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Tumor protein p63 (TP63) is a member of the TP53 protein family that are important for development and in tumor suppression. Unlike TP53, TP63 is rarely mutated in cancer, but instead different TP63 isoforms regulate its activity. TA isoforms (TAp63) act as tumor suppressors, whereas ΔN isoforms are strong drivers of squamous or squamous-like cancers. Many of these tumors become addicted to ΔN isoforms and removal of ΔN isoforms result in cancer cell death. Furthermore, some TP53 conformational mutants (TP53CM) gain the ability to interact with TAp63 isoforms and inhibit their antitumorigenic function, while indirectly promoting tumorigenic function of ΔN isoforms, but the exact mechanism of TP63–TP53CM interaction is unclear. The changes in the balance of TP63 isoform activity are crucial to understanding the transition between normal and tumor cells. Here, we modeled TP63–TP53CM complex using computational approaches. We then used our models to design peptides to disrupt the TP63–TP53CM interaction and restore antitumorigenic TAp63 function. In addition, we studied ΔN isoform oligomerization and designed peptides to inhibit its oligomerization and reduce their tumorigenic activity. We show that some of our peptides promoted cell death in a TP63 highly expressed cancer cell line, but not in a TP63 lowly expressed cancer cell line. Furthermore, we performed kinetic–binding assays to validate binding of our peptides to their targets. Our computational and experimental analyses present a detailed model for the TP63–TP53CM interaction and provide a framework for potential therapeutic peptides for the elimination of TP53CM cancer cells.

Supplementary Data from Computational Modeling of TP63–TP53 Interaction and Rational Design of Inhibitors: Implications for Therapeutics

Formation of Ras multimers, including dimers and nanoclusters, has emerged as an exciting, new front of research in the ‘old’ field of Ras biomedicine. With significant advances made in the past few years, we are beginning to understand the structure of Ras multimers and, albeit preliminary, mechanisms that regulate their formation in vitro and in cells. Here we aim to synthesize the knowledge accrued thus far on Ras multimers, particularly the presence of multiple globular (G-) domain interfaces, and discuss how membrane nanodomain composition and structure would influence Ras multimer formation. We end with some general thoughts on the potential implications of Ras multimers in basic and translational biology.

Proteins are dynamic, and this holds especially for their surfaces. They display ensembles of conformations, which allows them to interact with diverse partners, often via the same patch of surface, and execute their distinct functions. Binding a specific partner can stimulate - or suppress - a distinct signaling pathway. This diversity poses a challenge: how to reliably model a specific protein-protein interaction (PPI)? This problem is compounded in protein assemblies, which are typically large, involving multiple protein-protein interfaces. Integrative modeling (IM), which combines diverse data, has emerged as the most promising strategy; however, modeling dynamical interfaces, often at the detailed level, which are at the heart of reliable predictions of assemblies, still poses a challenge. Here we review hurdles and advances in integrative modeling of dynamical interfaces; while some could have been predicted or expected, others transformed modeling in unanticipated ways. We further comment on what we believe could be possible future advances.

p63 plays a central role in limb formation and epithelial differentiation. p63, reported to correlate with cancer formation, is rarely mutated in cancers. Different p63 isoforms can have anti-tumorigenic or pro-tumorigenic activity. TA isoforms act as tumor suppressor by promoting cell death, while deltaN isoforms are strong driver for squamous/squamous-like cancers including lung, head and neck, cervical, skin and pancreas. A large number of tumors are addicted to deltaN isoforms. Furthermore, cancer associated p53R175H mutant gains ability to interact with TAp63 isoforms and inhibit their anti-tumorigenic function, while indirectly promoting tumorigenic function of deltaN isoforms. However, the exact mechanism of p53R175H-p63 interaction is unclear. The changes in the balance of p63 isoform activity are likely to be crucial to understanding the transition between normal cell and tumor formation. Here, we modelled p53R175H-p63 complex using homology modelling, flexible/unbiased docking and molecular dynamics simulations. Moreover, we used our models to design peptides to disrupt p53R175H-p63 interaction and restore anti-tumorigenic function of TAp63 isoforms. Also, we studied deltaN isoform oligomerization and designed peptides to inhibit their oligomerization to reduce their tumorigenic activity. We showed that some of our peptides promoted cell death in a p63 enriched cancel cell line. We characterized our peptides performing kinetic binding assays to validate binding of our peptides to their designated targets. In this study, our computational and experimental analyses presented a detailed model for the p53R175H-p63 interaction. We also provided a framework for potential therapeutic peptides to outweigh the balance in the advantage of anti-tumorigenic p63 activity. Citation Format: E. Sila Ozdemir, Michelle Gomes, Jared Fischer. Targeting p63 isoforms in cancer to restore their anti-tumorigenic activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3358.

Ras recruits and activates effectors that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, Raf's activation requires dimerization, which can be facilitated by Ras dimerization. Previously, we showed that active K-Ras4B dimerizes in silico and in vitro through two major interfaces: (i) beta-interface, mapped to Switch I and effector-binding regions, (ii) alpha-interface at the allosteric lobe. Here, we chose constitutively active K-Ras4B as our control and two double mutants (K101D and R102E; and R41E and K42D) in the alpha- and beta-interfaces. Two of the mutations are from The Cancer Genome Atlas (TCGA) and the Catalogue Of Somatic Mutations In Cancer (COSMIC) data sets. R41 and R102 are found in several adenocarcinomas in Ras isoforms. We performed site-directed mutagenesis, cellular localization experiments, and molecular dynamics (MD) simulations to assess the impact of the mutations on K-Ras4B dimerization and function. alpha-interface K101D/R102E double mutations reduced dimerization but only slightly reduced downstream phosphorylated extracellular signal-regulated kinase (ERK) (pERK) levels. While beta-interface R41E/K42D double mutations did not interfere with dimerization, they almost completely blocked KRas4B-mediated ERK phosphorylation. Both double mutations increased downstream phosphorylated Akt (pAkt) levels in cells. Changes in pERK and pAkt levels altered ERK- and Akt-regulated gene expressions, such as EGR1, JUN, and BCL2L11. These results underscore the role of the alpha-interface in K-Ras4B homodimerization and the beta-surface in effector binding. MD simulations highlight that the membrane and hypervariable region (HVR) interact with both alpha- and beta-interfaces of K-Ras4B mutants, respectively, inhibiting homodimerization and probably effector binding. Mutations at both interfaces interfered with mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase signaling but in different forms and extents. We conclude that dimerization is not necessary but enhances downstream MAPK signaling. (C) 2020 Elsevier Ltd. All rights reserved.

Background: Senescent cells accumulate in tissues over time as part of the natural aging process and the removal of senescent cells has shown promise for alleviating many different age-related diseases in mice. Cancer is an age-associated disease and there are numerous mechanisms driving cellular senescence in cancer that can be detrimental to recovery. Thus, it would be beneficial to eliminate senescent cancer cells to prevent cancer recurrence or progression. Methods: We used molecular modeling to develop a series of rationally designed peptides to mimic and target FOXO4 disrupting the FOXO4-TP53 interaction and releasing TP53 to induce apoptosis. We then tested these peptides as senolytic agents for the elimination of senescent cancer cells both in cell culture and in vivo. Findings: Here we show that these peptides can act as senolytics for eliminating senescent human cancer cells both in cell culture and in orthotopic mouse models. We then further characterized one peptide, ES2, showing that it co-localizes with FOXO4 foci and binds FOXO4 to a greater extent compared to TP53. Finally, we show that concomitant intra-tumoral injection of ES2 plus a BRAF inhibitor results in a significant increase in apoptosis in a transgenic mouse model of melanoma. Interpretation: Taken together, our results reveal that peptides can be generated to specifically eliminate FOXO4+ senescent cancer cells, which has implications for eradicating residual disease and as a combination therapy for frontline treatment of cancer. Funding Statement: This work was supported by the Cancer Early Detection Advanced Research Center at Oregon Health & Science University. Declaration of Interests: S.S. Cinaroglu, Y. Ahiska, U. Sezerman, G.B. Akcapinar, E. Timucin have a patent for the ES2 structure and are members of a company, Eternans Ltd. that is working on ES2 for therapeutic use. No potential conflicts of interest were disclosed by the other authors. Ethics Approval Statement: All experiments were approved by Oregon Health and Science University (OHSU) Institutional Animal Care and Use Committee (IACUC) (TR01_IP00000674) and conformed to the guidelines set by United States Animal Welfare Act and the National Institutes of Health.

Motivation Single amino acid variations (SAVs) in protein-protein interaction (PPI) sites play critical roles in diseases. PPI sites (interfaces) have a small subset of residues called hot spots that contribute significantly to the binding energy, and they may form clusters called hot regions. Singlet hot spots are the single amino acid hot spots outside of the hot regions. The distribution of SAVs on the interface residues may be related to their disease association. Results We performed statistical and structural analyses of SAVs with literature curated experimental thermodynamics data, and demonstrated that SAVs which destabilize PPIs are more likely to be found in singlet hot spots rather than hot regions and energetically less important interface residues. In contrast, non-hot spot residues are significantly enriched in neutral SAVs, which do not affect PPI stability. Surprisingly, we observed that singlet hot spots tend to be enriched in disease-causing SAVs, while benign SAVs significantly occur in non-hot spot residues. Our work demonstrates that SAVs in singlet hot spot residues have significant effect on protein stability and function. Availability and implementation The dataset used in this paper is available as Supplementary Material. The data can be found at http://prism.ccbb.ku.edu.tr/data/sav/ as well. Supplementary information Supplementary data are available at Bioinformatics online.

Background: Senescent cells accumulate in tissues over time as part of the natural ageing process and the removal of senescent cells has shown promise for alleviating many different age-related diseases in mice. Cancer is an age-associated disease and there are numerous mechanisms driving cellular senescence in cancer that can be detrimental to recovery. Thus, it would be beneficial to develop a senolytic that acts not only on ageing cells but also senescent cancer cells to prevent cancer recurrence or progression. Methods: We used molecular modelling to develop a series of rationally designed peptides to mimic and target FOXO4 disrupting the FOXO4-TP53 interaction and releasing TP53 to induce apoptosis. We then tested these peptides as senolytic agents for the elimination of senescent cells both in cell culture and in vivo. Findings: Here we show that these peptides can act as senolytics for eliminating senescent human cancer cells both in cell culture and in orthotopic mouse models. We then further characterized one peptide, ES2, showing that it disrupts FOXO4-TP53 foci, activates TP53 mediated apoptosis and preferentially binds FOXO4 compared to TP53. Next, we show that intratumoural delivery of ES2 plus a BRAF inhibitor results in a significant increase in apoptosis and a survival advantage in mouse models of melanoma. Finally, we show that repeated systemic delivery of ES2 to older mice results in reduced senescent cell numbers in the liver with minimal toxicity. Interpretation: Taken together, our results reveal that peptides can be generated to specifically target and eliminate FOXO4+ senescent cancer cells, which has implications for eradicating residual disease and as a combination therapy for frontline treatment of cancer. Funding: This work was supported by the Cancer Early Detection Advanced Research Center at Oregon Health \& Science University. (C) 2021 The Authors. Published by Elsevier B.V.

Proper localization of Ras proteins at the plasma membrane (PM) is crucial for their functions. To get to the PM, KRas4B and some other Ras family proteins bind to the PDEδ shuttling protein through their farnesylated hypervariable regions (HVRs). The docking of their farnesyl (and to a lesser extent geranylgeranyl) in the hydrophobic pocket of PDEδ's stabilizes the interaction. At the PM, guanosine 5'-triphosphate (GTP)-bound Arf-like protein 2 (Arl2) assists in the release of Ras from the PDEδ. However, exactly how is still unclear. Using all-atom molecular dynamics simulations, we unraveled the detailed mechanism of Arl2-mediated release of KRas4B, the most abundant oncogenic Ras isoform, from PDEδ. We simulated ternary Arl2-PDEδ-KRas4B HVR complexes and observed that Arl2 binding weakens the PDEδ-farnesylated HVR interaction. Our detailed analysis showed that allosteric changes (involving β6 of PDEδ and additional PDEδ residues) compress the hydrophobic PDEδ pocket and push the HVR out. Mutating PDEδ residues that mediate allosteric changes in PDEδ terminates the release process. Mutant Ras proteins are enriched in human cancers, with currently no drugs in the clinics. This mechanistic account may inspire efforts to develop drugs suppressing oncogenic KRas4B release.

IQ motif-containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell-cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP-Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 "insert loop" was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis., Federal funds from the Frederick National Laboratory for Cancer Research, National Institutes of Health; Intramural Research Program of National Institutes of Health; Frederick National Laboratory; Center for Cancer Research; Intramural Research Program of the National Institutes of Health Clinical Center; Scientific and Technological Research Council of Turkey (TÜBİTAK)

Cataloged from PDF version of article. Here we report the synthesis of nanoparticles based on a conjugated oligomer which is synthesized through Heck-coupling of divinylfluorene and dibromobenzothiodiazole monomers. These water dispersible nanoparticles emit in the region of red tailing to the near-infrared region of the spectrum with high fluorescent quantum yield and brightness. The nanoparticles were found to be stable in water for a prolonged time without forming any aggregates and could carry camptothecin, an anticancer drug with high loading efficiency. MTT cell viability studies performed with breast cancer cell lines showed that half-maximal inhibitory concentration (IC50) values of nanoparticles for MCF7 and MDA-MB-231 were 44.7 μM and 24.8 μM, respectively. In order to further decrease the cytotoxicity and increase the stability of nanoparticles, amine groups were disguised by capping with cucurbit[7]uril (CB7). Drug release studies showed that drugs were released at low pH (at 5.0) faster than physiological pH (7.4) confirming the pH-responsive nature of the nanoparticles. On the other hand, CB7-capped drug-loaded nanoparticles regulated the release rate by providing slower release at pH 7.4 than the nanoparticles in the absence of CB7s. IC50 values for camptothecin in the presence of nanoparticles with or without CB7 were significantly reduced in MCF7 and MDA-MB-231 cells. © 2014 American Chemical Society.