Ovarian cancer (OC) is among the most common malignancies in the female reproductive system, marked by high rates of recurrence and mortality. Conventional chemotherapy, however, faces limitations due to the development of drug resistance, which hinders its effectiveness. Ferroptosis, an atypical form of programmed cell death distinct from autophagy, apoptosis, and necrosis, the relationship with tumors has become a hot research area in cancer studies in recent years. Anticancer therapies that target ferroptosis show strong potential in improving prognosis and counteracting chemotherapy resistance. Natural compounds, as a valuable source of novel targeted anticancer agents, its significant role in inhibiting tumor cell proliferation and metastasis and improving therapeutic sensitivity has been demonstrated in numerous existing studies. This review summarizes a range of natural compounds that target ferroptosis in OC cells, discussing their active components, mechanisms of action, and therapeutic potential, thereby providing useful insights for future targeted therapy and research in OC.

Wild-type p53 acts as a tumor suppressor, but p53 is frequently mutated and inactivated in tumor cells, promoting cancer progression, invasion, and metastasis. Thus, compounds that reactivate p53 may be leveraged for cancer treatment, and the development of drugs targeting p53 reactivation is actively progressing. Notably, natural products exhibit diverse structures and biological activities and are used as therapeutic agents for various diseases worldwide. This review discusses the natural products that inhibit p53 degradation through p53-Mdm2 interaction, promote p53 reactivation by inducing conformational changes, and exhibit p53-dependent growth inhibition.

Acute myeloid leukemia (AML) is a devastating disease characterized by extensive inter-patient and intra-patient heterogeneity. Despite the introduction of intensive chemotherapy in the 1970s as the standard treatment, the development of mechanism-based targeted therapies since 2017 has been broadening the therapeutic landscape. However, both chemotherapy and targeted therapies continue to face the challenges of primary and secondary resistance. This review summarizes the mechanisms underlying resistance to chemotherapy and targeted therapies in AML and discusses the opportunities and challenges brought by the transition from chemotherapy to precision medicine.

Understanding the tumor microenvironment (TME) is essential for the advancement of immunotherapy for ovarian cancer (OC). Nonetheless, predicting transcription factor (TF) regulation from the TME using single-cell RNA sequencing (scRNA-seq) data is challenging.

The OC scRNA-seq data were analyzed with a specialized scRNA-seq transcriptome analysis application. The OC TME was utilized to instruct the SCENIC procedure for TF regulation. We built a risk model using Lasso regression and identified immunological subgroups using ConsensusClusterPlus. To analyze the percentage of invading immune cells, the algorithms CIBERSORT, ESTIMATE, and xCell were used. We computed the stromal score, immunological score, estimate score, and tumor purity to evaluate the risk model's capacity to predict the tumor immune microenvironment. Additionally, the expression of immunological checkpoints was examined, and for pertinent evaluation, the imvigor 210 dataset of the immunotherapy cohort was used. pRophetic predicted the sensitivity of 138 GDSC database drugs. In addition, we examined the expression of unproven risk model genes using qPCR and immunohistochemistry (JCHAIN, UBD, and RARRES1). Cell proliferation was assessed by colony formation assays. Transwell experiments were used to examine the invasion and migration ability of OC cells.

Six immunologically malignant cell subpopulations have been identified within the cancer immune microenvironment (referred to as TC0-6). Unique in its immunological profile, TC0 demonstrates the most intimate interactions with immune cells. Following a meta gene screen in the TC0 subpopulation using the top 30 targets of 14 transcription factor (TF) factors, two distinct immunological molecular subtypes-the C1 and C2 subtypes-with notable survival differences were discovered. On the basis of nine genes whose expression differs between the C1 and C2 subtypes, a risk model was constructed. The risk model is an accurate method for forecasting the effectiveness of immunotherapies, clinicopathological characteristics, and survival. JCHAIN and UBD expression in OC tissues was found to be low according to qPCR and IHC analyses, whereas RARRES1 expression was found to be high. The functional experiment results indicated that downregulation of JCHAIN and UBD and overexpression of RARRES1 could suppress the proliferation, migration, and invasion of OC cells in vitro.

Based on TF regulatory networks in the tumor microenvironment, this study developed a 9-gene risk model for the prognosis of ovarian cancer. This model may aid in the future promotion of personalized OC immunotherapy. In addition, JCHAIN, UBD, and RARRES1 were identified as three novel immune-related biomarkers for OC.

The tumor suppressor p53 (Trp53), also known as p53, is the most commonly mutated gene in cancer. Canonical p53 DNA damage response pathways are well characterized and classically thought to underlie the tumor suppressive effect of p53. Challenging this dogma, mouse models have revealed that p53-driven apoptosis and cell cycle arrest are dispensable for tumor suppression. Here, we investigated the inverse context of a p53 mutation predicted to drive the expression of canonical targets but is detected in human cancer.

We established a novel mouse model with a single base pair mutation (GAG>GAT, p53E221D) in the DNA-Binding domain that has wild-type function in screening assays, but is paradoxically found in human cancer in Li-Fraumeni syndrome. Using mouse p53E221D and the analogous human p53E224D mutants, we evaluated expression, transcriptional activation, and tumor suppression in vitro and in vivo.

Expression of human p53E224D from cDNA translated to a fully functional p53 protein. However, p53E221D/E221D RNA transcribed from the endogenous locus is mis-spliced resulting in nonsense-mediated decay. Moreover, fibroblasts derived from p53E221D/E221D mice do not express a detectable protein product. Mice homozygous for p53E221D exhibited increased tumor penetrance and decreased life expectancy compared to p53WT/WT animals.

Mouse p53E221D and human p53E224D mutations lead to splice variation and a biologically relevant p53 loss of function in vitro and in vivo.

Fluorescent therapeutic molecules offer a unique platform to study cellular uptake and biological pathways of drug candidates. Inhibition of the p53-HDM2 protein complex with the reactivation of the p53 pathway leading to apoptosis is a promising way to overcome the barriers and challenges in cancer therapeutic design. Although p53 helix mimetics based on the 'hotspots' design using either helical or non-helical backbones are known, cell-permeable and biocompatible inherently fluorescent helix mimetics have not yet been described. We report theragnostic helix mimetics featuring both therapeutic and bioimaging properties in a cancer cell model for the first time. The solvatochromic phthalimide unit in the scaffold functions as a site to append the hotspot mimicking residues, helps in the intramolecular hydrogen bonding mediated pre-organization of side chains on one face, and importantly, exhibits intrinsic fluorescence. The design of the mimetics, synthesis, conformational studies, and molecular docking results are discussed. In vitro cytotoxicity studies were carried out on four cell lines: U87MG (human glioblastoma), A549 (human non-small cell lung cancer), MDA-MB-231 (human triple-negative breast cancer) and HEK293 (non-cancerous cell line). The molecules showed anticancer activity in the micromolar range. The fluorescence properties provided valuable insights into their cellular permeability, distribution, and selectivity towards cancer cells and can shed light on their mechanisms of action.

Immune function is highly controlled at the transcriptional level by the binding of transcription factors (TFs) to promoter and enhancer elements. Several TF families play major roles in immune gene expression, including NF-κB, STAT, IRF, AP-1, NRs, and NFAT, which trigger anti-pathogen responses, promote cell differentiation, and maintain immune system homeostasis. Aberrant expression, activation, or sequence of isoforms and variants of these TFs can result in autoimmune and inflammatory diseases as well as hematological and solid tumor cancers. For this reason, TFs have become attractive drug targets, even though most were previously deemed "undruggable" due to their lack of small molecule binding pockets and the presence of intrinsically disordered regions. However, several aspects of TF structure and function can be targeted for therapeutic intervention, such as ligand-binding domains, protein-protein interactions between TFs and with cofactors, TF-DNA binding, TF stability, upstream signaling pathways, and TF expression. In this review, we provide an overview of each of the important TF families, how they function in immunity, and some related diseases they are involved in. Additionally, we discuss the ways of targeting TFs with drugs along with recent research developments in these areas and their clinical applications, followed by the advantages and disadvantages of targeting TFs for the treatment of immune disorders.

Chemotherapy remains the cornerstone of cancer treatment; however, its efficacy is frequently compromised by the development of chemoresistance. Multidrug resistance (MDR), characterized by the refractoriness of cancer cells to a wide array of chemotherapeutic agents, presents a significant barrier to achieving successful and sustained cancer remission. One critical factor contributing to this chemoresistance is the overexpression of ATP-binding cassette (ABC) transporters. Furthermore, additional mechanisms, such as the malfunctioning of apoptosis, alterations in DNA repair systems, and resistance mechanisms inherent to cancer stem cells, exacerbate the issue. Intriguingly, microRNAs (miRNAs) have demonstrated potential in modulating chemoresistance by specifically targeting ABC transporters, thereby offering promising new avenues for overcoming drug resistance. This narrative review aims to elucidate the molecular underpinnings of drug resistance, with a particular focus on the roles of ABC transporters and the regulatory influence of miRNAs on these transporters.

Combining helical foldamers with α-peptides can produce α-helix mimetics with a reduced peptide character and enhanced resistance to proteolysis. Previously, we engineered a hybrid peptide-oligourea sequence replicating the N-terminal α-helical domain of p53 to achieve high affinity binding to hDM2. Here, we further advance this strategy by combining the foldamer approach with side chain cross-linking to create more constrained cell-permeable inhibitors capable of effectively engaging the target within cells. Starting from the crystal structure of the foldamer-hDM2 complex, we identified specific sites suitable for stapling, and generated a small library of macrocyclic foldamer-peptide hybrids. The most promising binders were subsequently optimized for cellular uptake and tested in a cellular assay. We observed that the introduction of a short segment of positively charged residues at the N-terminus of the sequence led to inhibitors that exhibited cytotoxic activity independently of p53. In contrast, neutral acetylated peptide-foldamer macrocycles demonstrated activity in a p53-dependent manner.

The p53 protein, encoded by the TP53 gene, serves as a critical tumor suppressor, playing a vital role in maintaining genomic stability and regulating cellular responses to stress. Dysregulation of p53 is frequently observed in hematological malignancies, significantly impacting disease progression and patient outcomes. This review aims to examine the regulatory mechanisms of p53, the implications of TP53 mutations in various hematological cancers, and emerging therapeutic strategies targeting p53. We conducted a comprehensive literature review to synthesize recent findings related to p53's multifaceted role in hematologic cancers, focusing on its regulatory pathways and therapeutic potential. TP53 mutations in hematological malignancies often lead to treatment resistance and poor prognosis. Current therapeutic strategies, including p53 reactivation and gene therapy, show promise in improving treatment outcomes. Understanding the intricacies of p53 regulation and the consequences of its mutations is essential for developing effective diagnostic and therapeutic strategies in hematological malignancies, ultimately enhancing patient care and survival.

Peptides possess a number of pharmacologically desirable properties, including greater chemical diversity than other biomolecule classes and the ability to selectively bind to specific targets with high potency, as well as biocompatibility, biodegradability, and ease and low cost of production. Consequently, there has been considerable interest in developing peptide-based therapeutics, including amyloid inhibitors. However, a major hindrance to the successful therapeutic application of peptides is their poor delivery to target tissues, cells or subcellular organelles. To overcome these issues, recent efforts have focused on engineering cell-penetrating peptide (CPP) antagonists of amyloidogenesis, which combine the attractive intrinsic properties of peptides with potent therapeutic effects (i.e., inhibition of amyloid formation and the associated cytotoxicity) and highly efficient delivery (to target tissue, cells, and organelles). This review highlights some promising CPP constructs designed to target amyloid aggregation associated with a diverse range of disorders, including Alzheimer's disease, transmissible spongiform encephalopathies (or prion diseases), Parkinson's disease, and cancer.

The p53 tumor suppressor is inactivated by mutations in about 50% of tumors. Rescuing the transcriptional function of mutant p53 has potential therapeutic benefits. Approximately 15% of p53 mutants are temperature sensitive (TS) and regain maximal activity at 32°C. Proof of concept study showed that induction of 32°C hypothermia in mice restored TS mutant p53 activity and inhibited tumor growth. However, 32°C is the lower limit of therapeutic hypothermia procedures for humans. Higher temperatures are preferable but result in suboptimal TS p53 activation. Recently, arsenic trioxide (ATO) was shown to rescue the conformation of p53 structural mutants by stabilizing the DNA binding domain. We examined the responses of 17 frequently observed p53 TS mutants to functional rescue by temperature shift and ATO. The results showed that ATO only rescued mild p53 TS mutants with high basal activity at 37°C. Mild TS mutants showed a common feature of regaining significant activity at the semi-permissive temperature of 35°C and could be further stimulated by ATO at 35°C. TS p53 rescue by ATO was antagonized by the cellular redox mechanism and was rapidly reversible. Inhibition of glutathione (GSH) biosynthesis enhanced ATO rescue efficiency and sustained p53 activity after ATO washout. The results suggest that mild TS p53 mutants are uniquely responsive to functional rescue by ATO due to small thermostability deficits and inherent potential to regain active conformation. Combining mild hypothermia and ATO may provide an effective and safe procedure for targeting tumors with p53 TS mutations.

Mutation of thetumor suppressor gene, TP53 (tumor protein 53), occurs in up to 15% of all patients with acute myeloid leukemia (AML) and is enriched within specific clinical subsets, most notably in older adults, and including secondary AML cases arising from preceding myeloproliferative neoplasm (MPN), myelodysplastic syndrome (MDS), patients exposed to prior DNA-damaging, cytotoxic therapies. In all cases, these tumors have remained difficult to effectively treat with conventional therapeutic regimens. Newer approaches fortreatmentofTP53-mutated AML have shifted to interventions that maymodulateTP53 function, target downstream molecular vulnerabilities, target non-p53 dependent molecular pathways, and/or elicit immunogenic responses. This review will describe the basic biology of TP53, the clinical and biological patterns of TP53 within myeloid neoplasms with a focus on elderly AML patients and will summarize newer therapeutic strategies and current clinical trials.

High expression of ubiquitin ligase MDM2 is a primary cause of p53 inactivation in many tumors, making it a promising therapeutic target. However, MDM2 inhibitors have failed in clinical trials due to p53-induced feedback that enhances MDM2 expression. This underscores the urgent need to find an effective adaptive genotype or combination of targets.

Kinome-wide CRISPR/Cas9 knockout screen was performed to identify genes that modulate the response to MDM2 inhibitor using TP53 wild type cancer cells and found ULK1 as a candidate. The MTT cell viability assay, flow cytometry and LDH assay were conducted to evaluate the activation of pyroptosis and the synthetic lethality effects of combining ULK1 depletion with p53 activation. Dual-luciferase reporter assay and ChIP-qPCR were performed to confirm that p53 directly mediates the transcription of GSDME and to identify the binding region of p53 in the promoter of GSDME. ULK1 knockout / overexpression cells were constructed to investigate the functional role of ULK1 both in vitro and in vivo. The mechanism of ULK1 depletion to activate GSMDE was mainly investigated by qPCR, western blot and ELISA.

By using high-throughput screening, we identified ULK1 as a synthetic lethal gene for the MDM2 inhibitor APG115. It was determined that deletion of ULK1 significantly increased the sensitivity, with cells undergoing typical pyroptosis. Mechanistically, p53 promote pyroptosis initiation by directly mediating GSDME transcription that induce basal-level pyroptosis. Moreover, ULK1 depletion reduces mitophagy, resulting in the accumulation of damaged mitochondria and subsequent increasing of reactive oxygen species (ROS). This in turn cleaves and activates GSDME via the NLRP3-Caspase inflammatory signaling axis. The molecular cascade makes ULK1 act as a crucial regulator of pyroptosis initiation mediated by p53 activation cells. Besides, mitophagy is enhanced in platinum-resistant tumors, and ULK1 depletion/p53 activation has a synergistic lethal effect on these tumors, inducing pyroptosis through GSDME directly.

Our research demonstrates that ULK1 deficiency can synergize with MDM2 inhibitors to induce pyroptosis. p53 plays a direct role in activating GSDME transcription, while ULK1 deficiency triggers upregulation of the ROS-NLRP3 signaling pathway, leading to GSDME cleavage and activation. These findings underscore the pivotal role of p53 in determining pyroptosis and provide new avenues for the clinical application of p53 restoration therapies, as well as suggesting potential combination strategies.

Macrocyclic peptides show promise in targeting high-value therapeutically relevant binding sites due to their high affinity and specificity. However, their clinical application is often hindered by low membrane permeability, which limits their effectiveness against intracellular targets. Previous studies focused on peptide conformations in various solvents, leaving a gap in understanding their interactions with and translocation through lipid bilayers. Addressing this, our study explores the membrane interactions of stapled peptides, a subclass of macrocyclic peptides, using solid-state nuclear magnetic resonance (ssNMR) spectroscopy and molecular dynamics (MD) simulations. We conducted ssNMR measurements on ATSP-7041M, a prototypical stapled peptide, to understand its interaction with lipid membranes, leading to an MD-informed model for peptide membrane permeation. Our findings reveal that ATSP-7041M adopts a stable α-helical structure upon membrane binding, facilitated by a cation-π interaction between its phenylalanine side chain and the lipid headgroup. This interaction makes the membrane-bound state energetically favorable, facilitating membrane affinity and insertion. The bound peptide displayed asymmetric insertion depths, with the C-terminus penetrating deeper (approximately 9 Å) than the N-terminus (approximately 4.3 Å) relative to the lipid headgroups. Contrary to expectations, peptide dynamics was not hindered by membrane binding and exhibited rapid motions similar to cell-penetrating peptides. These dynamic interactions and peptide-lipid affinity appear to be crucial for membrane permeation. MD simulations indicated a thermodynamically stable transmembrane conformation of ATSP-7041M, reducing the energy barrier for translocation. Our study offers an in silico view of ATSP-7041M's translocation from the extracellular to the intracellular region, highlighting the significance of peptide-lipid interactions and dynamics in enabling peptide transit through membranes.

Mutations resulting in decreased activity of p53 tumor suppressor protein promote tumorigenesis. P53 protein levels are tightly regulated through the Ubiquitin Proteasome System (UPS). Several E3 ligases were shown to regulate p53 stability, including MDM2. Here we report that the ubiquitin E3 ligase XIAP (X-linked Inhibitors of Apoptosis) is a direct ligase for p53 and describe a novel approach for modulating the levels of p53 by targeting the XIAP pathway. Using in vivo (live-cell) and in vitro (cell-free reconstituted system) ubiquitylation assays, we show that the XIAP-antagonist ARTS regulates the levels of p53 by promoting the degradation of XIAP. XIAP directly binds and ubiquitylates p53. In apoptotic cells, ARTS inhibits the ubiquitylation of p53 by antagonizing XIAP. XIAP knockout MEFs express higher p53 protein levels compared to wild-type MEFs. Computational screen for small molecules with high affinity to the ARTS-binding site within XIAP identified a small-molecule ARTS-mimetic, B3. This compound stimulates apoptosis in a wide range of cancer cells but not normal PBMC (Peripheral Blood Mononuclear Cells). Like ARTS, the B3 compound binds to XIAP and promotes its degradation via the UPS. B3 binding to XIAP stabilizes p53 by disrupting its interaction with XIAP. These results reveal a novel mechanism by which ARTS and p53 regulate each other through an amplification loop to promote apoptosis. Finally, these data suggest that targeting the ARTS binding pocket in XIAP can be used to increase p53 levels as a new strategy for developing anti-cancer therapeutics.

USP7 is an attractive therapeutic target for cancers, especially for acute lymphoblastic leukemia (ALL) with wild-type p53. Herein, we report the discovery of XM-U-14 as a highly potent, selective and efficacious USP7 proteolysis-targeting chimera degrader. XM-U-14 achieves DC50 values of 0.74 nM and Dmax of 93% in inducing USP7 degradation in RS4;11 cell lines, and also significantly inhibits ALL cell growth. XM-U-14 even at 5 mg/kg dosed daily effectively inhibits RS4;11 tumor growth with 64.7% tumor regressions and causes no signs of toxicity in mice. XM-U-14 is a promising USP7 degrader for further optimization for ALL treatment.

Inhibiting MDM2-p53 interaction is considered an efficient mode of cancer treatment. In our current study, Gaussian-accelerated molecular dynamics (GaMD), deep learning (DL), and binding free energy calculations were combined together to probe the binding mechanism of non-peptide inhibitors K23 and 0Y7 and peptide ones PDI6W and PDI to MDM2. The GaMD trajectory-based DL approach successfully identified significant functional domains, predominantly located at the helixes α2 and α2', as well as the β-strands and loops between α2 and α2'. The post-processing analysis of the GaMD simulations indicated that inhibitor binding highly influences the structural flexibility and collective motions of MDM2. Calculations of molecular mechanics-generalized Born surface area (MM-GBSA) and solvated interaction energy (SIE) not only suggest that the ranking of the calculated binding free energies is in agreement with that of the experimental results, but also verify that van der Walls interactions are the primary forces responsible for inhibitor-MDM2 binding. Our findings also indicate that peptide inhibitors yield more interaction contacts with MDM2 compared to non-peptide inhibitors. Principal component analysis (PCA) and free energy landscape (FEL) analysis indicated that the piperidinone inhibitor 0Y7 shows the most pronounced impact on the free energy profiles of MDM2, with the piperidinone inhibitor demonstrating higher fluctuation amplitudes along primary eigenvectors. The hot spots of MDM2 revealed by residue-based free energy estimation provide target sites for drug design toward MDM2. This study is expected to provide useful theoretical aid for the development of selective inhibitors of MDM2 family members.

The cavity-creating p53 cancer mutation Y220C is an ideal paradigm for developing small-molecule drugs based on protein stabilization. Here, we have systematically analyzed the structural and stability effects of all oncogenic Tyr-to-Cys mutations (Y126C, Y163C, Y205C, Y220C, Y234C, and Y236C) in the p53 DNA-binding domain (DBD). They were all highly destabilizing, drastically lowering the melting temperature of the protein by 8-17 °C. In contrast, two non-cancerous mutations, Y103C and Y107C, had only a moderate effect on protein stability. Differential stabilization of the mutants upon treatment with the anticancer agent arsenic trioxide and stibogluconate revealed an interesting proximity effect. Crystallographic studies complemented by MD simulations showed that two of the mutations, Y234C and Y236C, create internal cavities of different size and shape, whereas the others induce unique surface lesions. The mutation-induced pockets in the Y126C and Y205C mutant were, however, relatively small compared with that of the already druggable Y220C mutant. Intriguingly, our structural studies suggest a pronounced plasticity of the mutation-induced pocket in the frequently occurring Y163C mutant, which may be exploited for the development of small-molecule stabilizers. We point out general principles for reactivating thermolabile cancer mutants and highlight special cases where mutant-specific drugs are needed for the pharmacological rescue of p53 function in tumors.

p53 was discovered 45 years ago as an SV40 large T antigen binding protein, coded by the most frequently mutated TP53 gene in human cancers. As a transcription factor, p53 is tightly regulated by a rich network of post-translational modifications to execute its diverse functions in tumor suppression. Although early studies established p53-mediated cell-cycle arrest, apoptosis, and senescence as the classic barriers in cancer development, a growing number of new functions of p53 have been discovered and the scope of p53-mediated anti-tumor activity is largely expanded. Here, we review the complexity of different layers of p53 regulation, and the recent advance of the p53 pathway in metabolism, ferroptosis, immunity, and others that contribute to tumor suppression. We also discuss the challenge regarding how to activate p53 function specifically effective in inhibiting tumor growth without harming normal homeostasis for cancer therapy.

The USP7 deubiquitinase regulates proteins involved in the cell cycle, DNA repair, and epigenetics and has been implicated in cancer progression. USP7 inhibition has been pursued for the development of anti-cancer therapies. Here, we describe the discovery of potent and specific USP7 inhibitors exemplified by FX1-5303. FX1-5303 was used as a chemical probe to study the USP7-mediated regulation of p53 signaling in cells. It demonstrates mechanistic differences compared to MDM2 antagonists, a related class of anti-tumor agents that act along the same pathway. FX1-5303 synergizes with the clinically approved BCL2 inhibitor venetoclax in acute myeloid leukemia (AML) cell lines and ex vivo patient samples and leads to strong tumor growth inhibition in in vivo mouse xenograft models of multiple myeloma and AML. This work introduces new USP7 inhibitors, differentiates their mechanism of action from MDM2 inhibition, and identifies specific opportunities for their use in the treatment of AML.

Inhibition of MDM2/p53 interaction with small-molecule inhibitors stabilizes p53 from MDM2 mediated degradation, which is a promising strategy for the treatment of cancer. In this report, a novel series of 4-imidazolidinone-containing compounds have been synthesized and tested in MDM2/p53 and MDM4/p53 FP binding assays. Upon SAR studies, compounds 2 (TB114) and 22 were identified as the most potent inhibitors of MDM2/p53 but not MDM4/p53 interactions. Both 2 and 22 exhibited strong antiproliferative activities in HCT-116 and MOLM-13 cell lines harboring wild type p53. Mechanistic studies show that 2 and 22 dose-dependently activated p53 and its target genes and induced apoptosis in cells based on the Western blot, qPCR, and flow cytometry assays. In addition, the antiproliferative activities of 2 and 22 were dependent on wild type p53, while they were not toxic to HEK-293 kidney cells. Furthermore, the on-target activities of 2 were general and applicable to other cancer cell lines with wild type p53. These attributes make 2 a good candidate for future optimization to discover a potential treatment of wild-type p53 cancer.

Mitotic duration is tightly constrained, and extended mitosis is characteristic of problematic cells prone to chromosome missegregation and genomic instability. We show here that mitotic extension leads to the formation of p53-binding protein 1 (53BP1)-ubiquitin-specific protease 28 (USP28)-p53 protein complexes that are transmitted to, and stably retained by, daughter cells. Complexes assembled through a Polo-like kinase 1-dependent mechanism during extended mitosis and elicited a p53 response in G1 that prevented the proliferation of the progeny of cells that experienced an approximately threefold extended mitosis or successive less extended mitoses. The ability to monitor mitotic extension was lost in p53-mutant cancers and some p53-wild-type (p53-WT) cancers, consistent with classification of TP53BP1 and USP28 as tumor suppressors. Cancers retaining the ability to monitor mitotic extension exhibited sensitivity to antimitotic agents.

Cancer is a leading cause of death worldwide. Targeted therapies aimed at key oncogenic driver mutations in combination with chemotherapy and radiotherapy as well as immunotherapy have benefited cancer patients considerably. Tumor protein p53 (TP53), a crucial tumor suppressor gene encoding p53, regulates numerous downstream genes and cellular phenotypes in response to various stressors. The affected genes are involved in diverse processes, including cell cycle arrest, DNA repair, cellular senescence, metabolic homeostasis, apoptosis, and autophagy. However, accumulating recent studies have continued to reveal novel and unexpected functions of p53 in governing the fate of tumors, for example, functions in ferroptosis, immunity, the tumor microenvironment and microbiome metabolism. Among the possibilities, the evolutionary plasticity of p53 is the most controversial, partially due to the dizzying array of biological functions that have been attributed to different regulatory mechanisms of p53 signaling. Nearly 40 years after its discovery, this key tumor suppressor remains somewhat enigmatic. The intricate and diverse functions of p53 in regulating cell fate during cancer treatment are only the tip of the iceberg with respect to its equally complicated structural biology, which has been painstakingly revealed. Additionally, TP53 mutation is one of the most significant genetic alterations in cancer, contributing to rapid cancer cell growth and tumor progression. Here, we summarized recent advances that implicate altered p53 in modulating the response to various cancer therapies, including chemotherapy, radiotherapy, and immunotherapy. Furthermore, we also discussed potential strategies for targeting p53 as a therapeutic option for cancer.

Widespread adoption of mirror-image biological systems presents difficulties in accessing the requisite D-protein substrates. In particular, mirror-image phage display has the potential for high-throughput generation of biologically stable macrocyclic D-peptide binders with potentially unique recognition modes but is hindered by the individualized optimization required for D-protein chemical synthesis. We demonstrate a general mirror-image phage display pipeline that utilizes automated flow peptide synthesis to prepare D-proteins in a single run. With this approach, we prepare and characterize 12 D-proteins - almost one third of all reported D-proteins to date. With access to mirror-image protein targets, we describe the successful discovery of six macrocyclic D-peptide binders: three to the oncoprotein MDM2, and three to the E3 ubiquitin ligase CHIP. Reliable production of mirror-image proteins can unlock the full potential of D-peptide drug discovery and streamline the study of mirror-image biology more broadly.

For non-small-cell lung cancer (NSCLC), the ubiquitous occurrence of concurrent multiple genomic alterations poses challenges to single-gene therapy. To increase therapeutic efficacy, we used the branch-PCR method to develop a multigene nanovector, NP-TP53-BIM-PTEN, that carried three therapeutic gene expression cassettes for coexpression. NP-TP53-BIM-PTEN exhibited a uniform size of 104.8 ± 24.2 nm and high serum stability. In cell transfection tests, NP-TP53-BIM-PTEN could coexpress TP53, BIM, and PTEN in NCI-H1299 cells and induce cell apoptosis with a ratio of up to 94.9%. Furthermore, NP-TP53-BIM-PTEN also inhibited cell proliferation with a ratio of up to 42%. In a mouse model bearing an NCI-H1299 xenograft tumor, NP-TP53-BIM-PTEN exhibited a stronger inhibitory effect on the NCI-H1299 xenograft tumor than the other test vectors without any detectable side effects. These results exhibited the potential of NP-TP53-BIM-PTEN as an effective and safe multigene nanovector to enhance NSCLC therapy efficacy, which will provide a framework for genome therapy with multigene combinations.

Colorectal cancer (CRC) often involves wild-type p53 inactivation by MDM2 and MDM4 overexpression, promoting tumor progression and resistance to 5-fluoruracil (5-FU). Disrupting the MDM2/4 heterodimer can proficiently reactivate p53, sensitizing cancer cells to 5-FU. Herein, we developed 16 peptides based on Pep3 (1), the only known peptide acting through this mechanism. The new peptides, notably 3 and 9, showed lower IC50 values than 1. When incorporated into tumor-targeted biodegradable nanoparticles, these exhibited cytotoxicity against three different CRC cell lines. Notably, NPs/9 caused a significant increase in p53 levels associated with a strong increment of its main downstream target p21 inducing apoptosis. Also, the combined treatment of 9 with 5-FU caused the activation of nucleolar stress and a synergic apoptotic effect. Hence, the co-delivery of MDM2/4 heterodimer disruptors with 5-FU through nanoparticles might be a promising strategy to overcome drug resistance in CRC.

The tumor suppressor protein p53 is central to cancer biology, with its pathway reactivation emerging as a promising therapeutic strategy in oncology. This study introduced LZ22, a novel compound that selectively inhibits the growth, migration, and metastasis of tumor cells expressing wild-type p53, demonstrating ineffectiveness in cells devoid of p53 or those expressing mutant p53. LZ22's mechanism of action involves a high-affinity interaction with the histidine-96 pocket of the MDM2 protein. This interaction disrupted the MDM2-p53 binding, consequently stabilizing p53 by shielding it from proteasomal degradation. LZ22 impeded cell cycle progression and diminished cell proliferation by reinstating the p53-dependent suppression of the CDK2/Rb signaling pathway. Moreover, LZ22 alleviated the p53-dependent repression of Snail transcription factor expression and its consequent EMT, effectively reducing tumor cell migration and distal metastasis. Importantly, LZ22 administration in tumor-bearing mice did not manifest notable side effects. The findings position LZ22 as a structurally unique reactivator of p53, offering therapeutic promise for the management of human cancers with wild-type TP53.

MDM2-p53 inhibition may be effective in glioblastoma (GBM). This study evaluates the pharmacokinetics/pharmacodynamics of BI-907828, a potent antagonist of MDM2, in GBM, and demonstrates a translational paradigm with a focus on a unified "Delivery - Potency - Efficacy" relationship in drug development for central nervous system(CNS) tumors. BI-907828 was tested for cytotoxicity and MDM2-p53 pathway inhibition. Systemic pharmacokinetics and transport mechanisms controlling CNS distribution were evaluated in mice. BI-907828 free fractions in cell media, mouse and human specimens were measured to determine "active" unbound concentrations. Efficacy measures, including overall survival and target expression were assessed in mouse orthotopic GBM xenografts. BI-907828 exhibited potent inhibition of MDM2-p53 pathway and promoted cell death in GBM TP53 wild-type cells. MDM2-amplified cells are highly sensitive to BI-907828, with an effective unbound concentration of 0.1 nmol/L. The CNS distribution of BI-907828 is limited by blood-brain barrier (BBB) efflux mediated by P-gp, resulting in a Kp,uu_brain of 0.002. Despite this seemingly "poor" BBB penetration, weekly administration of 10 mg/kg BI-907828 extended median survival of orthotopic GBM108 xenografts from 28 to 218 days (P < 0.0001). This excellent efficacy can be attributed to high potency, resulting in a limited, yet effective, exposure in the CNS. These studies show that efficacy of BI-907828 in orthotopic models is related to high potency even though its CNS distribution is limited by BBB efflux. Therefore, a comprehensive understanding of all aspects of the "Delivery - Potency - Efficacy" relationship is warranted in drug discovery and development, especially for treatment of CNS tumors.

MDM2 and MDM4 cooperatively and negatively regulate p53, while this pathway is often hijacked by cancer cells in favor of their survival. Blocking MDM2/p53 interaction with small-molecule inhibitors liberates p53 from MDM2 mediated degradation, which is an attractive strategy for drug discovery. We reported herein structure-based discovery of highly potent spiroindoline-containing MDM2 inhibitor (-)60 (JN122), which also exhibited moderate activities against MDM4/p53 interactions. In a panel of cancer cell lines harboring wild type p53, (-)60 efficiently promoted activation of p53 and its target genes, inhibited cell cycle progression, and induced cell apoptosis. Interestingly, (-)60 also promoted degradation of MDM4. More importantly, (-)60 exhibited good PK properties and exerted robust antitumor efficacies in a systemic mouse xenograft model of MOLM-13. Taken together, our study showcases a class of potent MDM2 inhibitors featuring a novel spiro-indoline scaffold, which is promising for future development targeting cancer cells with wild-type p53.

Among tyrosine kinase inhibitors, quinazoline-based compounds represent a large and well-known group of multi-target agents. Our previous studies have shown interesting kinases inhibition activity for a series of 4-aminostyrylquinazolines based on the CP-31398 scaffold. Here, we synthesised a new series of styrylquinazolines with a thioaryl moiety in the C4 position and evaluated in detail their biological activity. Our results showed high inhibition potential against non-receptor tyrosine kinases for several compounds. Molecular docking studies showed differential binding to the DFG conformational states of ABL kinase for two derivatives. The compounds showed sub-micromolar activity against leukaemia. Finally, in-depth cellular studies revealed the full landscape of the mechanism of action of the most active compounds. We conclude that S4-substituted styrylquinazolines can be considered as a promising scaffold for the development of multi-kinase inhibitors targeting a desired binding mode to kinases as effective anticancer drugs.

The identification of novel candidate molecules with the potential to revolutionize the treatment of breast cancer holds profound clinical significance. Macropin (Mac)-1, derived from the venom of wild bees, emerges as an auspicious therapeutic agent for combating breast cancers. Nevertheless, linear peptides have long grappled with the challenges of traversing cell membranes and succumbing to protease hydrolysis. To address this challenge, the present study employed hydrocarbon stapling modification to synthesize a range of stapled Mac-1 peptides, which were comprehensively evaluated for their chemical and biological properties. Significantly, Mac-1-sp4 exhibited a remarkable set of improvements, including enhanced helicity, proteolytic stability, cell membrane permeability, induction of cell apoptosis, in vivo antitumor activity, and inhibition of tubulin polymerization. This study explores the significant impact of the hydrocarbon stapling technique on the secondary structure, hydrolase stability, and biological activity of Mac-1, shedding light on its potential as a revolutionary and potent anti-breast cancer therapy. The findings establish a strong basis for the development of innovative and highly effective anti-tumor treatments.

Stapled peptides are regarded as the promising next-generation therapeutics because of their improved secondary structure, membrane permeability and metabolic stability as compared with the prototype linear peptides. Usually, stapled peptides are obtained by a hydrocarbon stapling technique, anchoring from paired olefin-terminated unnatural amino acids and the consequent ring-closing metathesis (RCM). To investigate the adaptability of the rigid cyclobutane structure in RCM and expand the chemical diversity of hydrocarbon peptide stapling, we herein described the rational design and efficient synthesis of cyclobutane-based conformationally constrained amino acids, termed (E)-1-amino-3-(but-3-en-1-yl)cyclobutane-1-carboxylic acid (E7) and (Z)-1-amino-3-(but-3-en-1-yl)cyclobutane-1-carboxylic acid (Z7). All four combinations including E7-E7, E7-Z7, Z7-Z7 and Z7-E7 were proven to be applicable in RCM-mediated peptide stapling to afford the corresponding geometry-specific stapled peptides. With the aid of the combined quantum and molecular mechanics, the E7-E7 combination was proven to be optimal in both the RCM reaction and helical stabilization. With the spike protein of SARS-CoV-2 as the target, a series of cyclobutane-bearing stapled peptides were obtained. Among them, E7-E7 geometry-specific stapled peptides indeed exhibit higher α-helicity and thus stronger biological activity than canonical hydrocarbon stapled peptides. We believe that this methodology possesses great potential to expand the scope of the existing peptide stapling strategy. These cyclobutane-bearing restricted anchoring residues served as effective supplements for the existing olefin-terminated unnatural amino acids and the resultant geometry-specific hydrocarbon peptide stapling provided more potential for peptide therapeutics.

DNA hydroxymethylation is involved in many biological processes, including nuclear reprogramming, embryonic development, and tumor suppression. In this study, we report that an anticancer agent, nutlin-3, selectively stimulates global DNA hydroxymethylation in TP53 wild-type cancer cells as manifested by the elevation of 5-hydroxymethylcytosine (5hmC) in genomic DNA. In contrast, nutlin 3 fails to enhance DNA hydroxymethylation in TP53-mutated cancer cells. Consistently, nutlin-3 as a MDM2 antagonist only activates wild-type but not mutated TP53. Furthermore, nutlin-3 does not alter the expression of TET1 but slightly reduces the expression of TET2 and TET3 proteins. These TET family proteins are responsible for converting 5-methylcytosine (5mC) to 5hmC. Interestingly, TET1 knockdown could significantly block the nutlin-3-induced DNA hydroxymethylation as well as TP53 and P21 activation. Immunoprecipitation analysis supports that p53 strongly interacts with TET1 proteins. These results suggest that nutlin-3 activates TP53 and promotes p53-TET1 interaction. As positive feedback, the p53-TET1 interaction further enhances p53 activation and promotes apoptosis. Collectively, we demonstrate that nutlin-3 stimulates DNA hydroxymethylation and apoptosis via a positive feedback mechanism.

Allyl-isothiocyanate (AITC) is a common Isothiocyanates (ITC) and its chemo-preventive and anti-tumor effects are believed to be related to the activation of NF-E2 p45-related Factor 2 (Nrf2). However, its anti-tumor effects on colorectal cancer (CRC) are not well elucidated. Here, we investigated the therapeutic in vitro and/or in vivo effects and mechanisms of action (MOA) for AITC on CRC cell line HCT116 (human) and MC38 (mouse). AITC treatment in a low concentration range (1 mg/kg in vivo) significantly inhibited the tumor cell growth and increased the expression of p21 and Nrf2. The AITC-mediated induction of p21 was dependent on Nrf2 but independent on p53 in vitro and in vivo at low dose. In contrast, the high dose of AITC (5 mg/kg in vivo) failed to increase substantial levels of p21/MdmX, and impaired the total antioxidant capacity of tumors and subsequent anti-tumor effect in vivo. These results suggest that an optimal dose of AITC is important and required for the proper Nrf2 activation and its anti-CRC effects and thus, providing insights into the potential applications of AITC for the prevention and treatment of CRC.

The use of nanotechnology in medicine has important potential applications, including in anticancer strategies. Nanomedicine has made it possible to overcome the limitations of conventional monotherapies, in addition to improving therapeutic results by means of synergistic or cumulative effects. A highlight is the combination of gene therapy (GT) and photodynamic therapy (PDT), which are alternative anticancer approaches that have attracted attention in the last decade. In this review, strategies involving the combination of PDT and GT will be discussed, together with the role of nanocarriers (nonviral vectors) in this synergistic therapeutic approach, including aspects related to the design of nanomaterials, responsiveness, the interaction of the nanomaterial with the biological environment, and anticancer performance in studies in vitro and in vivo.

Tp53 is the most commonly mutated gene in cancer. Canonical Tp53 DNA damage response pathways are well characterized and classically thought to underlie the tumor suppressive effect of Tp53. Challenging this dogma, mouse models have revealed that p53 driven apoptosis and cell cycle arrest are dispensable for tumor suppression. Here, we investigated the inverse context of a p53 mutation predicted to drive expression of canonical targets, but is detected in human cancer.

We established a novel mouse model with a single base pair mutation (GAG>GAC, p53E221D) in the DNA-Binding domain that has wild-type function in screening assays, but is paradoxically found in human cancer in Li-Fraumeni syndrome. Using mouse p53E221D and the analogous human p53E224D mutant, we evaluated expression, transcriptional activation, and tumor suppression in vitro and in vivo.

Expression of human p53E224D from cDNA translated to a fully functional p53 protein. However, p53E221D/E221D RNA transcribed from the endogenous locus is mis-spliced resulting in nonsense mediated decay. Moreover, fibroblasts derived from p53E221D/E221D mice do not express a detectable protein product. Mice homozygous for p53E221D exhibited increased tumor penetrance and decreased life expectancy compared to p53 WT animals.

Mouse p53E221D and human p53E224D mutations lead to splice variation and a biologically relevant p53 loss of function in vitro and in vivo.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by the impaired differentiation and uncontrolled proliferation of myeloid blasts. Tumor suppressor p53 is often downregulated in AML cells via ubiquitination-mediated degradation. While the role of E3 ligase MDM2 in p53 ubiquitination is well-accepted, little is known about the involvement of deubiquitinases (DUBs). Herein, we found that the expression of YOD1, among several DUBs, is substantially reduced in blood cells from AML patients. We identified that YOD1 deubiqutinated and stabilized p53 through interaction via N-terminus of p53 and OTU domain of YOD1. In addition, expression levels of YOD1 were suppressed by elevated miR-221/222 in AML cells through binding to the 3' untranslated region of YOD1, as verified by reporter gene assays. Treatment of cells with miR-221/222 mimics and inhibitors yielded the expected effects on YOD1 expressions, in agreement with the negative correlation observed between the expression levels of miR-221/222 and YOD1 in AML cells. Finally, overexpression of YOD1 stabilized p53, upregulated pro-apoptotic p53 downstream genes, and increased the sensitivity of AML cells to FLT3 inhibitors remarkably. Collectively, our study identified a pathway connecting miR-221/222, YOD1, and p53 in AML. Targeting miR-221/222 and stimulating YOD1 activity may improve the therapeutic effects of FLT3 inhibitors in patients with AML.

As the guardian of the genome, p53 is well known for its tumor suppressor function in humans, controlling cell proliferation, senescence, DNA repair and cell death in cancer through transcriptional and non-transcriptional activities. p53 is the most frequently mutated gene in human cancer, but how its mutation or depletion leads to tumorigenesis still remains poorly understood. Recently, there has been increasing evidence that p53 plays a vital role in regulating cellular metabolism as well as in metabolic adaptation to nutrient starvation. In contrast, mutant p53 proteins, especially those harboring missense mutations, have completely different functions compared to wild-type p53. In this review, we briefly summarize what is known about p53 mediating anabolic and catabolic metabolism in cancer, and in particular discuss recent findings describing how metabolites regulate p53 functions. To illustrate the variability and complexity of p53 function in metabolism, we will also review the differential regulation of metabolism by wild-type and mutant p53.

Synthetic macrocyclic peptides are an emerging molecular class for both targeting intracellular protein-protein interactions (PPIs) and providing an oral modality for drug targets typically addressed by biologics. Display technologies, such as mRNA and phage display, often yield peptides that are too large and too polar to achieve passive permeability or oral bioavailability without substantial off-platform medicinal chemistry. Herein, we use DNA-encoded cyclic peptide libraries to discover a neutral nonapeptide, UNP-6457, that inhibits MDM2-p53 interaction with an IC50 of 8.9 nM. X-ray structural analysis of the MDM2-UNP-6457 complex revealed mutual binding interactions and identified key ligand modification points which may be tuned to enhance its pharmacokinetic profile. These studies showcase how tailored DEL libraries can directly yield macrocyclic peptides benefiting from low MW, TPSA, and HBD/HBA counts that are capable of potently inhibiting therapeutically relevant protein-protein interactions.