Future Frontiers in Small Molecule Inhibitors of Protein-Protein Interactions

Protein-protein interactions (PPIs) are ubiquitous in essential biological processes such as cell proliferation and differentiation, host-pathogen interactions, and signal transduction pathways [1]. Pioneering advances in the field of interactomics have uncovered new net-works of protein interactions within cells, with esti-mates for the size of the interactome ranging up to 650,000 PPIs [2]. However, targeting PPIs has histori-cally been considered to be a particularly challenging task due to their typically large size (>1,500 Å) and amorphous nature that lack well-defined crevices for recognition by small molecules. Not surprisingly, the pharmaceutical landscape over the last century has been dominated by programs for small molecule in-hibitors of enzymes (particularly kinases), G-protein-coupled receptors, protein transporters and ion chan-nels that account for the majority of known drugs

Mass Spectrometry Reveals Protein Kinase CK2 High-Order Oligomerization via the Circular and Linear Assembly.

CK2 is an intrinsically active protein kinase that is crucial for cellular viability. However, conventional kinase regulatory mechanisms do not apply to CK2, and its mode of regulation remains elusive. Interestingly, CK2 is known to undergo reversible ionic-strength-dependent oligomerization. Furthermore, a regulatory mechanism based on autoinhibitory oligomerization has been postulated on the basis of the observation of circular trimeric oligomers and linear CK2 assemblies in various crystal structures. Here, we employ native mass spectrometry to monitor the assembly of oligomeric CK2 species in an ionic-strength-dependent manner. A subsequent combination of ion mobility spectrometry-mass spectrometry and hydrogen-deuterium exchange mass spectrometry techniques was used to analyze the conformation of CK2 oligomers. Our findings support ionic-strength-dependent CK2 oligomerization, demonstrate the transient nature of the α/β interaction, and show that CK2 oligomerization proceeds via both the circular and linear assembly.This research was supported by the Wellcome Trust Strategic Award (090340/Z/09/Z), the Agency for Science Technology and Research (A*STAR) Singapore (Ph.D. sponsorship, W.G.S.), and the Croucher Foundation and the Cambridge Overseas Trust (Croucher Cambridge International Scholarship, D.S.-H.C).This is the author accepted manuscript. The final version is available from the American Chemical Society via http://dx.doi.org/10.1021/acschembio.6b0006

2-Bromo-5,7-dimeth­oxy-4-phenyl­quinoline

The title compound, C17H14BrNO2, was synthesized by the treatment of 5,7-dimeth­oxy-4-phenyl­quinolin-2-one with phosphoryl bromide in a Vilsmeier-type reaction. There are two independent mol­ecules (A and B) in the asymmetric unit which differ by 11.2° in the orientation of the 4-phenyl ring with respect to the planar quinoline ring system [dihedral angles = 55.15 (8) and 66.34 (8)° in mol­ecules A and B, respectively]. In the crystal structure, the independent mol­ecules are linked via C—H⋯N and C—H⋯O hydrogen bonds, forming centrosymmetric tetra­meric units which are cross-linked through C—H⋯π and C—Br⋯π inter­actions with Br⋯centroid distances of 3.4289 (8) and 3.5967 (8) Å

Covalent inactivation of Mycobacterium thermoresistibile inosine-5'-monophosphate dehydrogenase (IMPDH).

Inosine-5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme involved in nucleotide biosynthesis. Because of its critical role in purine biosynthesis, IMPDH is a drug design target for immunosuppressive, anticancer, antiviral and antimicrobial chemotherapy. In this study, we use mass spectrometry and X-ray crystallography to show that the inhibitor 6-Cl-purine ribotide forms a covalent adduct with the Cys-341 residue of Mycobacterium thermoresistibile IMPDH

Structural insights into Escherichia coli phosphopantothenoylcysteine synthetase by native ion mobility-mass spectrometry.

CoaBC, part of the vital coenzyme A biosynthetic pathway in bacteria, has recently been validated as a promising antimicrobial target. In this work, we employed native ion mobility-mass spectrometry to gain structural insights into the phosphopantothenoylcysteine synthetase domain of E. coli CoaBC. Moreover, native mass spectrometry was validated as a screening tool to identify novel inhibitors of this enzyme, highlighting the utility and versatility of this technique both for structural biology and for drug discovery

Luminescent detection of DNA-binding proteins

Transcription factors play a central role in cell development, differentiation and growth in biological systems due to their ability to regulate gene expression by binding to specific DNA sequences within the nucleus. The dysregulation of transcription factor signaling has been implicated in the pathogenesis of a number of cancers, developmental disorders, inflammation and autoimmunity. There is thus a high demand for convenient high-throughput methodologies able to detect sequence-specific DNA-binding proteins and monitor their DNA-binding activities. Traditional approaches for protein detection include gel mobility shift assays, DNA footprinting and enzyme-linked immunosorbent assays (ELISAs) which tend to be tedious, time-consuming, and may necessitate the use of radiographic labeling. By contrast, luminescence technologies offer the potential for rapid, sensitive and low-cost detection that are amenable to high-throughput and real-time analysis. The discoveries of molecular beacons and aptamers have spearheaded the development of new luminescent methodologies for the detection of proteins over the last decade. We survey here recent advances in the development of luminescent detection methods for DNA-binding proteins, including those based on molecular beacons, aptamer beacons, label-free techniques and exonuclease protection

Discutindo a educação ambiental no cotidiano escolar: desenvolvimento de projetos na escola formação inicial e continuada de professores

A presente pesquisa buscou discutir como a Educação Ambiental (EA) vem sendo trabalhada, no Ensino Fundamental e como os docentes desta escola compreendem e vem inserindo a EA no cotidiano escolar., em uma escola estadual do município de Tangará da Serra/MT, Brasil. Para tanto, realizou-se entrevistas com os professores que fazem parte de um projeto interdisciplinar de EA na escola pesquisada. Verificou-se que o projeto da escola não vem conseguindo alcançar os objetivos propostos por: desconhecimento do mesmo, pelos professores; formação deficiente dos professores, não entendimento da EA como processo de ensino-aprendizagem, falta de recursos didáticos, planejamento inadequado das atividades. A partir dessa constatação, procurou-se debater a impossibilidade de tratar do tema fora do trabalho interdisciplinar, bem como, e principalmente, a importância de um estudo mais aprofundado de EA, vinculando teoria e prática, tanto na formação docente, como em projetos escolares, a fim de fugir do tradicional vínculo “EA e ecologia, lixo e horta”.Facultad de Humanidades y Ciencias de la Educació

Mass spectrometry for fragment screening.

Fragment-based approaches in chemical biology and drug discovery have been widely adopted worldwide in both academia and industry. Fragment hits tend to interact weakly with their targets, necessitating the use of sensitive biophysical techniques to detect their binding. Common fragment screening techniques include differential scanning fluorimetry (DSF) and ligand-observed NMR. Validation and characterization of hits is usually performed using a combination of protein-observed NMR, isothermal titration calorimetry (ITC) and X-ray crystallography. In this context, MS is a relatively underutilized technique in fragment screening for drug discovery. MS-based techniques have the advantage of high sensitivity, low sample consumption and being label-free. This review highlights recent examples of the emerging use of MS-based techniques in fragment screening