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Dive into the research topics where Larisa Yurlova is active.

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Featured researches published by Larisa Yurlova.


ACS Chemical Biology | 2013

Stapled Peptides with Improved Potency and Specificity That Activate p53

Christopher J. Brown; Soo Tng Quah; Janice Jong; Amanda M. Goh; Poh C. Chiam; Kian Hoe Khoo; Meng Ling Choong; May A. Lee; Larisa Yurlova; Kourosh Zolghadr; Thomas L. Joseph; Chandra Verma; David P. Lane

By using a phage display derived peptide as an initial template, compounds have been developed that are highly specific against Mdm2/Mdm4. These compounds exhibit greater potency in p53 activation and protein-protein interaction assays than a compound derived from the p53 wild-type sequence. Unlike Nutlin, a small molecule inhibitor of Mdm2/Mdm4, the phage derived compounds can arrest cells resistant to p53 induced apoptosis over a wide concentration range without cellular toxicity, suggesting they are highly suitable for cyclotherapy.


PLOS ONE | 2013

Inhibition of Nutlin-Resistant HDM2 Mutants by Stapled Peptides

Siau Jia Wei; Thomas L. Joseph; Sharon Chee; Ling Li; Larisa Yurlova; Kourosh Zolghadr; Christopher J. Brown; David P. Lane; Chandra Verma; Farid J. Ghadessy

Pharmacological modulation of p53 activity is an attractive therapeutic strategy in cancers with wild-type p53. Presently in clinical trials, the small molecule Nutlin-3A competitively binds to HDM2, a key negative regulator of p53 and blocks its activity. We have described resistance mutations in HDM2 that selectively reduce affinity for Nutlin but not p53. In the present communication, we show that stapled peptides targeting the same region of HDM2 as Nutlin are refractory to these mutations, and display reduced discrimination between the wild-type and mutant HDM2s with regards to functional abrogation of interaction with p53. The larger interaction footprint afforded by stapled peptides suggests that this class of ligands may prove comparatively more resilient to acquired resistance in a clinical setting.


PLOS ONE | 2013

In Vitro Selection of Mutant HDM2 Resistant to Nutlin Inhibition

Siau Jia Wei; Thomas L. Joseph; Adelene Y. L. Sim; Larisa Yurlova; Kourosh Zolghadr; David P. Lane; Chandra Verma; Farid J. Ghadessy

HDM2 binds to the p53 tumour suppressor and targets it for proteosomal degradation. Presently in clinical trials, the small molecule Nutlin-3A competitively binds to HDM2 and abrogates its repressive function. Using a novel in vitro selection methodology, we simulated the emergence of resistance by evolving HDM2 mutants capable of binding p53 in the presence of Nutlin concentrations that inhibit the wild-type HDM2-p53 interaction. The in vitro phenotypes were recapitulated in ex vivo assays measuring both p53 transactivation function and the direct p53-HDM2 interaction in the presence of Nutlin. Mutations conferring drug resistance were not confined to the N-terminal p53/Nutlin–binding domain, and were additionally seen in the acidic, zinc finger and RING domains. Mechanistic insights gleaned from this broad spectrum of mutations will aid in future drug design and further our understanding of the complex p53-HDM2 interaction.


Journal of Biomolecular Screening | 2014

The fluorescent two-hybrid assay to screen for protein-protein interaction inhibitors in live cells: targeting the interaction of p53 with Mdm2 and Mdm4.

Larisa Yurlova; Maarten Derks; Andrea Buchfellner; Ian Hickson; Marc Janssen; Denise Morrison; Ian Stansfield; Christopher J. Brown; Farid J. Ghadessy; David P. Lane; Ulrich Rothbauer; Kourosh Zolghadr; Eberhard Krausz

Protein–protein interactions (PPIs) are attractive but challenging targets for drug discovery. To overcome numerous limitations of the currently available cell-based PPI assays, we have recently established a fully reversible microscopy-assisted fluorescent two-hybrid (F2H) assay. The F2H assay offers a fast and straightforward readout: an interaction-dependent co-localization of two distinguishable fluorescent signals at a defined spot in the nucleus of mammalian cells. We developed two reversible F2H assays for the interactions between the tumor suppressor p53 and its negative regulators, Mdm2 and Mdm4. We then performed a pilot F2H screen with a subset of compounds, including small molecules (such as Nutlin-3) and stapled peptides. We identified five cell-penetrating compounds as potent p53–Mdm2 inhibitors. However, none exhibited intracellular activity on p53–Mdm4. Live cell data generated by the F2H assays enable the characterization of stapled peptides based on their ability to penetrate cells and disrupt p53–Mdm2 interaction as well as p53–Mdm4 interaction. Here, we show that the F2H assays enable side-by-side analysis of substances’ dual Mdm2–Mdm4 activity. In addition, they are suitable for testing various types of compounds (e.g., small molecules and peptidic inhibitors) and concurrently provide initial data on cellular toxicity. Furthermore, F2H assays readily allow real-time visualization of PPI dynamics in living cells.


PLOS ONE | 2016

A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells

Andrea Buchfellner; Larisa Yurlova; Stefan Nüske; Armin M. Scholz; Jacqueline Bogner; Benjamin Ruf; Kourosh Zolghadr; Sophie E Drexler; Guido A. Drexler; Stefanie Girst; C. Greubel; Judith Reindl; Christian Siebenwirth; Tina Romer; Anna A. Friedl; Ulrich Rothbauer

Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.


The Journal of Steroid Biochemistry and Molecular Biology | 2017

The fluorescent two-hybrid assay for live-cell profiling of androgen receptor modulators

Jacqueline Bogner; Kourosh Zolghadr; Ian Hickson; Tina Romer; Larisa Yurlova

The androgen receptor (AR) is an important target for drug therapies combating prostate cancer. However, various acquired mutations within the AR sequence often render this receptor resistant to treatment. Ligand-induced interaction between the N- and C-termini of the AR marks the initial step in the AR signaling cascade and can thus serve as an early read-out for analysis of potential antagonists of wt and mutant AR. To measure changes of the N/C interaction in the wt and mutant AR variants upon the addition of inhibitors, we applied our recently developed Fluorescent Two-Hybrid (F2H) assay. The F2H method enables real-time monitoring and quantitative analysis of the interactions between GFP- and RFP-tagged proteins in live mammalian cells, where GFP-tagged proteins are tethered to a specific nuclear location. This anchoring approach provides a local signal enrichment suitable for direct visualization of protein-protein interactions as co-localizations by conventional epifluorescence microscopy. Since the F2H assay is fully reversible, we could monitor dynamics of AR N/C interactions in living cells in real time upon agonistic, as well as antagonistic treatments. In dose-response F2H experiments, we compared the potencies of abiraterone, bicalutamide, enzalutamide, flutamide, and galeterone/TOK-001 to prevent the dihydrotestosterone-induced N/C interaction in wt AR. We further applied the newly developed F2H assay to analyze how the AR N/C interaction is affected by the clinically relevant mutations W741L, F876L, T877A and F876L/T877A. We conclude that F2H is a reliable and technically undemanding approach for straightforward screening of new AR modulators, as well as for monitoring their activity in real time in living cells.


Oncotarget | 2016

Avoiding drug resistance through extended drug target interfaces: a case for stapled peptides

Siau Jia Wei; Sharon Chee; Larisa Yurlova; David P. Lane; Chandra Verma; Christopher J. Brown; Farid J. Ghadessy

Cancer drugs often fail due to the emergence of clinical resistance. This can manifest through mutations in target proteins that selectively exclude drug binding whilst retaining aberrant function. A priori knowledge of resistance-inducing mutations is therefore important for both drug design and clinical surveillance. Stapled peptides represent a novel class of antagonists capable of inhibiting therapeutically relevant protein-protein interactions. Here, we address the important question of potential resistance to stapled peptide inhibitors. HDM2 is the critical negative regulator of p53, and is often overexpressed in cancers that retain wild-type p53 function. Interrogation of a large collection of randomly mutated HDM2 proteins failed to identify point mutations that could selectively abrogate binding by a stapled peptide inhibitor (PM2). In contrast, the same interrogation methodology has previously uncovered point mutations that selectively inhibit binding by Nutlin, the prototypical small molecule inhibitor of HDM2. Our results demonstrate both the high level of structural p53 mimicry employed by PM2 to engage HDM2, and the potential resilience of stapled peptide antagonists to mutations in target proteins. This inherent feature could reduce clinical resistance should this class of drugs enter the clinic.


Biospektrum | 2013

Fluoreszierende Biomarker zum Nachweis nativer Zellstrukturen

Larisa Yurlova; Andrea Buchfellner; Jacqueline Gregor; Ulrich Rothbauer; Kourosh Zolghadr

Chromobodies are a new class of extremely small, highly stable fluorescent antibodies. The key elements of this technology are the antigen-binding domains of heavy chain antibodies from Camelidae, fused to a fluorescent protein. Chromobodies are efficiently introduced into any cell type as plasmid DNA by means of simple transfection and target and trace native, endogenous proteins in real-time.


Cancer Research | 2016

Abstract 2754: Antibody-based tools for in vitro and live cell analysis of endogenous PARP1, an essential human DNA repair enzyme

Andrea Buchfellner; Larisa Yurlova; Stephanie Dennison; Benjamin Ruf; Ulrich Rothbauer; Tina Romer

DNA damage caused by normal cell activity or exogenous genotoxic agents is a constant threat to the genome. The nuclear enzyme Poly(ADP-ribose) polymerase 1 (PARP1) is rapidly activated by DNA lesions such as single-strand breaks and signals their presence by attaching ADP-ribose units to chromatin-associated proteins. To improve our understanding of PARP1 within a disease context, there is an ongoing need to develop novel PARP1 detection systems. Here we describe the development of a VHH directed against human PARP1. The variable domain of single chain antibodies (VHH or nanobody) is a versatile research tool for a variety of applications. The high binding affinity of VHHs, their small size (15 kDa) and their robust expression in various cellular systems make them preferable to conventional antibodies. Moreover, VHH domains can be selected to recognize and bind their target structures within living cells. This highly specific PARP1 VHH was selected by phage display using a VHH library from an immunized alpaca. We characterized its affinity, selectivity and activity both in vitro and in live cells. To validate the in vitro interactions of this PARP1-specific VHH, we developed a PARP1 immunoprecipitation reagent by conjugating the PARP1-specific VHH to an immobilizing matrix (termed PARP1-Trap). This PARP1-Trap was shown to bind with high specificity to human PARP1 and not to other members of the PARP family. Importantly, the binding of the PARP1-Trap to PARP1 leaves the enzymatic activity of PARP1 unaffected. The epitope of the PARP1-Trap was localized to the N-terminal domain of PARP1 and consists of the three-dimensional motif of zinc fingers 2 and 3 together. To determine whether the PARP1 VHH recognizes its target structure also in a cellular environment, the VHH was genetically fused to a fluorescent protein and expressed in living cells (termed Chromobody). The interaction of the PARP1 Chromobody with PARP1 was visualized using a protein-protein interaction assay called fluorescent two-hybrid (F2H). The F2H principle is based on a tethering strategy: a GFP-tagged protein (here GFP-PARP1) is enriched at a protein interaction platform engineered into F2H-BHK cells and serves as bait, whereas the RFP-tagged PARP1 VHH serves as a prey. Using the F2H assay, we could show that the PARP1 Chromobody is functional within living cells and specifically recognizes its antigen in a cellular environment. Moreover, by monitoring the PARP1 Chromobody signal after microirradiation, we were for the first time able to follow the recruitment of endogenous PARP1 to sites of DNA damage in living cells. In summary, we developed a novel PARP1 VHH for both biochemical and live cell analysis of human PARP1. We anticipate that PARP1-VHH based reagents will provide new insights into the PARP1 enzyme. For example, the use of the PARP1-Trap coupled with mass spectrometry analysis may lead to the identification of hitherto unknown PARP1 interaction partners. Citation Format: Andrea Buchfellner, Larisa Yurlova, Stephanie Dennison, Benjamin Ruf, Ulrich Rothbauer, Tina Romer. Antibody-based tools for in vitro and live cell analysis of endogenous PARP1, an essential human DNA repair enzyme. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2754.


Cancer Research | 2015

Abstract 1231: Live cell and in vitro analysis of p53 interactions

Larisa Yurlova; Andrea Buchfellner; Benjamin Ruf; Sebastien Gabriel Michel Jo; Jean-Christophe Bourdon; Farid J. Ghadessy; Christopher J. Brown; David P. Lane; Tina Romer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Dysregulation of protein-protein interactions between the tumor suppressor p53 and its binding partners is implicated in the pathogenesis of various cancers. Here we describe several novel assays for analysis of p53 interactions and their inhibitors both in live mammalian cells and in vitro. To evaluate putative inhibitors of protein-protein interactions between p53 and its negative regulators Mdm2 and Mdm4, we recently developed two comparative live-cell Fluorescent-Two Hybrid (F2H) assays. The F2H principle is based on a tethering strategy: the GFP-tagged protein (here p53) is enriched at the protein interaction platform of the engineered F2H-BHK cells and serves as bait, whereas the RFP-tagged protein serves as a prey (here Mdm2 or Mdm4). By performing p53:Mdm2 and p53:Mdm4 F2H assays side-by-side, we could evaluate the dual inhibitory activity of the previously published stapled peptides. Furthermore, since F2H allows visualization of the dynamics of protein-protein interactions, we could compare the compounds kinetics with real-time imaging. We performed a mutant analysis with F2H and showed that several Nutlin-resistant mutants of Mdm2 are sensitive to inhibition with stapled peptides sMTide-02 and sMTide-02a. For in vitro validation of p53 interactions, we developed novel p53 immunoprecipitation reagents. We employed the single-domain antibody technology in conjunction with phage display to isolate two specific anti-p53 VHHs (also termed nanobodies) from immunized alpacas. When conjugated to agarose beads, these VHHs serve as highly efficient pull-down reagents (p53-Traps), specific exclusively against N- and C-terminus of p53 respectively. Using both p53-Traps, we could confirm our F2H results in immunoprecipitation followed by Western blotting. Furthermore, p53-Traps enabled us to extend our analysis of the p53 interaction network and evaluate interactions between the p53 isoforms. Taken together, we developed a toolbox for analysis of p53 interactions both biochemically and by fluorescence microscopy. Our fully reversible live-cell F2H assays can be applied for side-by-side profiling of new inhibitors of p53:Mdm2 and p53:Mdm4 interactions with respect to their intracellular activity, cell penetration and kinetics. Our N- and C-terminal p53-Traps complement p53 interaction analysis and enable highly efficient biochemical investigation of the p53 network and discovery of new interaction partners of p53. Citation Format: Larisa Yurlova, Andrea Buchfellner, Benjamin Ruf, Sebastien Gabriel Michel Jo, Jean-Christophe Bourdon, Farid J. Ghadessy, Christopher J. Brown, David P. Lane, Tina Romer. Live cell and in vitro analysis of p53 interactions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1231. doi:10.1158/1538-7445.AM2015-1231

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Benjamin Ruf

University of Tübingen

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