Stapled α−helical peptide drug development: A potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy

The p53 inhibitor MDMX is controlled by multiple stress signaling pathways. Using a proteolytic fragment release (PFR) assay, we detected an intramolecular interaction in MDMX that mechanistically mimics the interaction with p53, resulting in autoinhibition of MDMX. This mimicry is mediated by a hydrophobic peptide located in a long disordered central segment of MDMX that has sequence similarity to the p53 transactivation domain. NMR spectroscopy was used to show this hydrophobic peptide interacts with the N-terminal domain of MDMX in a structurally analogous manner to p53. Mutation of two critical tryptophan residues in the hydrophobic peptide disrupted the intramolecular interaction and increased p53 binding, providing further evidence for mechanistic mimicry. The PFR assay also revealed a second intramolecular interaction between the RING domain and central region that regulates MDMX nuclear import. These results establish the importance of intramolecular interactions in MDMX regulation, and validate a new assay for the study of intramolecular interactions in multidomain proteins with intrinsically disordered regions.T he p53 tumor suppressor is activated by numerous cellular and environmental signals and induces the expression of genes that regulate metabolism, cell growth, cell cycle, apoptosis, and senescence (1). MDM2 and MDMX are key regulators that control the cellular level and transcriptional activity of p53 through direct binding. Mouse knockout experiments showed that both MDM2 and MDMX are essential for controlling p53 activity during embryogenesis. Somatic knockout experiments showed that MDM2 is indispensable for regulating p53 in adult tissues, whereas MDMX deletion does not lead to cell death (2). MDM2 is a well-established p53 transcriptional target that forms a negative feedback loop by binding to the N-terminal transcriptional activation domain of p53 and, subsequently, ubiquitinating the C-terminal regulatory domain, which leads to degradation of p53 by the proteasome. p53 binding sites are also found in intron 1 of human MDMX, and p53 activation leads to moderate induction of MDMX transcription (3). Therefore, MDMX is a p53 target gene that may also provide dynamic feedback in response to p53 activation.MDMX alone does not have E3 ligase activity, but it is important for regulating p53 transcriptional function. MDMX expression and phosphorylation by the ATM/Chk2 pathway is important for the p53-mediated DNA damage response in mice (4, 5). MDMX levels are controlled by MDM2-mediated ubiquitination in a stress-dependent fashion (6, 7). Significant degradation of MDMX occurs after DNA damage through phosphorylation at several C-terminal sites (S342 and S367 by Chk2, S403 by ATM) (8). Furthermore, ribosomal stress promotes MDMX degradation through L11-MDM2 interaction (9), and oncogenic stress promotes MDMX degradation through ARF expression (10). Therefore, key signaling mechanisms that block p53 degradation simultaneously enhance MDMX degradation by MDM2. These observations underscore the co...

Section: Discussionmentioning

confidence: 99%

Autoinhibition of MDMX by intramolecular p53 mimicry

Chen¹,

Borcherds

Wu³

et al. 2015

“…By sampling alternative staple positions, interrogating cellular uptake, and correlating biochemical function with antitumor activity and mechanism of action, we have generated lead compounds that form the basis for therapeutic development (16,17,19,20). Here, we report the application of all-hydrocarbon stapling to recapitulate the native primary sequence and secondary structure of the RAS-interacting α-helix of SOS1.…”

mentioning

confidence: 99%

Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices

Leshchiner

Parkhitko²,

Bird

et al. 2015

148

121

Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of ∼30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequencespecific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85D V12 activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer.R AS signaling is a critical control point for a host of cellular functions ranging from cellular survival and proliferation to cellular endocytosis and motility (1). The on or off state of RAS is dictated by nucleotide exchange. GTP-bound RAS is the activated form that engages its downstream effectors with high avidity. The endogenous GTPase activity of RAS hydrolyzes GTP to GDP and inactivates signaling. This biochemical process is further regulated by GTPase-activating proteins (GAPs) that impair RAS signaling through increasing endogenous GTPase activity and guanine-nucleotide exchange factors (GEFs) that enhance RAS signaling by facilitating GDP release and, thus, GTP association. Given the central roles of RAS in cellular growth and metabolism, it is not surprising that cancer cells usurp its prosurvival activities to achieve immortality.Activating mutations in KRAS represent the most frequent oncogenic driving force among the RAS homologs K-, N-, and H-RAS, and are associated with poor prognosis and chemoresistance (2). KRAS mutations are present in ∼30% of human tumors and at even higher frequencies in cancers of the pancreas, lung, thyroid gland, colon, and liver. For example, in pancreatic ductal adenocarcinomas (PDAC) that carry a 5-y survival rate of less than 5%, activating KRAS mutations are present in more than 90% of tumors (3). Thus, therapeutic inhibition of RAS is among the highest priority goals of the cancer field. Because oncogenic forms of KRAS typically harbor single-point mutants that stabilize its active GTP-bound form, a host of recent small molecule and peptide development efforts have been aimed at disarming this pathologic biochemical state. The extremely high affinity of KRAS for its GTP substrate has hampered the development of competitive GTP inhibitors. However, a GDP mimetic that covalently modifies the mutant cysteine of KRAS G12C represents a promising approach to plugging the nucleotid...

“…Added to this difficulty has been the complexity of the ubiquitin system, with ligases having many substrates and many proteins being substrates for multiple ligases (6,9). Despite these problems, peptide and small molecule inhibitors have been successfully obtained that specifically target the substrate binding site of Mdm2, a RING ligase that acts on p53, and consequently activate the p53 pathway (10,11). However, only a few other inhibitors of RING ligases have been published (12,13), and none have been published that inhibit HECT ligases in vivo.…”

mentioning

confidence: 99%

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases

Mund

Lewis

Maslen

2014

129

133

The human genome encodes several hundred E3 ubiquitin ligases containing RING domains, and around 28 containing HECT domains. These enzymes catalyze the transfer of ubiquitin from E2 enzyme thioesters to a huge range of substrates and play crucial roles in many cellular functions. This makes them attractive potential therapeutic targets. However, they have proven difficult to inhibit: very few good inhibitors exist for RING domain ligases, and none have been described for HECT ligases. Here we show that bicyclic peptides isolated by phage display [Heinis C, Rutherford T, Freund S, Winter G (2009) Nat Chem Biol. 5(7):502-507] can target the E2 binding sites on the HECT domains of Smurf2, Nedd4, Mule/ Huwe1, and WWP1, and thus act as specific inhibitors of these enzymes in vitro. By screening for displacement of one of these peptides from Smurf2, we were able to identify a small molecule, heclin (HECT ligase inhibitor), which inhibits several HECT ligases in tissue culture cells. In vitro, heclin does not block E2 binding but causes a conformational change that results in oxidation of the active site Cys. This demonstrates that HECT domains are potentially druggable and provides molecules that may be of experimental use. Heclin kills HEK293 cells growing in culture, consistent with an essential role for HECT ligase activity in mammalian cells.inhibitor | ubiquitin | HECT ligase | phage display U biquitin ligases are involved in almost every important function of cells, including the removal of misfolded proteins, the regulation of signaling pathways, DNA repair, the cell cycle, and apoptosis (1-4). This broad range of functions is mediated by a large number of E3 ubiquitin ligases, which recognize a similarly large range of substrates. Ubiquitination begins with the ATP-dependent formation of a ubiquitin (Ub)-E2 enzyme thioester intermediate by the E1 enzyme. The E3 enzymes then transfer ubiquitin from this intermediate to a lysine residue on the substrate (3). There are two main classes of E3: the RING domain ligases and their relatives, of which there are several hundred in humans, and the HECT domain ligases, which number around 28 (5-7). Because of their diversity and numerous functions and the fact that some ligases are frequently overexpressed in cancer cells, ubiquitin ligases have attracted much attention as possible targets for therapeutic intervention (2,5,6,8).Despite this interest, it has proven challenging to make small molecule inhibitors of ubiquitin ligases. The action of these enzymes involves primarily protein-protein interactions among them, the Ub-E2 conjugate, and the substrate. For the RING enzymes, this is simultaneous, and the Ub moiety is transferred directly from E2 to substrate; for the HECT ligases, the interaction is sequential, with a thioester-linked Ub-E3 intermediate forming transiently. Such protein-protein interactions are considered difficult to block with small molecules. Added to this difficulty has been the complexity of the ubiquitin system, with ligases having many substrat...