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

Biochemical and structural characterization of SplD protease from Staphylococcus aureus.

Staphylococcus aureus is a dangerous human pathogen. A number of the proteins secreted by this bacterium are implicated in its virulence, but many of the components of its secretome are poorly characterized. Strains of S. aureus can produce up to six homologous extracellular serine proteases grouped in a single spl operon. Although the SplA, SplB, and SplC proteases have been thoroughly characterized, the properties of the other three enzymes have not yet been investigated. Here, we describe the biochemical and structural characteristics of the SplD protease. The active enzyme was produced in an Escherichia coli recombinant system and purified to homogeneity. P1 substrate specificity was determined using a combinatorial library of synthetic peptide substrates showing exclusive preference for threonine, serine, leucine, isoleucine, alanine, and valine. To further determine the specificity of SplD, we used high-throughput synthetic peptide and cell surface protein display methods. The results not only confirmed SplD preference for a P1 residue, but also provided insight into the specificity of individual primed- and non-primed substrate-binding subsites. The analyses revealed a surprisingly narrow specificity of the protease, which recognized five consecutive residues (P4-P3-P2-P1-P1') with a consensus motif of R-(Y/W)-(P/L)-(T/L/I/V)↓S. To understand the molecular basis of the strict substrate specificity, we crystallized the enzyme in two different conditions, and refined the structures at resolutions of 1.56 Å and 2.1 Å. Molecular modeling and mutagenesis studies allowed us to define a consensus model of substrate binding, and illustrated the molecular mechanism of protease specificity

Prevalence of genes encoding extracellular proteases in Staphylococcus aureus - important targets triggering immune response in vivo

Proteases of Staphylococcus aureus have long been considered to function as important virulence factors, although direct evidence of the role of particular enzymes remains incomplete and elusive. Here, we sought to provide a collective view of the prevalence of extracellular protease genes in genomes of commensal and pathogenic strains of S. aureus and their expression in the course of human and mouse infection. Data on V8 protease, staphopains A and B, aureolysin, and the recently described and poorly characterized group of six Spl proteases are provided. A phylogenetically diverse collection of 167 clinical isolates was analyzed, resulting in the comprehensive genetic survey of the prevalence of protease-encoding genes. No correlation between identified gene patterns with specific infections was established. Humoral response against the proteases of interest was examined in the sera derived from human patients and from a model mouse infection. The analysis suggests that at least some, if not all, tested proteases are expressed and secreted during the course of infection. Overall, the results presented in this study support the hypothesis that the secretory proteases as a group may contribute to the virulence of S. aureus

Formation of covalent adducts between compound 1 and MDMX.

<p><b>Panel a.</b> Mass spectrum arising from unmodified hMDMX (GST-tagged screening construct) showing unmodified mass of the protein. <b>Panel b.</b> Mass spectrum arising from treatment of 20 µM GST-hMDMX with 100 µM of compound <b>1</b> demonstrating multiple alkylation events. Note that 100 µM is well above the solubility limit of compound <b>1</b> and significant aggregation of compound exists. <b>Panel c.</b> Mass spectrum arising from treatment of 1 µM GST-hMDMX with 5 µM of compound <b>1</b> demonstrating no alkylation events. <b>Panel d.</b> Mass spectrum arising from unmodified hMDMX (untagged aa 23 to 111 construct) showing unmodified mass of the protein. <b>Panel e.</b> Mass spectrum arising from treatment of 20 µM hMDMX with 100 µM of compound <b>1</b> demonstrating partial alkylation. <b>Panel f.</b> Mass spectrum arising from treatment of 1 µM hMDMX with 5 µM of compound <b>1</b> demonstrating no alkylation.</p

Structures of relevant compounds.

<p><b>Panel A.</b> Structure of SJ-172550 (<b>1</b>) and a non-reactive analog (<b>2</b>). <b>Panel B.</b> The potential mechanism of covalent adduct formation.</p

Inhibition of MDMX-p53 peptide binding by compound 1 (IC₅₀ = 3 µM), compound 2 (IC₅₀>100 uM).

<p>Inhibition of MDMX-p53 peptide binding by compound 1 (IC₅₀ = 3 µM), compound 2 (IC₅₀>100 uM).</p

Thermal stability equilibria of MDMX.

<p><b>Panel a.</b> Thermal shift data for MDMX (23–111) showing a 7 degree stabilization of the protein’s melting point by addition of compound <b>1</b>. The panel shows individual data sampling points from 3 independent experiments from each condition. <b>Panel b.</b> Dose dependency and time dependency of the effect showing an apparent EC₅₀ of roughly 1 µM and minimal time dependency. <b>Panel c.</b> Dose dependent reversal of the effects of compound <b>1</b> by TCEP. <b>Panel d.</b> Dose dependent reversal of the effects of compound <b>1</b> by DTT.</p

Reversibility of the interaction of compound 1 with MDMX.

<p><b>Panel a.</b> SPR study of the binding of <b>1</b> (100 µM) to hMDMX (aa 23–111) under non-reducing conditions. While the off-rate is slow, the interaction is reversible. <b>Panel b.</b> SPR study of the binding of <b>1</b> (100 µM) to hMDMX (aa 23–111) under reducing conditions. No binding is observed.</p