Alternative modulation of protein-protein interactions by small molecules.

Protein-protein interactions (PPI) have become increasingly popular drug targets, with a number of promising compounds currently in clinical trials. Recent research shows, that PPIs can be modulated in more ways than direct inhibition, where novel non-competitive modes of action promise a solution for the difficult nature of PPI drug discovery. Here, we review recently discovered PPI modulators in light of their mode of action and categorise them as disrupting versus stabilising, orthosteric versus allosteric and by their ability to affect the proteins' dynamics. We also give recent examples of compounds successful in the clinic, analyse their physicochemical properties and discuss how to overcome the hurdles in discovering alternative modes of modulation.This work was supported by Wellcome Trust Strategic Award Grant (090340/Z/09/Z).This is the final published version. It first appeared with a CC BY licence at http://www.sciencedirect.com/science/article/pii/S0958166915000695#

Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2.

The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the 'Y pocket') that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases.We are grateful for the access and support at beamlines i02, i03 and i04-1 at Diamond Light Source at Harwell, UK (proposal MX9007 and MX9537) and at beamline Proxima1 at the SOLEIL Synchrotron, Gif-sur-Yvette, France. We are grateful for access and support from the X-ray and biophysics facilities (Dept. of Biochemistry) and the screening/imaging facility (MRC Cancer Unit). M.J. was supported by a Cancer Research UK studentship held in the labs of DS and ARV, PS and MR by a Wellcome Trust Strategic Award to ARV and MH, and DJH, BH, AJN and GM by grants from the UK Medical Research Council to ARV.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/srep2852

Development of a multifunctional benzophenone linker for peptide stapling and photoaffinity labeling

Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker, which serves as both a photocrosslinking motif and a peptide stapling reagent. Using a double-click stapling methodology, we attach the benzophenone to the peptide via the staple linker, rather than modifying the peptide sequence with a photocrosslinking amino acid. Applied to a p53-derived peptide, the resulting photoreactive stapled peptide is able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull-down assays, and can be used to investigate the target selectivity of stapled peptides.This work was supported by the EPSRC, BBSRC, MRC, Wellcome Trust and ERC (FP7/2007-2013; 279337/DOS). We thank Dr. Clemens Mayer for access to the UV crosslinker (University Chemical Laboratory, University of Cambridge), Weiyan Chen and Fran Kundel (University Chemical Laboratory, University of Cambridge) for assistance with the Typhoon imager and Dr. Laura Itzhaki and Wenshu Xu (Department of Pharmacology, University of Cambridge) for assistance with SDS-PAGE.This is the final version of the article. It first appeared from Wiley via https://doi.org/10.1002/cbic.20150064

Development of a Multifunctional Benzophenone Linker for Peptide Stapling and Photoaffinity Labelling.

Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker that serves as both a photo-crosslinking motif and a peptide stapling reagent. Using double-click stapling, we attached the benzophenone to the peptide via the staple linker, rather than by modifying the peptide sequence with a photo-crosslinking amino acid. When applied to a p53-derived peptide, the resulting photoreactive stapled peptide was able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull-down assays, and can be used to investigate the target selectivity of stapled peptides.This work was supported by the EPSRC, BBSRC, MRC, Wellcome Trust and ERC (FP7/2007-2013; 279337/DOS). We thank Dr. Clemens Mayer for access to the UV crosslinker (University Chemical Laboratory, University of Cambridge), Weiyan Chen and Fran Kundel (University Chemical Laboratory, University of Cambridge) for assistance with the Typhoon imager and Dr. Laura Itzhaki and Wenshu Xu (Department of Pharmacology, University of Cambridge) for assistance with SDS-PAGE.This is the final version of the article. It first appeared from Wiley via https://doi.org/10.1002/cbic.20150064

Computationally-guided optimization of small-molecule inhibitors of the Aurora A kinase-TPX2 protein-protein interaction.

Free energy perturbation theory, in combination with enhanced sampling of protein-ligand binding modes, is evaluated in the context of fragment-based drug design, and used to design two new small-molecule inhibitors of the Aurora A kinase-TPX2 protein-protein interaction

Strukturelle Untersuchungen von Proteinen des humanen Sphingolipid- Stoffwechsels

The amphiphilic saposins SapA, SapB, SapC, and SapD are glycoproteins acting at the lipid-water interface of intra-lysosomal lipid vesicles. They are required for the degradation of sphingolipids by glycosylceramidase and ceramidase, respectively, and lipid-antigen presentation by CD1 molecule. Despite the simple makeup of saposins, their mode of interaction with acidic phospholipid-containing membranes is not fully understood. The present work describes two high resolution crystal structures of human SapC that reveal an unusual homodimer with swapped monomers in an ‘open’ configuration. This novel form of SapC dimer provides new insights into protein-lipid interactions and supports the “clip-on” model for SapC-induced vesicle fusion. Small-angle X-ray scattering (SAXS) experiments with SapC have established the presence of SapC oligomers in solution supporting the mechanism in which SapC forms protein patches on the membrane surface and activates hydrolytic enzymes, of which one is a human acid ceramidase (ASAH) - a lysosomal enzyme indispensable for ceramide degradation in lysosomes as demonstrated by its association with the fatal sphingolipid storage disorder Farber disease. In the present work the X-ray crystal structure of the conjugated bile acid hydrolase (CBAH) from C. perfringens – a near bacterial homologue to the b-subunit of the human acid ceramidase – was determined at 1.6 Å resolution. Using CBAH structure, a homology model for acid ceramidase was generated, and residues responsible for the catalytic activity of the ASAH were proposed. The obtained 3D model of the ASAH provides a new tool to better understand Farber disease and the catalytic mechanism of the human acid ceramidase. On the basis of the crystal structure of CBAH determined here and prior works on related bacterial enzymes, the processing, catalytic mechanism, and substrate binding of this enzyme are discussed. The structures of CBAH in complex with reaction products are the first structures of a member of the choloylglycine-hydrolase family complexed with products and provide a working model for engineering substrate specifity of N-terminal nucleophillic hydrolases, a protein family employed in the industrial production of b-lactam antibiotics.Saposine SapA, SapB, SapC und SapD sind amphiphile Glykoproteine und agieren an der Lipid-Wasser-Phasengrenze. Sie sind unentbehrlich für den lysosomalen Abbau von Sphingolipiden durch spezifische Hydrolasen und für die Präsentation von Lipid-Antigenen durch CD1-Moleküle. Trotz des einfachen dreidimensionalen Aufbaus der Saposine ist deren Interaktion mit azidischen Phospholipid–Membranen noch nicht vollständig verstanden. Vorliegende Arbeit beschreibt zwei Kristallstrukturen von menschlichem Saposin C (SapC), die eine ungewöhnliche homodimere offene Konformation mit vertauschten Monomeren (domain swapping) aufweisen. Diese neuartige Form der SapC Dimerisierung bietet neue Einblicke in Protein-Lipid-Wechselwirkungen und unterstützt das so genannte "clip-on" Modell, das für die durch SapC induzierte Vesikel-Fusion vorgeschlagen wurde. Röntgen Klein-Winkel-Streuung (SAXS) Experimente mit SapC zeigten das Vorhandensein von SapC Oligomeren in Lösung auf und unterstützten so den Mechanismus, in dem SapC durch die Bildung von Protein-Pflastern an der Phasengrenze hydrolytische Enzyme in Lysosom aktiviert. Die menschliche saure Ceramidase (ASAH) gehört dazu als lysosomales Enzyms unentbehrlich für den Abbau der Ceramide, wie der Zusammenhang zwischen dem Ausfall von der saueren Ceramidase und der tödlichen Farber-Krankheit aufzeigt. In dieser Arbeit wurde die Kristallstruktur der konjugierten Gallensäure hydrolase (CBAH) von C. perfringens bestimmt, die homolog der -Untereinheit der ASAH ist. Die Struktur der CBAH wurde verwendet, um ein Homologie-Modell für die saure Ceramidase zu generieren sowie Aminosäuren vorzuschlagen, die verantwortlich für die katalytische Aktivität von ASAH sind. Das erhaltene 3D Modell bietet ein neues Werkzeug zum besseren Verständnis der Farber-Krankheit und des katalytischen Mechanismus der menschlichen saueren Ceramidase. Auf der Grundlage der in dieser Arbeit bestimmten CBAH-Kristallstruktur und anderen Arbeiten an ähnlichen bakteriellen Enzymen werden Prozessierung, katalytisches Mechanismus und Substrat-Bindung der CBAH diskutiert. Die Strukturen von CBAH- Komplexen mit Reaktionsprodukten sind die ersten Strukturen dieser Art von Choloylglycine-Hydrolasen. Sie bieten ein Modell für die Manipulation der Substrat-Spezifität von so genannten Ntn-Hydrolasen, einer Protein-Familie, die bei der industriellen Herstellung von -lactam Antibiotika verwendung findet

Intracellular calcium excess as one of the main factors in the etiology of prostate cancer

Numerous studies show that prostate cancer (PCa) incidence drastically increases with age, these malignant tumours are mainly formed in the peripheral zone of the prostate gland, and a high intake of calcium rich dairy products is associated with an increased risk of PCa. The main objective of this study was to identify a potential common pathophysiological factor associated with the PCa features mentioned above. We performed measurements of the intracellular Ca concentrations in the peripheral zones of nonhyperplastic prostate glands of 99 males aged 0–87 years. To clarify the age-related changes in the intracellular Ca, a quantitative morphometric and two analytical methods of Ca determination were employed. We found, that in 18–45 years old males intracellular Ca was maintained at a relatively high concentration, which steadily increased with age. The intracellular Ca accumulation increased after the age of 45. We found, that by the age of 55, Ca level in the prostatic cells of the peripheral zone reached concentration, which is two-to-four-fold higher than in the 18 year olds. Age-dependent accumulation of Ca in the peripheral zone of human prostate gland has been previously unrecognized and could play an important role in the etiology of PCa

A cryptic hydrophobic pocket in the polo-box domain of the polo-like kinase PLK1 regulates substrate recognition and mitotic chromosome segregation

Abstract: The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity