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

Development of a novel, high-affinity ssDNA trypsin inhibitor

Inhibitors of serine proteases are not only extremely useful in the basic research but are also applied extensively in clinical settings. Using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approach we developed a family of novel, single-stranded DNA aptamers capable of specific trypsin inhibition. Our most potent candidate (T24) and its short version (T59) were thoroughly characterised in terms of efficacy. T24 and T59 efficiently inhibited bovine trypsin with Ki of 176 nM and 475 nM, respectively. Interestingly, in contrast to the majority of known trypsin inhibitors, the selected aptamers have superior specificity and did not interact with porcine trypsin or any human proteases tested. These included plasmin and thrombin characterised by trypsin-like substrate specificity. Our results demonstrate that SELEX may be successfully employed in the development of potent and specific DNA based protease inhibitors

Mdm2 and MdmX inhibitors for the treatment of cancer:a patent review (2011-present)

Introduction: One of the hallmarks of cancer cells is the inactivation of the p53 pathway either due to mutations in the p53 gene or over-expression of negative regulators, Mdm2 and/or MdmX. Pharmacological disruption of the Mdm2/X-p53 interaction to restore p53 activity is an attractive concept, aiming at a targeted and non-toxic cancer treatment. Areas covered: The introduction covers the biological role of p53 pathway and its regulation by Mdm2 and MdmX in normal and cancer cells and the current repertoire and development status of inhibitors of the Mdm2/X-p53 interaction for the treatment of cancer. The main part of the article covers patents and patent applications describing small molecule inhibitors of the Mdm2/X-p53 interaction published from 2011 until 2012. Expert opinion: The area of small molecule Mdm2/X-p53 interaction inhibitor development is progressing fast. Several Phase I clinical studies and preclinical programs are now in progress, however, the clinical proof concept has yet to be demonstrated. Multiple available compounds inhibit Mdm2-p53 interaction with nanomolar affinities, but MdmX is still missing such potent binders. Since research points to a complementary mode of Mdm2 and MdmX action, the future compound classes will possibly want to include dual actions versus Mdm2 and MdmX

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

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

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

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

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