Achim Brinker

Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4.

Ectopic expression of defined transcription factors can reprogram somatic cells to induced pluripotent stem (iPS) cells, but the utility of iPS cells is hampered by the use of viral delivery systems. Small molecules offer an alternative to replace virally transduced transcription factors with chemical signaling cues responsible for reprogramming. In this report we describe a small-molecule screening platform applied to identify compounds that functionally replace the reprogramming factor Klf4. A series of small-molecule scaffolds were identified that activate Nanog expression in mouse fibroblasts transduced with a subset of reprogramming factors lacking Klf4. Application of one such molecule, kenpaullone, in lieu of Klf4 gave rise to iPS cells that are indistinguishable from murine embryonic stem cells. This experimental platform can be used to screen large chemical libraries in search of novel compounds to replace the reprogramming factors that induce pluripotency. Ultimately, such compounds may provide mechanistic insight into the reprogramming process.

Synthetic small molecules that control stem cell fate

In an attempt to better understand and control the processes that regulate stem cell fate, we have set out to identify small molecules that induce neuronal differentiation in embryonic stem cells (ESCs). A high-throughput phenotypic cell-based screen of kinase-directed combinatorial libraries led to the discovery of TWS119, a 4,6-disubstituted pyrrolopyrimidine that can induce neurogenesis in murine ESCs. The target of TWS119 was shown to be glycogen synthase kinase-3β (GSK-3β) by both affinity-based and biochemical methods. This study provides evidence that GSK-3β is involved in the induction of mammalian neurogenesis in ESCs. This and such other molecules are likely to provide insights into the molecular mechanisms that control stem cell fate, and may ultimately be useful to in vivo stem cell biology and therapy.

In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen

The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of ≈1.7 million compounds, we identified a diverse collection of ≈6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 μM). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities.

Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry

The reversible phosphorylation of tyrosine residues is an important mechanism for modulating biological processes such as cellular signaling, differentiation, and growth, and if deregulated, can result in various types of cancer. Therefore, an understanding of these dynamic cellular processes at the molecular level requires the ability to assess changes in the sites of tyrosine phosphorylation across numerous proteins simultaneously as well as over time. Here we describe a sensitive approach based on multidimensional liquid chromatography/mass spectrometry that enables the rapid identification of numerous sites of tyrosine phosphorylation on a number of different proteins from human whole cell lysates. We used this methodology to follow changes in tyrosine phosphorylation patterns that occur over time during either the activation of human T cells or the inhibition of the oncogenic BCR-ABL fusion product in chronic myelogenous leukemia cells in response to treatment with STI571 (Gleevec). Together, these experiments rapidly identified 64 unique sites of tyrosine phosphorylation on 32 different proteins. Half of these sites have been documented in the literature, validating the merits of our approach, whereas motif analysis suggests that a number of the undocumented sites are also potentially involved in biological pathways. This methodology should enable the rapid generation of new insights into signaling pathways as they occur in states of health and disease.

In vivo incorporation of unnatural amino acids to probe structure, dynamics and ligand binding in a large protein by Nuclear Magnetic Resonance spectroscopy

In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF 3Phe), (13)C/(15)N-labeled p-methoxyphenylalanine (OMePhe), and (15)N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in (1)H-(15)N HSQC, (1)H-(13)C HSQC, and (19)F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3Phe mutants of an active site tyrosine inhibited binding; incorporating (15)N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.

Crystal structures of human HSP90α-complexed with dihydroxyphenylpyrazoles

Abstract A series of dihydroxyphenylpyrazole compounds were identified as a unique class of reversible Hsp90 inhibitors. The crystal structures for two of the identified compounds complexed with the N-terminal ATP binding domain of human Hsp90α were determined. The dihydroxyphenyl ring of the compounds fits deeply into the adenine binding pocket with the C2 hydroxyl group forming a direct hydrogen bond with the side chain of Asp93. The pyrazole ring forms hydrogen bonds to the backbone carbonyl of Gly97, the hydroxyl group of Thr184 and to a water molecule, which is present in all of the published HSP90 structures. One of the identified compounds (G3130) demonstrated cellular activities (in Her-2 degradation and activation of Hsp70 promoter) consistent with the inhibition of cellular Hsp90 functions.

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

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.

Small molecule inhibitors of trans-translation have broad-spectrum antibiotic activity

The trans-translation pathway for protein tagging and ribosome release plays a critical role for viability and virulence in a wide range of pathogens but is not found in animals. To explore the use of trans-translation as a target for antibiotic development, a high-throughput screen and secondary screening assays were used to identify small molecule inhibitors of the pathway. Compounds that inhibited protein tagging and proteolysis of tagged proteins were recovered from the screen. One of the most active compounds, KKL-35, inhibited the trans-translation tagging reaction with an IC50 = 0.9 µM. KKL-35 and other compounds identified in the screen exhibited broad-spectrum antibiotic activity, validating trans-translation as a target for drug development. This unique target could play a key role in combating strains of pathogenic bacteria that are resistant to existing antibiotics.

Identification of small molecule and genetic modulators of AON-induced dystrophin exon skipping by high-throughput screening.

One therapeutic approach to Duchenne Muscular Dystrophy (DMD) recently entering clinical trials aims to convert DMD phenotypes to that of a milder disease variant, Becker Muscular Dystrophy (BMD), by employing antisense oligonucleotides (AONs) targeting splice sites, to induce exon skipping and restore partial dystrophin function. In order to search for small molecule and genetic modulators of AON-dependent and independent exon skipping, we screened ∼10,000 known small molecule drugs, >17,000 cDNA clones, and >2,000 kinase- targeted siRNAs against a 5.6 kb luciferase minigene construct, encompassing exon 71 to exon 73 of human dystrophin. As a result, we identified several enhancers of exon skipping, acting on both the reporter construct as well as endogenous dystrophin in mdx cells. Multiple mechanisms of action were identified, including histone deacetylase inhibition, tubulin modulation and pre-mRNA processing. Among others, the nucleolar protein NOL8 and staufen RNA binding protein homolog 2 (Stau2) were found to induce endogenous exon skipping in mdx cells in an AON-dependent fashion. An unexpected but recurrent theme observed in our screening efforts was the apparent link between the inhibition of cell cycle progression and the induction of exon skipping.

A biochemical screen for GroEL/GroES inhibitors

High-throughput screening of 700,000 small molecules has identified 235 inhibitors of the GroEL/GroES-mediated refolding cycle. Dose-response analysis of a subset of these hits revealed that 21 compounds are potent inhibitors of GroEL/GroES-mediated refolding (IC50 <10 μM). The screening results presented herein represent the first steps in a broader aim of developing molecular probes to study chaperonin biochemistry and physiology.