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Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac((R)))or homologous recombination transfection within insect cells (e.g. flashBAC (TM)). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204kDa; human ABL1 wildtype, 126kDa; human FMRP, 68 kDa; viral vNSl-Hl, 76kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.

Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies.

ATR, a protein kinase in the PIKK family, plays a critical role in the cell DNA-damage response and is an attractive anticancer drug target. Several potent and selective inhibitors of ATR have been reported showing significant antitumor efficacy, with most advanced ones entering clinical trials. However, due to the absence of an experimental ATR structure, the determinants contributing to ATR inhibitors' potency and specificity are not well understood. Here we present the mutations in the ATP-binding site of PI3Kα to progressively transform the pocket to mimic that of ATR. The generated PI3Kα mutants exhibit significantly improved affinity for selective ATR inhibitors in multiple chemical classes. Furthermore, we obtained the X-ray structures of the PI3Kα mutants in complex with the ATR inhibitors. The crystal structures together with the analysis on the inhibitor affinity profile elucidate the roles of individual amino acid residues in the binding of ATR inhibitors, offering key insights for the binding mechanism and revealing the structure features important for the specificity of ATR inhibitors. The ability to obtain structural and binding data for these PI3Kα mutants, together with their ATR-like inhibitor binding profiles, makes these chimeric PI3Kα proteins valuable model systems for structure-based inhibitor design.

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., PLoS ONE, 12 (4), ISSN:1932-6203

Phosphoinositide-3-kinases (PI3Ks) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein we describe the structure guided optimization of a series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines where the pharmacokinetic properties were improved by modulating the electronics of the 6-position heterocycle, and the overall druglike properties were fine-tuned further by modification of the 4-position substituent. The resulting 2,4-bismorpholino 6-heterocyclic pyrimidines are potent class I PI3K inhibitors showing mechanism modulation in PI3K dependent cell lines and in vivo efficacy in tumor xenograft models with PI3K pathway deregulation (A2780 ovarian and U87MG glioma). These efforts culminated in the discovery of 15 (NVP-BKM120), currently in Phase II clinical trials for the treatment of cancer.

Midkine (MDK) belongs to a class of heparin-binding growth factors and is highly expressed in a number of cancers. MDK is a cysteine-rich 13 kDa protein containing five disulfide bonds. In this study, we expressed recombinant human MDK (rhMDK) in Escherichia coli Origami 2 (DE3) strain, which carries a ( trxB − /gor 522 − ) double mutation. Soluble rhMDK was expressed at a high-level in this strain and the protein was purified by a two-step purification using heparin affinity and gel filtration chromatography. Seven milligrams of rhMDK with high purity was obtained from a 3 L culture. All 10 cysteines were confirmed to be engaged in correct disulfide bond linkages by mass spectrometry analysis. Activity of purified rhMDK was confirmed by a neurite outgrowth assay using rat cerebellar granule cells. Active rhMDK is a critical reagent for cancer drug discovery studies.

Identification of constructs suitable for the recombinant protein production pipeline is a bottleneck for structural genomics efforts, as most methods require purified proteins and/or are labor-intensive. Here, we present a novel high-throughput approach, Binding Rate Screen, that can alleviate this bottleneck by screening expression constructs in crude soluble lysate. This functional screen utilizes the frequently employed hexahistidine (His(6)) tag as a reporter, and measures its binding rate to an affinity matrix as a metric to reflect aggregation, concentration, and purifiability of the target protein. The constructs with the highest binding rates also exhibit high expression of soluble monomeric protein as judged by analytical size-exclusion chromatography. Constructs expressing variations of the target protein can be prioritized on a time scale of minutes, which is at least 10-100 times faster than any other technologies currently available.

Mutated forms of KRAS are no longer able to switch effectors between “on” and “off” states. It is known that the function of KRAS is controlled by key parts in the C-terminus, including six consecutive lysines, a terminal prenyl moiety and a terminal carboxymethyl functional group. We set out to discover compounds which would inhibit the function of mutated KRAS as an activator for effectors. This campaign yielded several compounds that blocked biochemical and cellular functions of KRAS with low micromolar activity while not affecting markers outside of KRAS pathways in cells. In order to understand the mode of binding of these compounds to KRAS, we generated different forms of the protein, including unprenylated truncated and fully processed full-length protein. NMR studies with truncated protein (amino acids 1-169) identified a site at which compound binding stabilized the inactive conformation of KRAS. This site is located adjacent to switch-II and is similar to sites described by others. The Kd determined for this binding event is almost 3 orders of magnitude higher than the IC50 and EC50 values measured in biochemical and cellular assays. In order to understand this difference, we developed a biophysical assay using the Fortebio system which enabled binding studies in a system with full-length prenylated protein in the presence of lipids, to match the context of the biochemical and cellular assays. Micromolar binding to the full-length prenylated KRAS protein was observed in the Fortebio assay and binding was not observed in the absence of prenylation, consistent with the near millimolar Kd observed by NMR for truncated KRAS. Curiously, similar micromolar binding was seen to a peptide derived from the C-terminus of KRAS (amino acids 168-185) with and without prenyl modification while related compounds that do not bind to the full-length prenylated KRAS also do not bind to these peptides. It is still unclear whether binding to the terminal peptide in lipid context is related to the binding site adjacent to switch-II. From a drug discovery perspective, it remains to be confirmed whether current inhibitors can be optimized. Citation Format: Johanna Jansen, Wolfgang Jahnke, Susan Fong, Laura Tandeske, Charles Wartchow, Keith Pfister, Tatiana Zavorotinskaya, Anke Blechschmidt, Dirksen Bussiere, Yumin Dai, Jeff Dove, Eric Fang, David Farley, Jean-Michel Florent, John Fuller, Simona Gokhin, Alvar Gossert, Mohammad Hekmat-Nejad, Chrystèle Henry, Julia Klopp, Bill Lenahan, Andreas Lingel, Arndt Meyer, Jamie Narberes, Gwynn Pardee, C Gregory Paris, Savithri Ramurthy, Paul Renhowe, Sebastien Rieffel, Kevin Shoemaker, Sharadha Subramanian, Tiffany Tsang, Stephania Widger, Armin Widmer, Isabel Zaror, Stephen Hardy. Inhibiting mutated KRAS, a broken switch of effector pathways. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B38. doi: 10.1158/1557-3125.RASONC14-B38

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.

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.

Immunoscreening of a Salmonella typhi cosmid library in E. coli allowed the detection of clones producing a 36 kDa porin from S. typhi. The gene is efficiently expressed in an E. coli porin-less mutant and the protein is exported to the outer membrane envelope. Two clones which markedly differ in their level of expression have been isolated.