective or mutant-truncated o2 polypeptides in endosperm cells, their subcellular localization, participation in complex formation, and involvement in functional activity. Besides wild type, four mutant alleles (o2T, o2-52, o2It, and o2-676) producing o2 polypeptides and a null transcript allele (o2R) were considered. Observing the effects of these mutations, multiphosphorylation events in O2 or o2 proteins were confirmed and further investigated, and the involvement of both the nuclear localization signal (NLS)-B and Leu-zipper domains in proper targeting to the nucleus was ascertained. The absence of these domains in the o2T and o2It-S mutant-truncated forms holds them within the cytoplasm, where they are partially phosphorylated, whereas the presence of NLS-B and a partial Leu-zipper domain in o2-52 distributes this mutant-truncated form in both cytoplasm and nucleus. Although mutated in the NLS-B domain, the o2It-L and o2-676 mutant-defective forms are, respectively, partially or completely distributed into the nucleus. Only wild-type O2 and mutant-defective o2 polypeptides bearing the Leu-zipper are able to form complexes whose components were proven to bind the O2-zein target site by in vitro analyses. The transcription of a subset of H-zein genes as well as H-zein polypeptide accumulation in several o2-mutant-defective genotypes indicate the in vivo involvement of o2-mutant-defective proteins in O2-zein target site recognition. The gathered information broadens our knowledge on O2 functional activity and our view on possible quality protein maize trait manipulation or plant transformation via the utilization of cisgenic elements.

The maize O2 and PBF proteins act additively to promote transcription from storage protein gene promoters in rice endosperm cells.

A transient expression assay system was employed to investigate the possible use of the maize Opaque 2 (O2) and prolamin box binding factor (PBF) proteins as transcriptional activators of rice and wheat storage protein gene promoters. When assayed in developing rice endosperm cells, either O2 or PBF alone could increase transcription from the promoter of the rice glutelin gene, Gt1. However, mutant forms of O2 and PBF that are defective in DNA binding could not. Co-transfection with both transcriptional activators resulted in an additive increase in transactivation of the Gt1 promoter. Co-bombardment of a Gt1::GUS construct with plasmids expressing the DNA binding domains of O2 and PBF in antisense orientation resulted in a decrease of GUS expression below background levels. Similar stimulatory and additive effects of O2 and PBF could be observed on the promoters from other storage protein genes including rice globulin (Glb), prolamins (RP6 and PG5a) and a wheat glutenin (Bx7). However, responsiveness of the promoters from non-storage protein genes like rice actin and CaMV 35S to O2 and PBF was insignificant. Our results indicate that the maize O2 and PBF proteins can act singly or additively as effective stimulators of heterologous storage protein promoters in developing rice endosperm cells. These data support the use of well-characterized transcription factors from maize as an effective means of increasing the expression level of recombinant proteins in developing rice seeds.

Conservation of B-class floral homeotic gene function between maize and Arabidopsis.

The ABC model of flower development, established through studies in eudicot model species, proposes that petal and stamen identity are under the control of B-class genes. Analysis of B- and C-class genes in the grass species rice and maize suggests that the C- and B-class functions are conserved between monocots and eudicots, with B-class genes controlling stamen and lodicule development. We have undertaken a further analysis of the maize B-class genes Silky1, the putative AP3 ortholog, and Zmm16, a putative PI ortholog, in order to compare their function with the Arabidopsis B-class genes. Our results show that maize B-class proteins interact in vitro to bind DNA as an obligate heterodimer, as do Arabidopsis B-class proteins. The maize proteins also interact with the appropriate Arabidopsis B-class partner proteins to bind DNA. Furthermore, we show that maize B-class genes are capable of rescuing the corresponding Arabidopsis B-class mutant phenotypes. This demonstrates B-class activity of the maize gene Zmm16, and provides compelling evidence that B-class gene function is conserved between monocots and eudicots.