Showing papers by "Peter G. Schultz published in 2011"

Oxysterols direct immune cell migration via EBI2

Abstract: The EBI2 receptor (Epstein–Barr virus-induced gene 2, also known as GPR183) was recently shown to be linked to autoimmune disease, and is a critical regulator of the humoral immune response. It is a G-protein-coupled receptor, and its natural ligand has been unknown. Two groups now bring an end to the 'orphan' status of this receptor with identification of specific oxysterols as its natural ligands. The most potent ligand and activator is 7a,25-dihydroxycholesterol, and the EBI2–oxysterol signalling pathway has an important role in the adaptive immune response. Epstein–Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases1,2,3. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H)4. Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2–oxysterol signalling pathway has an important role in the adaptive immune response.

Imaging of Plasmodium Liver Stages to Drive Next-Generation Antimalarial Drug Discovery

Abstract: Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

An Evolved Aminoacyl-tRNA Synthetase with Atypical Polysubstrate Specificity,

Abstract: We have employed a rapid fluorescence-based screen to assess the polyspecificity of several aminoacyl-tRNA synthetases (aaRSs) against an array of unnatural amino acids. We discovered that a p-cyanophenylalanine specific aminoacyl-tRNA synthetase (pCNF-RS) has high substrate permissivity for unnatural amino acids, while maintaining its ability to discriminate against the 20 canonical amino acids. This orthogonal pCNF-RS, together with its cognate amber nonsense suppressor tRNA, is able to selectively incorporate 18 unnatural amino acids into proteins, including trifluoroketone-, alkynyl-, and halogen-substituted amino acids. In an attempt to improve our understanding of this polyspecificity, the X-ray crystal structure of the aaRS-p-cyanophenylalanine complex was determined. A comparison of this structure with those of other mutant aaRSs showed that both binding site size and other more subtle features control substrate polyspecificity.

Optimized clinical performance of growth hormone with an expanded genetic code

Abstract: The ribosomal incorporation of nonnative amino acids into polypeptides in living cells provides the opportunity to endow therapeutic proteins with unique pharmacological properties. We report here the first clinical study of a biosynthetic protein produced using an expanded genetic code. Incorporation of p-acetylphenylalanine (pAcF) at distinct locations in human growth hormone (hGH) allowed site-specific conjugation with polyethylene glycol (PEG) to produce homogeneous hGH variants. A mono-PEGylated mutant hGH modified at residue 35 demonstrated favorable pharmacodynamic properties in GH-deficient rats. Clinical studies in GH-deficient adults demonstrated efficacy and safety comparable to native human growth hormone therapy but with increased potency and reduced injection frequency. This example illustrates the utility of nonnative amino acids to optimize protein therapeutics in an analogous fashion to the use of medicinal chemistry to optimize conventional natural products, low molecular weight drugs, and peptides.

Chemical Control of Stem Cell Fate and Developmental Potential

Abstract: Potential applications of stem cells in medicine range from their inclusion in disease modeling and drug discovery to cell transplantation and regenerative therapies. However, before this promise can be realized several obstacles must be overcome, including the control of stem cell differentiation, allogeneic rejection and limited cell availability. This will require an improved understanding of the mechanisms that govern stem cell potential and the development of robust methods to efficiently control their fate. Recently, a number of small molecules have been identified that can be used both in vitro and in vivo as tools to expand stem cells, direct their differentiation, or reprogram somatic cells to a more naive state. These molecules have provided a wealth of insights into the signaling and epigenetic mechanisms that regulate stem cell biology, and are already beginning to contribute to the development of effective treatments for tissue repair and regeneration.

Evolution of cyclic peptide protease inhibitors

Abstract: We report a bacterial system for the evolution of cyclic peptides that makes use of an expanded set of amino acid building blocks. Orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs, together with a split intein system were used to biosynthesize a library of ribosomal peptides containing amino acids with unique structures and reactivities. This peptide library was subsequently used to evolve an inhibitor of HIV protease using a selection based on cellular viability. Two of three cyclic peptides isolated after two rounds of selection contained the keto amino acid p-benzoylphenylalanine (pBzF). The most potent peptide (G12: GIXVSL; X = pBzF) inhibited HIV protease through the formation of a covalent Schiff base adduct of the pBzF residue with the ϵ-amino group of Lys 14 on the protease. This result suggests that an expanded genetic code can confer an evolutionary advantage in response to selective pressure. Moreover, the combination of natural evolutionary processes with chemically biased building blocks provides another strategy for the generation of biologically active peptides using microbial systems.

Probing protein-protein interactions with a genetically encoded photo-crosslinking amino acid

Abstract: Considerable effort has been devoted to mapping the complex interactions that make up the molecular circuitry of living cells. For weak or transient interactions that are not easily identified through affinity-based approaches, methods such as yeast two-hybrid screening, protein-fragment complementation, chemical crosslinking, and photo-crosslinking are particularly useful. Photo-crosslinkers can be directly incorporated into proteins, 7] nucleic acids, 9] and carbohydrates 11] by exogenously supplying cells with appropriate precursors. Alternatively, photo-crosslinking moieties can be site-specifically introduced into proteins chemically or enzymatically, or in response to nonsense codons by means of orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pairs. For example, amino acids containing benzophenone, diazirine, and aryl azide side chains have all been genetically encoded in prokaryotic and eukaryotic organisms, and used to crosslink protein–protein and protein–DNA interactions in vitro and in living cells. However, the size and photochemical reactivity of these photo-crosslinkers might not be ideal for a given site in a target protein of interest. Recently, an aliphatic diazirine amino acid, 3’-azibutyl-N-carbamoyl-lysine (AbK, Figure 1 A) was introduced into proteins by using the wild-type M. barkeri pyrrolysyl tRNA/tRNA synthetase pair (wt-mbPylRS/tRNA). Site-specific incorporation of AbK into glutathione S-transferase allowed covalent crosslinking of the two subunits of the dimeric protein in E. coli by using UV light. Inspired by this success, we sought to solve another limitation: in mammalian cells, photo-crosslinking amino acids typically have a low efficiency of incorporation in response to amber (TAG) codons. 25] Here we report that a new aminoacyl-tRNA synthetase engineered from wt-mbPylRS, when coupled with tRNA, can significantly increase the coding efficiency of AbK in both E. coli and mammalian cells. Moreover, when Abk was substituted for Asp144 in cyclin-dependent kinase 5 (Cdk5), the diazirine moiety photo-crosslinked Cdk5 to its substrate, p21-activated kinase 1 (Pak1). Previously, wild-type M. maize PylRS and mbPylRS have been used to incorporate a variety of carbamoyl lysine derivatives into proteins. We also confirmed that the wt-mbPylRS/ tRNA pair can indeed suppress the TAG codon in the presence of AbK. A plasmid encoding wt-mbPylRS (pBK-PylRS) was used to cotransform E. coli DH10B cells along with a second plasmid pMyo-Lys99TAG (harboring a His6-tagged myoglobin gene containing a Lys99TAG mutation, and one copy of the pyrrolysyl-tRNA gene). Protein expression was induced in the presence of 1 mm AbK. Ni-NTA affinity chromatography was used to purify full-length myoglobin, and eluted protein was analyzed by SDS-PAGE. Although full-length protein was observed when AbK was present in culture media, the yield was low (0.7 0.2 mg myoglobin per liter of culture; Figure 1 B). Previous work has also made use of mutant PylRS/tRNA pairs to encode lysine analogues. We postulated that the wt-mbPylRS/tRNA pair could be subjected to directed evolution to optimize its amber suppression efficiency in the presence of AbK. We therefore used a mbPylRS library randomized at residues Leu270, Tyr271, Leu274, and Cys313 (and an additional Tyr349Phe mutation) to select synthetases that can efficiently charge AbK to tRNA. A series of positive and negative selections were performed in E. coli strain DH10B as previously described. In brief, in the positive selection, PylRS variants were introduced into E. coli containing a plasmid encoding chloramphenicol acetyltransferase with the Asp112TAG mutation, and cells were selected for chloramphenicol resistance in the presence of 1 mm AbK; the negative selection was carried out with a toxic barnase gene with amber mutations at multiple sites (Gln2TAG, Asp44TAG, and Gly65TAG), and the selection was carried out in the absence of AbK. After three positive and two negative rounds of selection, single E. coli colonies containing PylRS mutants were obtained. The sequences of mutant synthetases converged to a unique clone (AbKRS; mbPylRS-Leu274Met/Cys313Ala/Tyr349Phe). Next, we tested amber suppression of pMyo-Lys99TAG by the AbKRS/tRNA Figure 1. A) The chemical structure of 3’-azibutyl-N-carbamoyl-lysine (AbK). B) SDS-PAGE of Ni-NTA-purified mutant myoglobin (lane 1: AbKRS in the presence of 1 mm AbK; lane 2: AbKRS in the absence of AbK; lane 3: wtmbPylRS in the presence of 1 mm AbK; lane 4: wt-mbPylRS in the absence of AbK).

A Chemical Genomic Analysis of Decoquinate, a Plasmodium falciparum Cytochrome b Inhibitor

Abstract: Decoquinate has single-digit nanomolar activity against in vitro blood stage Plasmodium falciparum parasites, the causative agent of human malaria. In vitro evolution of decoquinate-resistant parasites and subsequent comparative genomic analysis to the drug-sensitive parental strain revealed resistance was conferred by two nonsynonymous single nucleotide polymorphisms in the gene encoding cytochrome b. The resultant amino acid mutations, A122T and Y126C, reside within helix C in the ubiquinol-binding pocket of cytochrome b, an essential subunit of the cytochrome bc(1) complex. As with other cytochrome bc(1) inhibitors, such as atovaquone, decoquinate has low nanomolar activity against in vitro liver stage P. yoelii and provides partial prophylaxis protection when administered to infected mice at 50 mg kg(-1). In addition, transgenic parasites expressing yeast dihydroorotate dehydrogenase are >200-fold less sensitive to decoquinate, which provides additional evidence that this drug inhibits the parasite's mitochondrial electron transport chain. Importantly, decoquinate exhibits limited cross-resistance to a panel of atovaquone-resistant parasites evolved to harbor various mutations in cytochrome b. The basis for this difference was revealed by molecular docking studies, in which both of these inhibitors were shown to have distinctly different modes of binding within the ubiquinol-binding site of cytochrome b.

Two-dimensional IR spectroscopy of protein dynamics using two vibrational labels: a site-specific genetically encoded unnatural amino acid and an active site ligand.

Abstract: Protein dynamics and interactions in myoglobin (Mb) were characterized via two vibrational dynamics labels (VDLs): a genetically incorporated site-specific azide (Az) bearing unnatural amino acid (AzPhe43) and an active site CO ligand. The Az-labeled protein was studied using ultrafast two-dimensional infrared (2D IR) vibrational echo spectroscopy. CO bound at the active site of the heme serves as a second VDL located nearby. Therefore, it was possible to use Fourier transform infrared (FT-IR) and 2D IR spectroscopic experiments on the Az in unligated Mb and in Mb bound to CO (MbAzCO) and on the CO in MbCO and MbAzCO to investigate the environment and motions of different states of one protein from the perspective of two spectrally resolved VDLs. A very broad bandwidth 2D IR spectrum, encompassing both the Az and CO spectral regions, found no evidence of direct coupling between the two VDLs. In MbAzCO, both VDLs reported similar time scale motions: very fast homogeneous dynamics, fast, ∼1 ps dynamics, and dynamics on a much slower time scale. Therefore, each VDL reports independently on the protein dynamics and interactions, and the measured dynamics are reflective of the protein motions rather than intrinsic to the chemical nature of the VDL. The AzPhe VDL also permitted study of oxidized Mb dynamics, which could not be accessed previously with 2D IR spectroscopy. The experiments demonstrate that the combined application of 2D IR spectroscopy and site-specific incorporation of VDLs can provide information on dynamics, structure, and interactions at virtually any site throughout any protein.

Incorporation of fluorotyrosines into ribonucleotide reductase using an evolved, polyspecific aminoacyl-tRNA synthetase.

Abstract: Tyrosyl radicals (Y·s) are prevalent in biological catalysis and are formed under physiological conditions by the coupled loss of both a proton and an electron. Fluorotyrosines (FnYs, n = 1–4) are promising tools for studying the mechanism of Y· formation and reactivity, as their pKa values and peak potentials span four units and 300 mV, respectively, between pH 6 and 10. In this manuscript, we present the directed evolution of aminoacyl-tRNA synthetases (aaRSs) for 2,3,5-trifluorotyrosine (2,3,5-F3Y) and demonstrate their ability to charge an orthogonal tRNA with a series of FnYs while maintaining high specificity over Y. An evolved aaRS is then used to incorporate FnYs site-specifically into the two subunits (α2 and β2) of Escherichia coli class Ia ribonucleotide reductase (RNR), an enzyme that employs stable and transient Y·s to mediate long-range, reversible radical hopping during catalysis. Each of four conserved Ys in RNR is replaced with FnY(s), and the resulting proteins are isolated in good yield...

Incorporation of Fluorotyrosines into Ribonucleotide Reductase Using an Evolved, Polyspecific Aminoacyl-tRNA Synthetase

Abstract: Tyrosyl radicals (Y·s) are prevalent in biological catalysis and are formed under physiological conditions by the coupled loss of both a proton and an electron. Fluorotyrosines (FnYs, n = 1–4) are promising tools for studying the mechanism of Y· formation and reactivity, as their pKa values and peak potentials span four units and 300 mV, respectively, between pH 6 and 10. In this manuscript, we present the directed evolution of aminoacyl-tRNA synthetases (aaRSs) for 2,3,5-trifluorotyrosine (2,3,5-F3Y) and demonstrate their ability to charge an orthogonal tRNA with a series of FnYs while maintaining high specificity over Y. An evolved aaRS is then used to incorporate FnYs site-specifically into the two subunits (α2 and β2) of Escherichia coli class Ia ribonucleotide reductase (RNR), an enzyme that employs stable and transient Y·s to mediate long-range, reversible radical hopping during catalysis. Each of four conserved Ys in RNR is replaced with FnY(s), and the resulting proteins are isolated in good yield...

A Small Molecule Modulates Circadian Rhythms through Phosphorylation of the Period Protein

Abstract: Time shift A high-throughput cell-based screen identified a benzothiazole analogue, LH846, which induces period lengthening of the circadian rhythm. Affinity chromatography coupled with mass spectrometry and genomic analysis identified protein kinase CKIδ as the biological target of LH846 (see picture).

Pan-Src family kinase inhibitors replace Sox2 during the direct reprogramming of somatic cells.

Abstract: Ectopic expression of the four transcription factors Oct4, Klf4, Sox2 and c-Myc reprograms adult somatic cells to induced pluripotent stem (iPS) cells.[1] Although iPS cells hold considerable promise as tools in research and drug discovery, the clinical application of iPS cells is hindered by the use of viruses that deliver the exogenous factors and modify the host genome. It is therefore of great interest to replace virally transduced factors with either proteins or small molecules. To date a number of compounds have been identified that facilitate reprogramming of somatic cells. Among these are kenpaullone[2], valproic acid[3] and inhibitors of TGFβ-signaling.[4] Here we have exploited a reporter based screen[2] to identify a new class of compounds that functionally replace Sox2: inhibitors of the Src family of kinases. These molecules provide novel tools to study the molecular mechanism of Sox2 in reprogramming. To screen for small molecule replacements of Sox2, mouse embryonic fibroblasts (MEFs) harboring the firefly luciferase (Fluc) gene in the Nanog locus[2] (NL-MEFs) were transduced with Oct4, Klf4 and c-Myc (OKM), seeded into 1536-well plates in standard growth media and assayed against a large chemical library[5] (750,000 compounds; 2.2 μM). Compounds that reproducibly and dose-dependently activated the NL reporter >2.5-fold over vehicle-treated controls (Figure 1a) were then counter-screened in a cell based SV40-driven Fluc assay to rule out false positives that directly and non-specifically induce luciferase signal.[2, 6] Figure 1 Chemical complementation of Sox2 To confirm that filtered hit compounds which activate Nanog gene expression also replace Sox2, iPS cell colony formation was used as a secondary assay. Specifically, Klf4 and c-Myc were delivered retrovirally to O4NR-MEFs[1b] (cells harboring a Doxycycline (Dox)-inducible Oct4 cDNA in the collagen locus and the neomycin-resistance gene in the Oct4 locus), and Oct4 expression was induced by addition of Dox to the culture media (day 0). Two days later, positive screen hits (1-10 μM) were added to OKM-expressing MEFs in place of Sox2. After 10 days of compound treatment, growth media was supplemented with neomycin to select for colonies that reactivated the endogenous Oct4 locus. The reactivation of epigenetically silenced pluripotency-associated genes is required for somatic cells to transition to the iPS cell state.[7] Dox-independent, neomycin resistant colonies were not observed in DMSO-treated (0.1%, v/v) controls, indicating that vehicle-treated cells had not removed the epigenetic silencing marks from the Oct4 promoter (which drives NeoR) and were thus not pluripotent. Among the compounds tested, one compound, iPYrazine (iPY; 10 μM), promoted the formation of neomycin-resistant iPS cell colonies (Figure 1b, blue bars) that survived and could be cultured in the absence of Dox. Transgenic Oct4 independent (minus Dox) growth of the iPY-treated iPS cells demonstrated that they had reactivated and relied on endogenous Oct4 to maintain the pluripotent state. In addition, OKM transduction combined with iPY treatment of MEFs carrying a GFP reporter under control of the endogenous Oct4 locus[8] also gave rise to stable, GFP-positive iPS cell lines (Figure S1, Supporting Information). iPS cells derived from O4NR-MEFs with iPY, Dox and KM-transduction grew as pluripotent stem cell colonies in the absence of Dox and iPY. Moreover, these cells were indistinguishable from ES cells by morphological criteria and expressed the pluripotency-associated markers Oct4 and SSEA1 (Figure 1c). We next tested the differentiation potential of the iPY-derived iPS cells in a teratoma assay by injecting 106 cells subcutaneously into NOD-SCID mice. Tumors were isolated 3 weeks later and histological analyses demonstrated that cell types of all three germ layers were present; these included neural tissues, bone, cartilage and ciliated epithelium (Figure 1d). Furthermore, iPY-derived iPS cells contributed to live chimeras, as shown in Figure 1d. The results from this series of analyses indicate that the iPY-derived, Sox2-free iPS cells are pluripotent. In order to identify the biological target of iPY, we profiled the compound against a biochemical panel of tyrosine kinases (51 kinases; Table S1). From this analysis, we found that iPY potently inhibited a number of tyrosine kinases at 5 μM. Commercially available inhibitors (Figures 2a-b and Table S2) of these candidate kinase targets were then assayed for their ability to replace Sox2 in the iPS cell colony formation assay. As shown in Figure 2b, the pan-Src family kinase (SFK) inhibitors Dasatinib[9] and PP1[10] (Figure 2b) were able to recapitulate the activity of iPY. Interestingly, both Dasatinib and PP1 were >2-fold more active than iPY and efficiently replaced Sox2 (Figure 2b). Moreover, the pan-SFK inhibitors gave rise to colonies with a similar efficiency to TGFβ inhibitors (SB-431542 and LY-364947). The latter have been reported to replace Sox2 and served as a positive control in this study.[4] In addition to TGFβ inhibitors, Ichida et al. have also reported that the SFK inhibitor PP1 is able to replace Sox2.[4a] Together with our work, these results indicate that iPY is likely playing a role in reprogramming by inhibiting Src kinases, although additional mechanisms cannot be excluded. Figure 2 Src family kinase and TGFβ-inhibitors recapitulate the Sox2 replacement activity of iPY SFKs are a class of proto-oncogene tyrosine kinases that include nine mammalian members (i.e., c-Src, Yes, Fyn, Fgr, Lck, Hck, Blk, Lyn and Frk).[11] Several members of the SFK family have been reported to influence embryonic stem (ES) cell self-renewal and differentiation.[12] For example, activation of c-Src signaling promotes ES cell differentiation.[13] Consistent with this observation we find that the activation of Src signaling in MEFs with JK239[14] potently inhibits 4-factor reprogramming (Figure 2c). Together, our results suggest that SFK signaling is an important mediator of somatic cell reprogramming, where activation of the SFK pathway prevents reprogramming and inhibition allows for reprogramming in the absence of exogenous Sox2. Previously, Ichida et al. demonstrated that small molecule mediated inhibition of TGFβ-signaling with LY-364947 or E-616452 can replace Sox2 through the activation of Nanog expression.[4a] The results from our screen, which rely on Nanog activation as a surrogate for the replacement of Sox2, suggest that the inhibition of SFK- and TGFβ-signaling may converge on a similar mechanism; that is, the function of Sox2 can be replaced during direct reprogramming by activating Nanog expression. Another potential scenario comes from the observation that both Nanog[15] and SFK inhibition[13] are capable of maintaining the self-renewing pluripotent state in ES cells. Thus, TGFβ inhibitor-mediated Nanog activation and pan-SFK inhibition may instead converge on a common mechanism in which the differentiation of newly formed iPS cells is prevented, thereby assisting in the transition to an undifferentiated state. In either case, it is interesting to note that inhibition of distinct signaling responses converge on a common end point. In summary, we applied a cell-based, high-throughput chemical screen to identify small molecules that replace Sox2 during somatic cell reprogramming. The identification of novel SFK inhibitors provides new chemical tools to study the mechanisms underlying direct reprogramming and may ultimately help to bring iPS cell technology one step closer to clinical application.

Subcellular protein localization by using a genetically encoded fluorescent amino acid.

Abstract: The use of fluorescent protein fusions has revolutionized cell biology by allowing exploration of proteins in their native context. However, the utilization of current techniques is limited by the size and placement of the fused fluorescent protein. This is especially true for proteins that oligomerize or assemble into large complexes in which the fluorescent protein fusion can lead to improper assembly and/or function. To circumvent this problem, we report a novel technique for fluorescent labeling of proteins, in vivo. The unique potential of this technique lies in the ability to place a very small fluorescent tag virtually anywhere along a chosen protein sequence, thereby minimizing the risk of affecting protein function. To illustrate the utility of this method, we have genetically encoded a single unnatural fluorescent amino acid in the sequence of the bacterial tubulin, FtsZ. This resulted in the production of a functional protein that could be visualized, in vivo. The aim of this study was to explore novel methods of labeling proteins for in vivo localization studies that do not perturb protein function or structure. To this end, we have exploited a technique that allows unnatural amino acids with novel properties, in our case a fluorescent, coumarin-derived amino acid (CouAA; Figure 1A), to be encoded into a given sequence. The technique uses an orthogonal tRNA/aminoacyl-tRNA synthetase pair that transfers a defined unnatural amino acid to a growing polypeptide chain when nonsense amber codons (UAG) are present in the coding sequence. Specifically, we have used an archaebacteria amber suppressor tRNA (MjtRNA)/aminoacyl-tRNA synthetase (Mj-aaRS) pair (from Methanococcus jannaschi) that does not interact with endogenous E. coli aminoacyl-tRNA synthetases or tRNAs. This aaRS (CouRS) was then evolved by using a two-step selection process to specifically recognize CouAA and not an endogenous host amino acid. As a consequence, by simply inserting an amber stop codon in the desired gene sequence, an unnatural amino acid will be introduced in the corresponding translated protein (see cartoon in the Supporting Information). Conveniently, endogenous amber codons in E. coli and other organisms are likely poorly recognized by the Mj-tRNA, so that the expression of genes terminated by amber codons is apparently not affected. Although the exact mechanisms are not understood, it is known that the termination and suppression processes are influenced by sequences adjacent to the amber codon. To demonstrate that this system can be effectively used as a means to visualize the in vivo subcellular location of a CouAAlabeled protein in bacteria, we chose to label the bacterial tubulin homologue FtsZ, which has been extensively studied both in vitro and in vivo. FtsZ assembles into a contractile ring (visible by fluorescence microscopy as a midcell band called Zring) during cytokinesis. But fusion of FtsZ to a fluorescent protein impairs its cellular function, a well-known problem for cytoskeletal proteins. Consequently, to date, all the reported FtsZ–fluorescent protein fusions have been shown to be nonfunctional on their own and must be produced in the presence of an untagged FtsZ copy for normal cell function. Thus, in this context, the FtsZ–fluorescent protein fusions merely label the endogenous, untagged FtsZ structure. While this approach has proven to be sufficient to generate considerable insight into FtsZ cellular function, it remains an imperfect artifice that might fail in the case of other oligomer-forming proteins or proteins forming macromolecular complexes. To visualize FtsZ in E. coli, we substituted the tenth amino acid (Asp10) for CouAA (FtsZ10CouAA). Asp10 is located in a disordered N-terminal segment with no known function and is likely to be surface exposed. To characterize the specificity of CouAA incorporation, Histagged FtsZ10CouAA was expressed in BL21(DE3) E. coli cells transformed with two plasmids: one constitutively expressed the evolved Mj-tRNA (pBKcouRS) and the other one (pBADJYftsZH6D10TAG) constitutively expressed the evolved tRNA synthetase (CouRS) and the mutated ftsZ gene under an arabinose inducible promoter. SDS-PAGE analysis (Figure 1B) of total cell extract of cells grown in the absence or presence of CouAA (1 mm) in the growth medium to express FtsZ10CouAA, showed a unique band at approximately 40 kDa only with [a] Dr. G. Charbon, Prof. Dr. C. Jacobs-Wagner Department of Molecular, Cellular, and Developmental Biology KBT 1032, Yale University, New Haven, CT 06520 (USA) [b] Dr. G. Charbon, Prof. Dr. A. Lobner-Olesen Department of Science, Systems and Models, Roskilde University Building 18.1, 4000 Roskilde (Denmark) [c] Dr. E. Brustad, Prof. Dr. J. Wang, Prof. Dr. P. G. Schultz Department of Chemistry, The Scripps Research Institute SR202, 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA) [d] K. A. Scott, Prof. Dr. E. Chapman Department of Molecular Biology, The Scripps Research Institute MB46, 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA) E-mail : echapman@scripps.edu [e] Prof. Dr. C. Jacobs-Wagner Howard Hughes Medical Institute New Haven, CT 06510 (USA) [f] Prof. Dr. C. Jacobs-Wagner Microbial Pathogenesis Section, Yale School of Medicine New Haven, CT 06510 (USA) [g] Prof. Dr. J. Wang Present address: National Key Laboratory of Biomacromolecules Institute of Biophysics, Chinese Academy of Sciences Beijing 100101 (China) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201100282.

A Chemically Induced Vaccine Strategy for Prostate Cancer

Abstract: Here we report the design and evaluation of a bifunctional, small molecule switch that induces a targeted immune response against tumors in vivo. A high affinity ligand for prostate specific membrane antigen (PSMA) was conjugated to a hapten that binds dinitrophenyl (DNP)-specific antibodies. When introduced into hu-PBL-NOD/SCID mice previously immunized with a KLH-DNP immunogen, this conjugate induced a targeted antibody-dependent cellular cytotoxicity (ADCC) response to PSMA-expressing tumor cells in a mouse xenograft model. The ability to create a small molecule inducible antibody response against self-antigens using endogenous non-autoreactive antibodies may provide advantages over the autologous immune response generated by conventional vaccines in certain therapeutic settings.

Loss of CD4 T-cell–dependent tolerance to proteins with modified amino acids

Abstract: The site-specific incorporation of the unnatural amino acid p-nitrophenylalanine (pNO2Phe) into autologous proteins overcomes self-tolerance and induces a long-lasting polyclonal IgG antibody response. To determine the molecular mechanism by which such simple modifications to amino acids are able to induce autoantibodies, we incorporated pNO2Phe, sulfotyrosine (SO3Tyr), and 3-nitrotyrosine (3NO2Tyr) at specific sites in murine TNF-α and EGF. A subset of TNF-α and EGF mutants with these nitrated or sulfated residues is highly immunogenic and induces antibodies against the unaltered native protein. Analysis of the immune response to the TNF-α mutants in different strains of mice that are congenic for the H-2 locus indicates that CD4 T-cell recognition is necessary for autoantibody production. IFN-γ ELISPOT analysis of CD4 T cells isolated from vaccinated mice demonstrates that peptides with mutated residues, but not the wild-type residues, are recognized. Immunization of these peptides revealed that a CD4 repertoire exists for the mutated peptides but is lacking for the wild-type peptides and that the mutated residues are processed, loaded, and presented on the I-Ab molecule. Overall, our results illustrate that, although autoantibodies are generated against the endogenous protein, CD4 cells are activated through a neo-epitope recognition mechanism. Therefore, tolerance is maintained at a CD4 level but is broken at the level of antibody production. Finally, these results suggest that naturally occurring posttranslational modifications such as nitration may play a role in antibody-mediated autoimmune disorders.

Regenerative phenotype in mice with a point mutation in transforming growth factor β type I receptor (TGFBR1)

Abstract: Regeneration of peripheral differentiated tissue in mammals is rare, and regulators of this process are largely unknown. We carried out a forward genetic screen in mice using N-ethyl-N-nitrosourea mutagenesis to identify genetic mutations that affect regenerative healing in vivo. More than 400 pedigrees were screened for closure of a through-and-through punch wound in the mouse ear. This led to the identification of a single pedigree with a heritable, fast, and regenerative wound-healing phenotype. Within 5 wk after ear-punch, a threefold decrease in the diameter of the wound was observed in the mutant mice compared with the wild-type mice. At 22 wk, new cartilage, hair follicles, and sebaceous glands were observed in the newly generated tissue. This trait was mapped to a point mutation in a receptor for TGF-β, TGFBR1. Mouse embryonic fibroblasts from the affected mice had increased expression of a subset of TGF-β target genes, suggesting that the mutation caused partial activation of the receptor. Further, bone marrow stromal cells from the mutant mice more readily differentiated to chondrogenic precursors, providing a plausible explanation for the enhanced development of cartilage islands in the regenerated ears. This mutant mouse strain provides a unique model to further explore regeneration in mammals and, in particular, the role of TGFBR1 in chondrogenesis and regenerative wound healing.

Dual regulation of hepatitis C viral RNA by cellular RNAi requires partitioning of Ago2 to lipid droplets and P-bodies.

Abstract: The antiviral role of RNA interference (RNAi) in humans remains to be better understood. In RNAi, Ago2 proteins and microRNAs (miRNAs) or small interfering RNAs (siRNAs) form endonucleolytically active complexes which down-regulate expression of target mRNAs. P-bodies, cytoplasmic centers of mRNA decay, are involved in these pathways. Evidence exists that hepatitis C virus (HCV) utilizes host cellular RNAi machinery, including miRNA-122, Ago1-4, and Dicer proteins for replication and viral genome translation in Huh7 cells by, so far, nebulous mechanisms. Conversely, synthetic siRNAs have been used to suppress HCV replication. Here, using a combination of biochemical, transfection, confocal imaging, and digital image analysis approaches, we reveal that replication of HCV RNA depends on recruitment of Ago2 and miRNA-122 to lipid droplets, while suppression of HCV RNA by siRNA and Ago2 involves interaction with P-bodies. Such partitioning of Ago2 proteins into different complexes and separate subcellular domains likely results in modulation of their activity by different reaction partners. We propose a model in which partitioning of host RNAi and viral factors into physically and functionally distinct subcellular compartments emerges as a mechanism regulating the dual interaction of cellular RNAi with HCV RNA.

Small-molecule regulators of human stem cell self-renewal.

Abstract: Agents that selectively control the self-renewal and differentiation of human hematopoietic stem cells (hHSCs) can serve as useful tools for understanding the basic developmental biology of hematopoiesis. 2] The identification of molecules that regulate HSC fate might also lead to new therapies for the treatment of cancer and genetic blood diseases. For example, the ability to use cord-blood (CB)-derived HSCs would greatly facilitate the finding of matched donors for allogenic bone marrow transplants. 4] However, because engraftment depends on HSC number, and the number of human HSCs available from individual cord blood units is small, the widespread application of cord-blood HSCs is currently limited to pediatric transplants. 6] Thus, the ability to expand HSCs, especially CBderived HSCs, during ex vivo culture would have a major impact on the field of HSC transplantation. We recently identified the purine derivative stem regenin1 (SR1), which expands CB-derived HSCs ex vivo by selectively antagonizing the aryl hydrocarbon receptor (AhR). CB-derived HSCs expanded with SR1 maintain full multilineage potential and engraft efficiently in the NOD Scid Gamma (NSG) mouse transplant model. To identify alternative chemical scaffolds that induce HSC self-renewal through similar or distinct mechanisms, we screened additional chemical libraries (>100 000 discrete small heterocyclic chemical compounds) in a cell-based phenotypic screen, and now report a series of benzimidazoles, diarylamides, and flavonoids that induce HSC self-renewal (Scheme 1). This assay takes advantage of advances in screening technology developed in our group that permit lowvolume (~40 mL) screens to be conducted in a massively parallel fashion with the use of advanced automation. The primary screen was a seven-day assay using mobilized blood-derived CD34 cells in which the loss of CD34 expression (phenotypic hematopoietic stem and progenitor marker) was monitored by high-throughput flow cytometry. 8] A variety of heterocyclic compounds such as flavonoid, benzimidazole, phenylbenzamide, quinoxaline, benzoxazole, and benzothiazole derivatives were identified as potential hits. Secondary assays were then conducted to confirm the phenotypic changes observed in the primary screen. First, compounds were tested for their ability to significantly increase (up to twofold) the number of CD34-expressing cells in a dose-dependent fashion. Next, we determined whether compounds expand CD34 cells isolated from human cord blood. One thousand CB-derived CD34 cells were cultured with each compound, and the cell number and phenotype were measured weekly. After three weeks of culture, three compounds (SR2, SR3, and SR4) were able to expand the number of CD34 cells. Nucleated cell numbers in the cultures increased on average sevento tenfold in the compound-treated culture (SR2, SR3, and SR4) compared to the vehicle-treated culture (DMSO, 0.01 %; see the Supporting Information). Most importantly, 28– 48 % of compound-treated cells (SR2, SR3, and SR4) were CD34 compared to 13 % in vehicle control cultures (see Figure 1). These results suggest that SR2, SR3, and SR4 are expanding phenotypic hematopoietic stem and progenitor cells. Notably, when cultured in the absence of cytokines, these compounds did not induce proliferation of CD34 cells, neither did they have an effect on murine HSCs (LSK: Lin Sca-1 Kit). We next carried out a preliminary structure–activity study of SR2, SR3 and SR4, beginning with the benzimidazole (SR2) scaffold. A collection of benzimidazole derivatives was prepared in an expedient manner from 3-nitro-4-fluorobenzoic acid. The fluoro-group was displaced by nucleophilic aromatic substitution, thus allowing facile incorporation of the amine function at C4. The acid moiety was readily transformed into Scheme 1. Structures of SR1 and compounds identified in the primary screen that expand phenotypic HSCs (SR2–5).

Dorsomorphin promotes human embryonic stem cell self-renewal

Abstract: Human embryonic stem cells (hESCs), derived from the inner cell mass (ICM) of the blastocyst, have the capacity for longterm undifferentiated growth in culture and the theoretical potential to differentiate into all somatic cell types. These hESCs offer not only a model system for human development, but also a potentially unlimited source of graft material for transplantation-based therapies. Long-term hESCs can be supported using feeder-fibroblast conditioned medium (CM), or high concentrations of basic FGF, PEDF, or TGF-b/activin/Nodal proteins. Recently, small molecules that can support mouse embryonic stem cell self-renewal and hESC self-renewal have been reported. Unfortunately, none of the reported small molecules is able to support longterm hESC self-renewal (five or more passages) under defined culture conditions without bFGF or TGF-b/activin/ Nodal proteins. Consequently, unbiased cellular screens for small molecules that regulate long-term hESC self-renewal may provide new insights into stem cell biology, and also facilitate practical applications of ES cells in research and therapy. Herein, we report the identification of a small molecule that promotes long-term hESC self-renewal. To screen for small-molecules that promote self-renewal, H9 hESCs were seeded into matrigel coated 384-well plates at 2000 cellswell 1 in UM medium (DMEM/F12 + 1X N2/B27 supplement) and screened against a diverse chemical library of 50 000 heterocyclic compounds. Compounds were added a day after plating at a final concentration of 2 mm in 0.1% DMSO. After treatment with compound for 7 days, the cells were fixed and stained with OCT-4 antibodies (OCT-4 is highly expressed in ES cells and downregulated upon differentiation), and analyzed using an Opera high-content confocal image system. Of 11 confirmed screen hits (Supporting Information Figure S1), dorsomorphin, (Figure 1a), an inhibitor of bone morphogenic protein (BMP) type I receptors (ALK2, ALK3, and ALK6), maintained the highest percentage of OCT4 positive cells in a dose-dependent manner, with an EC50 of 1 mm (Figure 1b,c). Further immunohistochemical analysis indicated that greater than 90% of the cells maintain expression of the self-renewal associated proteins NANOG, SOX2, SSEA-4, and Tra-1-80 after five passages in UM medium plus DORSO (2 mm) (Figure 2), compared to less than 20% of the hESC cells grown in UM plus 0.1% DMSO (Figure 1b and Supporting Information Figure S2). To determine whether hESCs expanded in the presence of DORSO retain pluripotency, cells expanded through five passages in the presence of 2 mm DORSO were assessed for their ability to differentiate into multiple lineages in vitro. hESCs-H9 cells treated with DORSO for 7 days formed embryoid bodies in suspension culture supplemented with Figure 1. a) Chemical structure of dorsomorphin (DORSO); b) After 7 days of culture in UM medium without bFGF, hESC-H9 cells treated with DMSO (0.1%) or DORSO (2 mm) were fixed, stained, and imaged by confocal microscopy for OCT4 protein expression. Cell nuclei were stained with DAPI (blue); c) After 7 days of culture, DORSO maintains OCT4 expression in HESC-H9 and H1 cell lines in a dose-dependent manner. Values are the mean SD for three measurements.

Identification and Characterization of Small-Molecule Inducers of Fetal Hemoglobin

Abstract: A single nucleotide change from GAG to GTG in the b-globin gene leads to an amino acid mutation (glutamate to valine) that causes SCD. 2] Hemoglobin with the mutated b-globin (sickle hemoglobin, HbS, a2b s 2) polymerizes at low oxygen tension, resulting in a sickling of erythrocytes. This morphological change significantly affects the oxygen delivering ability of erythrocytes and leads to various pathological conditions, including vaso-occlusive crisis, anemia, ischemia, and severe infections. Approximately 1 in 500 newborn African Americans has the disease. Humans carry eight globin genes with distinct developmental expression patterns. While gamma (g) globin is expressed during gestation to generate fetal hemoglobin (HbF), which has a stronger affinity for molecular oxygen than maternal hemoglobin, its expression is silenced postnatally. Beta (b) globin expression commences shortly after birth and replaces gglobin in the formation of adult hemoglobin (HbA) tetramers. It has been shown that increased expression of HbF (a2g2) reduces polymerization of HbS in SCD patients and relieves vaso-occlusive crises and other symptoms. Induction of gglobin, therefore, has been a central strategy for the treatment of SCD; 7] it has been postulated that only a 20–30 % increase in g-globin synthesis is needed to alleviate symptoms. However, a few inducers of g-globin expression have been reported. To this end, we carried out a high-throughput screen (HTS) using luciferase-based reporter cells, and herein we report the discovery and preliminary characterization of a potent, smallmolecule HbF inducer, TN1. Human leukemia KU812 cells were stably transfected with a band g-globin dual reporter LCRprRluc–prFluc, which contains a 1.4 kb segment of the gglobin gene promoter. Using this assay, a large library of structurally diverse heterocycles (~2 million compounds) was screened in 1536-well format at 1.25 mm compound concentration to identify g-globin inducing compounds. The screen parameters, coefficient of variation (CV) value of 8 %, signal-tonoise (S/N) ratio of >100, and a z-factor value of 0.6, confirmed the robustness of the assay, with hemin and DMSO as positive and negative controls, respectively. Compounds that increased g-globin-driven firefly luciferase activity by more than twofold of the plate mean were selected for further testing. Compounds were tested in dose–response experiments, and strong hits with EC50 values smaller than 150 nm were identified. To exclude compounds that nonspecifically increase luciferase activity, 12] compounds were tested using HEK293 cells transfected with a pGL3 luciferase reporter. Compounds that increased reporter signal more than threefold were triaged. The majority of hit compounds were negative in this assay. Toxic compounds that had a CC50 value less than threefold the EC50 value were also excluded. Of the compounds screened, 65 derivatives met these efficacy, toxicity, and specificity criteria and were chosen for further analysis (Supporting Information S2) To confirm compounds increased the levels of endogenous g-globin, HbF levels were determined in compound-treated KU812 cells by Western blot. Of the 65 hit compounds, most were weaker HbF inducers compared with the positive controls (hemin and hydroxyurea). TN1 (structure given in Figure 1; see Supporting Information S1 for synthesis) was selected for further study because it increased HbF protein in both leukemic KU812 and K562 cells in a dose-dependent manner (Figure 2 a and b). At 100 nm concentration, Western blot analysis indicated that TN1 increased g-globin expression (2.9and 3.7fold increase in KU812 cell and K562 cell, respectively) to higher levels than 50–100 mm HU (1.8and 1.9-fold increase in KU812 cell and K562 cell, respectively), the first drug approved for the treatment of SCD. 15] The EC50 value for TN1-mediated HbF induction is approximately three orders of magnitude lower than that of HU (HU: EC50 = 50–100 mm ; TN1: EC50 = 100 nm). In addition, TN1 is more potent than a number of previously reported small-molecule HbF inducers including sodium butyrate and other histone deacetylase (HDAC) inhibitors (Supporting Information S3), although somewhat less

Pan-Src Family Kinase Inhibitors Replace Sox2 during the Direct Reprogramming of Somatic Cells

Abstract: Ectopic expression of the four transcription factors Oct4, Klf4, Sox2 and c-Myc reprograms adult somatic cells to induced pluripotent stem (iPS) cells.[1] Although iPS cells hold considerable promise as tools in research and drug discovery, the clinical application of iPS cells is hindered by the use of viruses that deliver the exogenous factors and modify the host genome. It is therefore of great interest to replace virally transduced factors with either proteins or small molecules. To date a number of compounds have been identified that facilitate reprogramming of somatic cells. Among these are kenpaullone[2], valproic acid[3] and inhibitors of TGFβ-signaling.[4] Here we have exploited a reporter based screen[2] to identify a new class of compounds that functionally replace Sox2: inhibitors of the Src family of kinases. These molecules provide novel tools to study the molecular mechanism of Sox2 in reprogramming. To screen for small molecule replacements of Sox2, mouse embryonic fibroblasts (MEFs) harboring the firefly luciferase (Fluc) gene in the Nanog locus[2] (NL-MEFs) were transduced with Oct4, Klf4 and c-Myc (OKM), seeded into 1536-well plates in standard growth media and assayed against a large chemical library[5] (750,000 compounds; 2.2 μM). Compounds that reproducibly and dose-dependently activated the NL reporter >2.5-fold over vehicle-treated controls (Figure 1a) were then counter-screened in a cell based SV40-driven Fluc assay to rule out false positives that directly and non-specifically induce luciferase signal.[2, 6] Figure 1 Chemical complementation of Sox2 To confirm that filtered hit compounds which activate Nanog gene expression also replace Sox2, iPS cell colony formation was used as a secondary assay. Specifically, Klf4 and c-Myc were delivered retrovirally to O4NR-MEFs[1b] (cells harboring a Doxycycline (Dox)-inducible Oct4 cDNA in the collagen locus and the neomycin-resistance gene in the Oct4 locus), and Oct4 expression was induced by addition of Dox to the culture media (day 0). Two days later, positive screen hits (1-10 μM) were added to OKM-expressing MEFs in place of Sox2. After 10 days of compound treatment, growth media was supplemented with neomycin to select for colonies that reactivated the endogenous Oct4 locus. The reactivation of epigenetically silenced pluripotency-associated genes is required for somatic cells to transition to the iPS cell state.[7] Dox-independent, neomycin resistant colonies were not observed in DMSO-treated (0.1%, v/v) controls, indicating that vehicle-treated cells had not removed the epigenetic silencing marks from the Oct4 promoter (which drives NeoR) and were thus not pluripotent. Among the compounds tested, one compound, iPYrazine (iPY; 10 μM), promoted the formation of neomycin-resistant iPS cell colonies (Figure 1b, blue bars) that survived and could be cultured in the absence of Dox. Transgenic Oct4 independent (minus Dox) growth of the iPY-treated iPS cells demonstrated that they had reactivated and relied on endogenous Oct4 to maintain the pluripotent state. In addition, OKM transduction combined with iPY treatment of MEFs carrying a GFP reporter under control of the endogenous Oct4 locus[8] also gave rise to stable, GFP-positive iPS cell lines (Figure S1, Supporting Information). iPS cells derived from O4NR-MEFs with iPY, Dox and KM-transduction grew as pluripotent stem cell colonies in the absence of Dox and iPY. Moreover, these cells were indistinguishable from ES cells by morphological criteria and expressed the pluripotency-associated markers Oct4 and SSEA1 (Figure 1c). We next tested the differentiation potential of the iPY-derived iPS cells in a teratoma assay by injecting 106 cells subcutaneously into NOD-SCID mice. Tumors were isolated 3 weeks later and histological analyses demonstrated that cell types of all three germ layers were present; these included neural tissues, bone, cartilage and ciliated epithelium (Figure 1d). Furthermore, iPY-derived iPS cells contributed to live chimeras, as shown in Figure 1d. The results from this series of analyses indicate that the iPY-derived, Sox2-free iPS cells are pluripotent. In order to identify the biological target of iPY, we profiled the compound against a biochemical panel of tyrosine kinases (51 kinases; Table S1). From this analysis, we found that iPY potently inhibited a number of tyrosine kinases at 5 μM. Commercially available inhibitors (Figures 2a-b and Table S2) of these candidate kinase targets were then assayed for their ability to replace Sox2 in the iPS cell colony formation assay. As shown in Figure 2b, the pan-Src family kinase (SFK) inhibitors Dasatinib[9] and PP1[10] (Figure 2b) were able to recapitulate the activity of iPY. Interestingly, both Dasatinib and PP1 were >2-fold more active than iPY and efficiently replaced Sox2 (Figure 2b). Moreover, the pan-SFK inhibitors gave rise to colonies with a similar efficiency to TGFβ inhibitors (SB-431542 and LY-364947). The latter have been reported to replace Sox2 and served as a positive control in this study.[4] In addition to TGFβ inhibitors, Ichida et al. have also reported that the SFK inhibitor PP1 is able to replace Sox2.[4a] Together with our work, these results indicate that iPY is likely playing a role in reprogramming by inhibiting Src kinases, although additional mechanisms cannot be excluded. Figure 2 Src family kinase and TGFβ-inhibitors recapitulate the Sox2 replacement activity of iPY SFKs are a class of proto-oncogene tyrosine kinases that include nine mammalian members (i.e., c-Src, Yes, Fyn, Fgr, Lck, Hck, Blk, Lyn and Frk).[11] Several members of the SFK family have been reported to influence embryonic stem (ES) cell self-renewal and differentiation.[12] For example, activation of c-Src signaling promotes ES cell differentiation.[13] Consistent with this observation we find that the activation of Src signaling in MEFs with JK239[14] potently inhibits 4-factor reprogramming (Figure 2c). Together, our results suggest that SFK signaling is an important mediator of somatic cell reprogramming, where activation of the SFK pathway prevents reprogramming and inhibition allows for reprogramming in the absence of exogenous Sox2. Previously, Ichida et al. demonstrated that small molecule mediated inhibition of TGFβ-signaling with LY-364947 or E-616452 can replace Sox2 through the activation of Nanog expression.[4a] The results from our screen, which rely on Nanog activation as a surrogate for the replacement of Sox2, suggest that the inhibition of SFK- and TGFβ-signaling may converge on a similar mechanism; that is, the function of Sox2 can be replaced during direct reprogramming by activating Nanog expression. Another potential scenario comes from the observation that both Nanog[15] and SFK inhibition[13] are capable of maintaining the self-renewing pluripotent state in ES cells. Thus, TGFβ inhibitor-mediated Nanog activation and pan-SFK inhibition may instead converge on a common mechanism in which the differentiation of newly formed iPS cells is prevented, thereby assisting in the transition to an undifferentiated state. In either case, it is interesting to note that inhibition of distinct signaling responses converge on a common end point. In summary, we applied a cell-based, high-throughput chemical screen to identify small molecules that replace Sox2 during somatic cell reprogramming. The identification of novel SFK inhibitors provides new chemical tools to study the mechanisms underlying direct reprogramming and may ultimately help to bring iPS cell technology one step closer to clinical application.