T he phosphoinositide-3-kinase (PI3K) family of lipid kinases is involved in a diverse set of cellular functions, including cell growth, proliferation, motility, differentiation, glucose transport, survival, intracellular trafficking, and membrane ruffling. 1 PI3K's can be categorized into class I, II, or III, depending on their subunit structure, regulation, and substrate selectivity. 2 Class IA PI3K's are activated by receptor tyrosine kinases and consist of a regulatory subunit (p85) and a catalytic subunit (p110). There are three catalytic isoforms: p110R, β, and δ. A single class IB PI3K, activated by GPCRs, consists of only one member: a p110γ catalytic subunit and a p101 regulatory subunit. The primary in vivo substrate of the class I PI3K's is phosphatidylinositol (4,5) diphosphate (PtdIns(4,5)P2), which upon phosphorylation at the 3-position of the inositol ring to form phosphatidylinositol triphosphate (3,4,5)P3 (PIP3) serves as a second messenger by activating a series of downstream effectors that mediate the cellular functions mentioned above. The PI3K isoforms have different distributions and share similar cellular functions, which are context dependent. In particular, p110R pathway deregulation has been demonstrated in ovarian, breast, colon, and brain cancers. 3,4 Inhibitors of PI3KR represent an intriguing therapeutic modality for these indications, and as such, there is much interest in generating suitable molecules to test this hypothesis in the clinic. 5À10 We have previously reported on a series of 6-hydroxyphenyl-2-morpholino pyrimidines, 11 as potent pan class I PI3K inhibitors that exhibit high selectivity toward protein kinases (serine/threonine and tyrosine kinases). We have further reported on non-phenol containing heterocyclic, morpholino pyrimidines 12 such as compound 1 which demonstrate in vivo PI3K pathway modulation and modest tumor growth inhibition. Described herein are our efforts to identify potent morpholino pyrimidinyl inhibitors of class I PI3Ks that exhibit potency and pharmacokinetic properties which allow for maximal pathway modulation in vivo and have druglike properties suitable for clinical development. These efforts culminated in the identification of 15, NVP-BKM120.Aminopyrimidine 1 and analogues such as 3 (Figure 1) exhibit low or sub-nanomolar biochemical potency and sub-micromolar cellular potency against PI3KR. Even with high rodent CL values, such analogues can demonstrate PI3K pathway modulation in mouse xenograft models. 12 During our exploration of the C 6 position, it was noted that C 6 aminopyridine analogue 4, while being less potent than 3 against PI3KR (>10Â potency loss), exhibited a markedly reduced (>9Â) rat CL value, increased %F, and increased oral AUC. Thus, superior pharmacokinetic properties were achievable within this scaffold and the challenge remaining was to retain this kind of pharmacokinetic profile while optimizing all the other attributes (potency, solubility, permeability, safety) necessary for advancement. To address this challenge, ...

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.

Erwinia chrysanthemi exports degradative enzymes by using a type I protein secretion system. The proteases secreted by this system lack an N-terminal signal peptide but contain a C-terminal secretion signal. To explore the substrate specificity of this system, we have expressed the E. chrysanthemi transporter system (prtDEF genes) in Escherichia coli and tested the ability of this ABC transporter to export hybrid proteins carrying C-terminal fragments of E. chrysanthemi protease B. The C terminus contains six glycine-rich repeated motifs, followed by two repeats of the sequences DFLV and DIIV. Two types of hybrid proteins were assayed for transport, proteins with the 93-residue-protease-B C terminus containing one glycine-rich repeat and both hydrophobic terminal repeats and proteins with the 181-residue C terminus containing all repeat motifs. Although the shorter C terminus is unable to export the hybrids, the longer C terminus can promote the secretion of hybrid proteins with N termini as large as 424 amino acids, showing that the glycine-rich motifs are required for the efficient secretion of these hybrids. However, the secretion of hybrids occurs only if these proteins do not carry disulfide bonds in their mature structures. These latter results suggest that disulfide bond formation can occur prior to or during the secretion. Disulfide bonds may prevent type I secretion of hybrids. One simple hypothesis to explain these results is that the type I channel is too narrow to permit the export of proteins with secondary structures stabilized by disulfide bonds.Nonpathogenic strains of Escherichia coli, such as the laboratory strain E. coli K-12, export few proteins to the external medium (53). The secretion of proteins in E. coli depends on a type II (Sec-dependent) mechanism in which unfolded proteins carrying an N-terminal signal sequence are transported across the inner membrane to the periplasm and then processed and folded in the periplasm prior to their translocation across the outer membrane. To initiate type II secretion, it is thought that a polypeptide and its signal peptide must be fully extended to cross the inner membrane (3). After crossing the inner membrane, proteins are folded in the periplasm in a process assisted by chaperones. In addition, the periplasm contains the enzymes required for the correct assembly of disulfide bonds to complete protein folding (5, 48).In contrast, many gram-negative pathogens including enteropathogenic E. coli (38), Yersinia spp. (36), Salmonella enterica serovar Typhimurium (39), and Vibrio cholerae (35) export virulence factors required for host colonization and survival. These virulence factors are secreted by mechanisms that operate independently from the type II system, mechanisms that involve specialized membrane transport apparatuses (64). A subset of virulence factors is exported by a type I mechanism in which three proteins assemble to form a transmembrane structure that couples the export of protein substrates with ATP hydrolysis. These ABC transporters in...

Influenza virus uses a unique mechanism to initiate viral transcription named cap-snatching. The PB2 subunit of the viral heterotrimeric RNA polymerase binds the cap structure of cellular pre-mRNA to promote its cleavage by the PA subunit. The resulting 11–13 capped oligomer is used by the PB1 polymerase subunit to initiate transcription of viral proteins. VX-787 is an inhibitor of the influenza A virus pre-mRNA cap-binding protein PB2. This clinical stage compound was shown to bind the minimal cap-binding domain of PB2 to inhibit the cap-snatching machinery. However, the binding of this molecule in the context of an extended form of the PB2 subunit has remained elusive. Here we generated a collection of PB2 truncations to identify a PB2 protein representative of its structure in the viral heterotrimeric protein. We present the crystal structure of VX-787 bound to a PB2 construct that recapitulates VX-787's biological antiviral activity in vitro. This co-structure reveals more extensive interactions than previously identified and provides insight into the observed resistance profile, affinity, binding kinetics, and conformational rearrangements induced by VX-787.

The PI3K/Akt/mTor signaling pathway plays an important role in controlling cell growth, proliferation and survival. Through various mechanisms, the pathway is frequently dysregulated in human cancers, suggesting the use of PI3K inhibitors as novel targeted anticancer therapeutic agents. To this end, substantial drug discovery efforts have been devoted both in pharmaceutical companies and in academia to identify and develop therapeutic agents able to specifically down regulate PI3K or other components of this pathway in tumors cells. Following the discovery of NVP-BEZ235, our first dual pan-PI3K/mTOR clinical compound, we sought to identify additional PI3K inhibitors from different chemical classes with more stringent selectivity profiles. The key to achieve these objectives was to pursue a structure-based design approach coupled with intensive pharmacological evaluation of selected compounds during the medicinal chemistry optimization process. Here we report on the biological characterization of the pan-PI3K pyrimidine-derived inhibitor NVP-BKM120. This compound inhibits all four Class I PI3K isoforms (IC50 values in the 35 to 248 nM range) with at least 50-fold selectivity (compared to p110α) towards protein kinases. The compound is also active against the most common somatic PI3Kα mutations (H1047R, E542K and E545K). NVP-BKM120 does not significantly inhibit the related Class III (Vps34) and Class IV (mTOR, DNA-PK) PI3K kinases. Consistent with its mechanism of action, NVP-BKM120 decreases the cellular levels of p-Akt in mechanistic and relevant tumor cell lines (e.g., IC50 for S473P-Akt in Rat1-p110α cells of 93 nM). This biological activity correlates with inhibition of various Akt downstream signaling pathway components, and with its anti-proliferative activity. Thus, the compound demonstrates significant, concentration dependent cell growth inhibition and induction of apoptosis in a variety of tumor cancer cells, particularly for those harboring p110α mutants and/or over-expressing erbB2. In addition, NVP-BKM120 demonstrates significant, dose dependent in vivo pharmacodynamic activity as measured by inhibition of p-Akt in relevant xenograft models. The pharmacological, biological and preclinical safety profile of NVP-BKM120 supports its clinical development and the compound is currently undergoing Phase 1/II clinical trials in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4498.