On the Mechanism of Action of SJ-172550 in Inhibiting the Interaction of MDM4 and p53

SJ-172550 (1) was previously discovered in a biochemical high throughput screen for inhibitors of the interaction of MDMX and p53 and characterized as a reversible inhibitor (J. Biol. Chem. 2010; 285∶10786). Further study of the biochemical mode of action of 1 has shown that it acts through a complicated mechanism in which the compound forms a covalent but reversible complex with MDMX and locks MDMX into a conformation that is unable to bind p53. The relative stability of this complex is influenced by many factors including the reducing potential of the media, the presence of aggregates, and other factors that influence the conformational stability of the protein. This complex mechanism of action hinders the further development of compound 1 as a selective MDMX inhibitor

Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy

Peer reviewe

Heterodimerization of the Sialidase NEU1 with the Chaperone Protective Protein/Cathepsin A Prevents Its Premature Oligomerization

Lysosomal neuraminidase-1 (NEU1) forms a multienzyme complex with beta-galactosidase and protective protein/cathepsin A (PPCA). Because of its association with PPCA, which acts as a molecular chaperone, NEU1 is transported to the lysosomal compartment, catalytically activated, and stabilized. However, the mode(s) of association between these two proteins both en route to the lysosome and in the multienzyme complex has remained elusive. Here, we have analyzed the hydrodynamic properties of PPCA, NEU1, and a complex of the two proteins and identified multiple binding sites on both proteins. One of these sites on NEU1 that is involved in binding to PPCA can also bind to other NEU1 molecules, albeit with lower affinity. Therefore, in the absence of PPCA, as in the lysosomal storage disease galactosialidosis, NEU1 self-associates into chain-like oligomers. Binding of PPCA can reverse self-association of NEU1 by causing the disassembly of NEU1-oligomers and the formation of a PPCA-NEU1 heterodimeric complex. The identification of binding sites between the two proteins allowed us to create innovative structural models of the NEU1 oligomer and the PPCA-NEU1 heterodimeric complex. The proposed mechanism of interaction between NEU1 and its accessory protein PPCA provides a rationale for the secondary deficiency of NEU1 in galactosialidosis.</p

Design, Synthesis and Evaluation of 2,5-Diketopiperazines as Inhibitors of the MDM2-p53 Interaction

<div><p>The transcription factor p53 is the main tumour suppressor in cells and many cancer types have p53 mutations resulting in a loss of its function. In tumours that retain wild-type p53 function, p53 activity is down-regulated by MDM2 (human murine double minute 2) <i>via</i> a direct protein—protein interaction. We have designed and synthesised two series of 2,5-diketopiperazines as inhibitors of the MDM2-p53 interaction. The first set was designed to directly mimic the α-helical region of the p53 peptide, containing key residues in the <i>i</i>, <i>i+4</i> and <i>i+7</i> positions of a natural α-helix. Conformational analysis indicated that 1,3,6-trisubstituted 2,5-diketopiperazines were able to place substituents in the same spatial orientation as an α-helix template. The key step of the synthesis involved the cyclisation of substituted dipeptides. The other set of tetrasubstituted 2,5-diketopiperazines were designed based on structure-based docking studies and the Ugi multicomponent reaction was used for the synthesis. This latter set comprised the most potent inhibitors which displayed micromolar IC₅₀-values in a biochemical fluorescence polarisation assay.</p></div

Intratumoural Heterogeneity Underlies Distinct Therapy Responses and Treatment Resistance in Glioblastoma

Glioblastomas are the most common and lethal neoplasms of the central nervous system. Neighbouring glioma cells maintain extreme degrees of genetic and phenotypic variation that form intratumoural heterogeneity. This genetic diversity allows the most adaptive tumour clones to develop treatment resistance, ultimately leading to disease recurrence. We aimed to model this phenomenon and test the effectiveness of several targeted therapeutic interventions to overcome therapy resistance. Heterogeneous tumour masses were first deconstructed into single tumour cells, which were expanded independently as single-cell clones. Single nucleotide polymorphism arrays, whole-genome and RNA sequencing, and CpG methylation analysis validated the unique molecular profile of each tumour clone, which displayed distinct pathologic features, including cell morphology, growth rate, and resistance to temozolomide and ionizing radiation. We also identified variable sensitivities to AURK, CDK, and EGFR inhibitors which were consistent with the heterogeneous molecular alterations that each clone harboured. These targeted therapies effectively eliminated the temozolomide- and/or irradiation-resistant clones and also parental polyclonal cells. Our findings indicate that polyclonal tumours create a dynamic environment that consists of diverse tumour elements and treatment responses. Designing targeted therapies based on a range of molecular profiles can be a more effective strategy to eradicate treatment resistance, recurrence, and metastasis

Alteration of RNA Splicing by Small-Molecule Inhibitors of the Interaction between NHP2L1 and U4

Splicing is an important eukaryotic mechanism for expanding the transcriptome and proteome, influencing a number of biological processes. Understanding its regulation and identifying small molecules that modulate this process remain a challenge. We developed an assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) to detect the interaction between the protein NHP2L1 and U4 RNA, which are two key components of the spliceosome. We used this assay to identify small molecules that interfere with this interaction in a high-throughput screening (HTS) campaign. Topotecan and other camptothecin derivatives were among the top hits. We confirmed that topotecan disrupts the interaction between NHP2L1 and U4 by binding to U4 and inhibits RNA splicing. Our data reveal new functions of known drugs that could facilitate the development of therapeutic strategies to modify splicing and alter gene function