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technology to select for DARPins with novel binding specificities. However, like thioredoxin, DARPins cannot be efficiently exported to the periplasm using the signal sequences encoded in commonly available cloning vectors, such as the PhoA signal sequence, a model signal sequence that has been extensively studied, and the PelB signal sequence, found in many pET vectors (Fig. 1a). To get around this problem, the authors tested several of the SRP-dependent signal sequences (Fig. 1b). In every case, expression of the DARPins on the phage surface was dramatically improved. This report is not only a significant step forward for phage display technology, but also suggests that SRP-dependent signal sequences may be useful for efficient periplasmic expression of proteins for a wide variety of purposes.

Premature cytoplasmic folding may have been the major obstacle in many failed attempts to export proteins to the periplasm. Indeed, that E. coli has evolved so many mechanisms to prevent exported proteins from folding in the cytoplasm suggests that protein folding is an issue for translocation of native substrates. For example, some translocated proteins, such as alkaline phosphatase, require disulfide bonds, which can be formed only in the periplasm, to fold stably8. Other proteins, such as maltose-binding protein, are maintained in an unfolded state in the cytoplasm by chaperones (such as SecB)9. Indeed, SRP targeting itself may pre-vent cytoplasmic folding of native proteins.

Drugging the PI3 kinomePaul Workman, Paul A Clarke, Sandrine Guillard & Florence I Raynaud

Inhibitors of the PI3 kinase family of enzymes show promise for treating brain tumors.

Paul Workman, Paul A Clarke, Sandrine Guillard and Florence I Raynaud are at the Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey, UK. e-mail: paul.workman@icr.ac.uk

Protein kinases have proved their mettle as drug targets in oncology. The strategy of “drugging the cancer kinome”1 has led to trastuzumab (Herceptin), targeted to ERBB2/HER2; ima-tinib (Gleevec), targeted to BRC-ABL, KIT and PDFGR; and gefitinib/erlotinib (Iressa/Tarceva), targeted to EGFR. Yet the spotlight on protein kinases has overshadowed another

important potential kinase target, the phos-phatidylinositol 3-kinase (PI3 kinase) family of lipid kinases, which has been implicated by a wealth of evidence in the etiology of can-cer, inflammation, autoimmune conditions, thrombosis and viral infection2. This situation is about to change. Recent papers by Knight et al.3 in Cell and by Fan et al.4 in Cancer Cell describe the use of a library of small molecules to define patterns of inhibitory selectivity within the PI3 kinase family. As part of this tour de force of chemical biology, the papers demon-strate proof of concept for the exciting poten-tial of compounds that simultaneously inhibit PI3 kinase p110α and mTOR for the treat-ment of malignant brain tumors (gliomas).

Although the same chemical probe approach revealed that p110α is critical for insulin sig-naling3 (also demonstrated in ref. 5), the dual p110α/mTOR inhibitor showed clear evidence of a therapeutic window without undue toxic-ity in an animal model of human glioma4.

Cancer treatment is the arena in which information from the human genome project is being translated most readily into person-alized treatment1. Malignant progression is driven by molecular abnormalities, many of which result in the hijacking of signal trans-duction pathways2. This causes cancer cells to become highly dependent upon particular sig-naling pathways, a process known as “oncogene addiction”. Hence, pharmacologic inhibitors of oncogenic pathways show therapeutic selectiv-ity towards cancer versus normal cells.

The PI3 kinase pathway controls a range of cellular processes, including cell growth, sur-vival, differentiation, chemotaxis and metabo-lism. Responding to receptor tyrosine kinases and Ras, PI3 lipid kinases activate many down-stream signaling pathways by generating second messengers, particularly phosphatidylinositol-3,4,5-trisphosphate (PIP3) (Fig. 1). The over-all family of around 16 PI3 kinases6 includes the four class I lipid kinase isoforms p110α, p110β, p110δ and p110γ, which generate PIP3. The class IV isoforms—known as PIKKS—are protein kinases that monitor genomic integrity (through DNA-PK, ATM, ATR and hSmg-1) or integrate nutrient signaling to regulate cell growth (through mTOR).

The importance of each of these PI3 kinase family members in health and disease contin-ues to be defined. Of particular note, p110α is frequently overexpressed and mutated in many cancers, including gliomas and colon, breast, prostate and gynecological tumors, among others6. Moreover, additional players in the PI3 kinase signaling pathway are also commonly deregulated in malignancy. For example, loss of the PTEN phosphatase and overexpression and activation of the upstream receptor tyro-sine kinases and the downstream serine/threo-nine kinase PKB/Akt have been associated with tumorigenesis7 (Fig. 1).

Knight et al. and Fan et al. applied libraries of chemical probes to help determine the biological functions of particular PI3 kinase isoforms and to identify therapeutic opportunities for the cognate inhibitors. The paper by Knight et al. is especially innovative in two ways. First, it applies a diverse matrix of chemical structures to probe the function of PI3 kinases using a range of techniques. These include enzyme, cell and whole-animal assays, X-ray crystallography and molecular modeling, as well as chemical synthesis and chemoinformatics. Second, it applies these technologies to help understand the

Nevertheless, the use of SRP-dependent signal sequences may not suffice to achieve protein export in every case, as protein folding is only one of several factors that can inter-fere with protein translocation. Other possible factors—which pertain to both post-transla-tional and cotranslational translocation—include positive charges in the N-terminal region of the protein attached to the signal sequence10, signal sequence processing defects and the length of the protein11. Systematic study of these factors should be fruitful both for researchers wishing to understand the details of these processes and for those wish-ing to exploit these processes for biotechno-logical ends.

1. Steiner, D., Forrer, P., Stumpp, M.T. & Plückthun, A. Nat. Biotechnol. 24, 823–831 (2006).

2. Blobel, G. & Dobberstein, B. J. Cell Biol. 67, 835–851 (1975).

3. Takeishi, K., Yasumura, M., Pirtle, R. & Inouye, M. J. Biol. Chem. 251, 6259–6266 (1976).

4. Inouye, H. & Beckwith, J. Proc. Natl. Acad. Sci. USA 74, 1440–1444 (1977).

5. Huber, D. et al. J. Bacteriol. 187, 2983–2991 (2005).

6. Huber, D. et al. Proc. Natl. Acad. Sci. USA 102, 18872–18877 (2005).

7. Schierle, C.F. et al. J. Bacteriol. 185, 5706–5713 (2003).

8. Sone, M., Kishigami, S., Yoshihisa, T. & Ito, K. J. Biol. Chem. 272, 6174–6178 (1997).

9. Kumamoto, C.A. & Gannon, P.M. J. Biol. Chem. 263, 11554–11558 (1988).

10. Li, P., Beckwith, J. & Inouye, H. Proc. Natl. Acad. Sci. USA 85, 7685–7689 (1988).

11. Andersson, H. & von Heijne, G. Embo. J. 12, 683–691 (1993).

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biology and pathophysiology of the PI3 kinase family, revealing the similarities and differences between family members and demonstrating their potential for therapeutic application.

Knight et al. synthesized a battery of PI3 kinase inhibitors identified from the academic and patent literature and screened them for selectivity against 15 purified PI3 kinase iso-forms and 40 additional kinases. Principal com-ponent analysis revealed intriguing patterns of PI3 kinase inhibition that could not be predicted from either the kinase sequences or the chemical structure of the inhibitor (chemotype). Analysis identified pairs of PI3 kinases, referred to as ‘pharmalogs,’ which were inhibited by the same chemotypes. For example, p110α and p110γ tended to be inhibited by the same compounds, as did p110β and p110δ. Also, p110α inhibitors tended to block DNA-PK activity. The molecu-lar basis of some of the selectivity patterns could be explained by binding information revealed by X-ray crystallography and molecular model-ing. Potency and selectivity were controlled by a compound-induced conformational switch in the ATP site and also by binding to a deeper pocket.

Next, Knight et al. used the molecularly characterized inhibitors as probes to explore the roles of PI3 kinase isoforms in insulin sig-naling. Studies with cultured adipocytes and

myotubes showed, in contrast to some previous findings, that p110α but not p110β was pri-marily responsible for insulin signaling. This was confirmed by insulin tolerance studies in mice. The results show the power of molecu-larly characterized chemical probes as tools both for interrogating biological function and for validating targets. This strategy comple-ments genetic approaches, such as that in the recent p110α mouse knock-in studies5, which also demonstrated the critical role of p110α in growth and metabolic regulation.

Using the same panel of PI3 kinase inhibi-tors, Fan et al. found that pyridofuropyrimidine PI-1031,8 (inset in Fig. 1) strongly inhibits PKB/Akt phosphorylation and blocks the prolifera-tion of cultured malignant glioma cells, which are known to have an activated PI3 kinase pathway. In the presence of 10 µM ATP, PI-103 exhibits 50% inhibition (IC50 concentration) at 8 nM for p110α, 88 nM for p110β, 48 nM for p110δ and 150 nM for p110γ. Additionally, in the presence of 100 µM ATP, mTORC1 (rapamycin-sensitive mTOR-raptor complex), mTORC2 (rapamycin-insensitive mTOR-ric-tor complex) and DNA-PK were inhibited by PI-103 with IC50s of 20 nM, 83 nM and 2 nM, respectively. Although PI-103 was described as a multi-targeted PI3 kinase inhibitor (in contrast to other inhibitors with narrower selectivity

profiles), it showed a remarkable lack of activity against a broad range of other lipid and protein kinases. An exception was the class II PI3 kinase PI3KC2β, which had an IC50 of 26 nM.

Comparison of various compounds for their ability to inhibit glioma cell growth showed that the dual competitive kinase inhibitor PI-103 was the most effective, suggesting that p110α cooperates with mTOR in malignant glioma. The importance of this combinatorial inhibi-tion may be explained by the fact that mTOR inhibition alone by rapamycin analogs prevents p70S6K-mediated serine phosphorylation of the insulin receptor substrate-1 (IRS-1), blocks IRS-1 degradation and increases the associa-tion of IRS-1 with insulin and IGF receptors. This abrogates the feedback inhibition of the PI3 kinase pathway, resulting in an undesirable activation of PKB/Akt in cancer models and in patients treated with rapamycin analogs9.

According to this model, the distinct advan-tage of dual p110α/mTOR inhibitors like PI-103 is that they can abrogate this compen-satory effect and deliver a powerful two-hit inhibition of the pathway. However, things may be even more complicated. It is possible that particular combinatorial benefit may be gained from the direct inhibition by PI-103 of p110α, mTORC1, mTORC2 and possibly DNA-PK. Phosphorylation of PKB/Akt on Ser473 by both mTORC210 and DNA-PK11 have been reported. Confirming the therapeutic potential of the approach, PI-103 was shown to inhibit growth of a human glioma xenograft in nude mice. Antitumor activity has also been seen in other tumor types in which activation of the PI3 kinase pathway is important1,8.

These findings will fuel the growing enthusiasm of the pharmaceutical industry

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Figure 1 Sites of action of PI-103 in the PI3 kinase signaling pathway. Class I PI3 kinase isoforms (e.g., p110α) are activated downstream of ligand-bound receptor tyrosine kinases (e.g., insulin and IGF-1 receptors) through interactions involving Ras and p85 regulatory subunits that are often promoted by adaptor proteins such as IRS-1. Activated PI3 kinase catalyzes the production of the second messenger PIP3, which recruits the PKB/Akt protein kinase to the membrane where it is activated through dual phosphorylation (denoted by ‘P’) by PDK1 and the mTOR-rictor complex (mTORC2)10. Active PKB/Akt phosphorylates multiple substrates resulting in the regulation of several cellular processes, many of which are often deregulated in cancer. Negative regulation of the PI3 kinase pathway is mediated by the PTEN phosphatase (depleting PIP3 levels) and reduced expression of IRS-1 mediated by p70S6K. PI-103 (inset) inhibits p110α and both mTORC2 and the mTOR-raptor complex (mTORC1). This inhibits the PI3 kinase pathway at multiple sites and potentially accounts for the anticancer effects of PI-103 in malignant glioma4.

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796 VOLUME 24 NUMBER 7 JULY 2006 NATURE BIOTECHNOLOGY

The enthusiasm surrounding RNA interfer-ence1 (RNAi) as a new therapeutic modality has been tempered by a growing awareness of its potential to cause unwanted side effects. RNAi has been found variously to induce ‘off-target’ gene silencing, to trigger an inter-feron response leading to global inhibition of translation, to stimulate nonspecific RNA deg-radation by RNase L and to activate toll-like receptors and retinoic acid–inducible gene-1 protein, both of which promote adverse immune reactions. A recent paper by Grimm et al.2 in Nature adds another concern to this list: excessive RNAi can kill mice by clogging up the natural RNAi pathway.

RNAi is a ubiquitous biological phenom-enon in which small noncoding RNAs silence gene expression. It is triggered by double-stranded RNA (dsRNA) from a variety of sources (Fig. 1). One source is transcription of cellular genes encoding self-complementary RNA that forms imperfect hairpins, termed

primary microRNA (pri-miRNA), which are then trimmed by a specialized nuclear RNaseIII, Drosha, to generate pre-miRNA. The pre-miRNA is exported out of the nucleus by exportin-5, a Ran-GTP-dependent nucleocy-toplasmic transporter of the karyopherin fam-ily3. A second source of dsRNA is transcription of viral genomes.

Once in the cytoplasm, essentially any dsRNA that is ~26 base pairs or longer is cleaved by a cytoplasmic RNaseIII, Dicer, generating a 21- to 23-bp miRNA or a short interfering RNA (siRNA). In the final, execu-tion steps of RNAi, the antisense strand of the si/miRNA latches on to the complementary target mRNA as part of a large ribonucleo-protein complex, known as the RNA-induced silencing complex (RISC), either degrading the target or repressing its translation.

Researchers can exploit RNAi by introduc-ing dsRNA into cells (Fig. 1). Transcription of an inverted repeat from engineered, recom-binant DNA generates short hairpin RNA (shRNA) that follows the pre-miRNA path-way to produce designer miRNA. A segment of DNA cloned between two mutually facing promoters generates dsRNA that is diced by the cytoplasmic pathway to generate siRNA. Alternatively, mi/siRNAs are chemically syn-

thesized and directly transfected into the cell, bypassing the need for processing.

The net outcome of RNAi, then, is post-transcriptional silencing or ‘knockdown’ of gene expression, a property that is being har-nessed to silence disease-causing genes. But regardless of its utility in science and medicine, it is important to remember that RNAi is an essential process of normal cellular physiology. Mammalian genomes encode a few hundred miRNA genes that are regulated in health and disease and expressed in a tissue-specific man-ner4. Each miRNA silences multiple cellular mRNAs, and together, they regulate a large variety of important genes involved in cell death, differentiation and development.

Whereas synthetic mi/siRNA can provide short-term relief, for example, in acute viral infections5, therapy of chronic ailments such as genetic diseases, cancer or AIDS calls for long-term, sustained RNAi. To this end, Grimm et al. optimized an adeno-associated virus (AAV) vector and delivered the recom-binant AAV-shRNA clones into mouse liver by intravenous injection. The transfected liver cells expressed shRNAs that were processed into miRNAs (Fig. 1). Surprisingly, out of 49 different shRNA sequences tested, 36 resulted in liver injury, with 23 ultimately killing the mice within a month. A detailed study led to the discovery that the overexpressed recombi-nant pre-miRNAs overwhelmed and saturated exportin-5. The resultant traffic jam abrogated the exit of normal cellular pre-miRNAs, pre-venting their cytoplasmic maturation, and hence, function. The global shutdown of the cellular miRNA pathway led to lethality.

The authors performed a variety of control experiments to exclude other scenarios. Lethality did not require the presence of a target mRNA, thus ruling out gene silencing or formation of RISC as a contributing factor. Simultaneous disappearance of two mature miRNAs of the liver, miR-122 and let-7a, suggested that miRNA had been repressed globally, although other miRNAs were not tested. Loss of mir-122 function was confirmed by the expression of green fluorescent protein from a mir-122-repressible engineered message. Finally, overexpression of recombinant exportin-5 largely restored sh/miRNA function, suggesting a relief of saturation and extending similar conclusions made in cell culture6. In stark contrast to the high-producer shRNA clones, those that produced only modest amounts of shRNA showed no oversaturation or lethality, as expected. In fact, one such shRNA, designed against hepatitis B virus, protected mice against hepatitis B challenge with no signs of morbidity.

Sailen Barik is in the Department of Biochemistry and Molecular Biology, University of South Alabama, College of Medicine, 307 University Blvd., Mobile, Alabama 36688-0002, USA. e-mail: sbarik@jaguar1.usouthal.edu

RNAi in moderationSailen Barik

A new animal study shows that too much RNAi is lethal.

for developing PI3 kinase inhibitors as well as drugs that target other intermediates in the pathway, for example, PDK1, PKB/Akt and p70S6K (Fig. 1). Many upstream receptor tyrosine kinases are already effectively targeted by inhibitors that are in clinical use or in development. For example, rapamycin and its analogs have been used to inhibit the activity of mTORC1 in the clinic. Current PI3 kinase inhibitors such as PI-103 are pharmacological tools that require further optimization to become drugs. Of interest, an orally active s-triazine PI3 kinase inhibitor has been identified by screening compounds for those with an antiproliferative fingerprint similar to that of the prototype inhibitors LY294002 and wortmannin12. The compound ZSTK474 inhibited at least 3 class I lipid PI3 kinase enzymes and none of a range of protein kinases tested, although the activity on mTOR is not clear. Further work is needed to define

the optimal PI3 kinase selectivity profile for particular tumor types and mutation patterns. Nevertheless, it seems that the time is ripe for PI3 kinases to join their protein kinase relatives as targets for therapies based on the molecular profile of individual cancers.

1. Workman, P. Cold Spring Harbor Quant. Biol. 70, 1–18 (2005).

2. Shaw, R.J. & Cantley, L.C. Nature 441, 424–430 (2006).

3. Knight, Z.A. et al. Cell 125, 1–15 (2006).4. Fan Q.W et al. Cancer Cell 9, 341–349 (2006).5. Foukas, L.C. et al. Nature 441, 366–370 (2006).6. Samuels, Y. et al. Science 23, 304 (2004).7. Vivanco, I. & Sawyers, C.L. Nat. Rev. Cancer 2, 489–

501 (2002).8. Workman, P. et al. Eur. J. Cancer 2, 8 (Suppl) (2004).9. O’Reilly, K.E. et al. Cancer Res. 66, 1500–1508

(2006).10. Sabassov, D.D. et al. Science 307, 1098–1101

(2005).11. Feng, J. et al. J. Biol. Chem. 279, 41189–41196

(2004).12 Yaguchi, S. et al. J. Natl. Cancer Inst. 98, 545–556

(2006).

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