A Top-Down Design Approach for Generating a Peptide PROTAC Drug Targeting Androgen Receptor for Androgenetic Alopecia Therapy.

While large-scale artificial intelligence (AI) models for protein structure prediction and design are advancing rapidly, the translation of deep learning models for practical macromolecular drug development remains limited. This investigation aims to bridge this gap by combining cutting-edge methodologies to create a novel peptide-based PROTAC drug development paradigm. Using ProteinMPNN and RFdiffusion, we identified binding peptides for androgen receptor (AR) and Von Hippel-Lindau (VHL), followed by computational modeling with Alphafold2-multimer and ZDOCK to predict spatial interrelationships. Experimental validation confirmed the designed peptide's binding ability to AR and VHL. Transdermal microneedle patching technology was seamlessly integrated for the peptide PROTAC drug delivery in androgenic alopecia treatment. In summary, our approach provides a generic method for generating peptide PROTACs and offers a practical application for designing potential therapeutic drugs for androgenetic alopecia. This showcases the potential of interdisciplinary approaches in advancing drug development and personalized medicine.

CK2-dependent degradation of CBX3 dictates replication fork stalling and PARP inhibitor sensitivity.

DNA replication is a vulnerable cellular process, and its deregulation leads to genomic instability. Here, we demonstrate that chromobox protein homolog 3 (CBX3) binds replication protein A 32-kDa subunit (RPA2) and regulates RPA2 retention at stalled replication forks. CBX3 is recruited to stalled replication forks by RPA2 and inhibits ring finger and WD repeat domain 3 (RFWD3)-facilitated replication restart. Phosphorylation of CBX3 at serine-95 by casein kinase 2 (CK2) kinase augments cadherin 1 (CDH1)-mediated CBX3 degradation and RPA2 dynamics at stalled replication forks, which permits replication fork restart. Increased expression of CBX3 due to gene amplification or CK2 inhibitor treatment sensitizes prostate cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors while inducing replication stress and DNA damage. Our work reveals CBX3 as a key regulator of RPA2 function and DNA replication, suggesting that CBX3 could serve as an indicator for targeted therapy of cancer using PARP inhibitors.

Development of a PAK4-targeting PROTAC for renal carcinoma therapy: concurrent inhibition of cancer cell proliferation and enhancement of immune cell response.

BACKGROUND: Finding the oncogene, which was able to inhibit tumor cells intrinsically and improve the immune answers, will be the future direction for renal cancer combined treatment. Following patient sample analysis and signaling pathway examination, we propose p21-activated kinase 4 (PAK4) as a potential target drug for kidney cancer. PAK4 exhibits high expression levels in patient samples and plays a regulatory role in the immune microenvironment. METHODS: Utilizing AI software for peptide drug design, we have engineered a specialized peptide proteolysis targeting chimera (PROTAC) drug with selectivity for PAK4. To address challenges related to drug delivery, we developed a nano-selenium delivery system for efficient transport of the peptide PROTAC drug, termed PpD (PAK4 peptide degrader). FINDINGS: We successfully designed a peptide PROTAC drug targeting PAK4. PpD effectively degraded PAK4 with high selectivity, avoiding interference with other homologous proteins. PpD significantly attenuated renal carcinoma proliferation in vitro and in vivo. Notably, PpD demonstrated a significant inhibitory effect on tumor proliferation in a fully immunocompetent mouse model, concomitantly enhancing the immune cell response. Moreover, PpD demonstrated promising tumor growth inhibitory effects in mini-PDX and PDO models, further underscoring its potential for clinical application. INTERPRETATION: This PAK4-targeting peptide PROTAC drug not only curtails renal cancer cell proliferation but also improves the immune microenvironment and enhances immune response. Our study paves the way for innovative targeted therapies in the management of renal cancer. FUNDING: This work is supported by Research grants from non-profit organizations, as stated in the Acknowledgments.

Adverse events reporting of XPO1 inhibitor - selinexor: a real-word analysis from FAERS database.

As the world's first oral nuclear export inhibitor, selinexor is increasingly being used in clinical applications for malignant tumors. However, there is no extensive exploration on selinexor's adverse events (ADEs), necessitating a real-word assessment of its clinical medication safety. FAERS data (July 2019-June 2023) were searched for selinexor ADE reports across all indications. Use the system organ class (SOC) and preferred terms (PT) from the medical dictionary for regulatory activities (MedDRA) to describe, categorize, and statistic ADEs. Disproportionality analysis was employed through calculation of reporting odds ratio (ROR) and proportional reporting ratio (PRR). Based on total of 4392 selinexor related ADE reports as the primary suspect (PS), of which 2595 instances were severe outcomes. The predominant ADEs included gastrointestinal disorders, myelosuppression symptoms, and various nonspecific manifestations. 124 signals associated with selinexor ADE were detected, and 10 of these top 15 signals were not included into the instructions. Our study provides real-world evidence regarding the drug safety of selinexor, which is crucial for clinicians to safeguard patients' health.

Development of a Double-Stapled Peptide Stabilizing Both α-Helix and ß-Sheet Structures for Degrading Transcription Factor AR-V7.

Peptide drugs offer distinct advantages in therapeutics; however, their limited stability and membrane penetration abilities hinder their widespread application. One strategy to overcome these challenges is the hydrocarbon peptide stapling technique, which addresses issues such as poor conformational stability, weak proteolytic resistance, and limited membrane permeability. Nonetheless, while peptide stapling has successfully stabilized α-helical peptides, it has shown limited applicability for most ß-sheet peptide motifs. In this study, we present the design of a novel double-stapled peptide capable of simultaneously stabilizing both α-helix and ß-sheet structures. Our designed double-stapled peptide, named DSARTC, specifically targets the androgen receptor (AR) DNA binding domain and MDM2 as E3 ligase. Serving as a peptide-based PROTAC (proteolysis-targeting chimera), DSARTC exhibits the ability to degrade both the full-length AR and AR-V7. Molecular dynamics simulations and circular dichroism analysis validate the successful constraint of both secondary structures, demonstrating that DSARTC is a "first-in-class" heterogeneous-conformational double-stapled peptide drug candidate. Compared to its linear counterpart, DSARTC displays enhanced stability and an improved cell penetration ability. In an enzalutamide-resistant prostate cancer animal model, DSARTC effectively inhibits tumor growth and reduces the levels of both AR and AR-V7 proteins. These results highlight the potential of DSARTC as a more potent and specific peptide PROTAC for AR-V7. Furthermore, our findings provide a promising strategy for expanding the design of staple peptide-based PROTAC drugs, targeting a wide range of "undruggable" transcription factors.

MYC induces CDK4/6 inhibitors resistance by promoting pRB1 degradation.

CDK4/6 inhibitors (CDK4/6i) show anticancer activity in certain human malignancies, such as breast cancer. However, their application to other tumor types and intrinsic resistance mechanisms are still unclear. Here, we demonstrate that MYC amplification confers resistance to CDK4/6i in bladder, prostate and breast cancer cells. Mechanistically, MYC binds to the promoter of the E3 ubiquitin ligase KLHL42 and enhances its transcription, leading to RB1 deficiency by inducing both phosphorylated and total pRB1 ubiquitination and degradation. We identify a compound that degrades MYC, A80.2HCl, which induces MYC degradation at nanomolar concentrations, restores pRB1 protein levels and re-establish sensitivity of MYC high-expressing cancer cells to CDK4/6i. The combination of CDK4/6i and A80.2HCl result in marked regression in tumor growth in vivo. Altogether, these results reveal the molecular mechanisms underlying MYC-induced resistance to CDK4/6i and suggest the utilization of the MYC degrading molecule A80.2HCl to potentiate the therapeutic efficacy of CDK4/6i.

A GPX4 non-enzymatic domain and MDM2 targeting peptide PROTAC for acute lymphoid leukemia therapy through ferroptosis induction.

Ferroptosis, an emerging form of programmed cell death, has garnered substantial attention as a potential target for cancer therapy. However, despite the potential promise, no ferroptosis-related therapies have progressed to clinical trials. Identifying disease types sensitive to ferroptosis and developing specific ferroptosis-targeting drugs are critical focal points in the field of ferroptosis-based treatment. In this study, we conducted a comprehensive database analysis and presented compelling evidence indicating a high expression of GPX4 in patients with acute lymphoblastic leukemia (ALL), significantly correlating with poor prognosis. Notably, elevated GPX4 expression is closely associated with ALL relapse, a major challenge in the treatment of this disease. Building upon these findings, we devised a novel peptide-based Proteolysis Targeting Chimeras (PROTAC) drug targeting GPX4 through computer-aided design. In contrast to existing drugs that target the conjugative enzyme active site, our design focused on a peptide drug targeting the non-active site of GPX4. Furthermore, we strategically selected MDM2, an E3 ligase highly expressed in ALL, for the PROTAC drug design. This deliberate choice amplifies the drug's effect on cancer cells while minimizing its impact on normal cells, achieving desirable selectivity for cancer cells. Leveraging nanogold delivery, we successfully facilitated intracellular action of the GPX4-targeting peptide PROTAC drug, denoted as Au-PGPD (peptide GPX4 PROTAC drug). Au-PGPD effectively induced GPX4 degradation and inhibited ALL cell proliferation. Remarkably, Au-PGPD exhibited significantly less efficacy on normal cells, underscoring the selectivity and safety of our design.

TRABID overexpression enables synthetic lethality to PARP inhibitor via prolonging 53BP1 retention at double-strand breaks.

53BP1 promotes nonhomologous end joining (NHEJ) over homologous recombination (HR) repair by mediating inactivation of DNA end resection. Ubiquitination plays an important role in regulating dissociation of 53BP1 from DNA double-strand breaks (DSBs). However, how this process is regulated remains poorly understood. Here, we demonstrate that TRABID deubiquitinase binds to 53BP1 at endogenous level and regulates 53BP1 retention at DSB sites. TRABID deubiquitinates K29-linked polyubiquitination of 53BP1 mediated by E3 ubiquitin ligase SPOP and prevents 53BP1 dissociation from DSBs, consequently inducing HR defects and chromosomal instability. Prostate cancer cells with TRABID overexpression exhibit a high sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors. Our work shows that TRABID facilitates NHEJ repair over HR during DNA repair by inducing prolonged 53BP1 retention at DSB sites, suggesting that TRABID overexpression may predict HR deficiency and the potential therapeutic use of PARP inhibitors in prostate cancer.

EZH2-regulated immune risk score prognostic model predicts outcome of clear cell renal cell carcinoma.

Background: The enhancer of zeste homolog 2 (EZH2) plays an important role in the tumor microenvironment (TME), and EZH2 in shaping the epigenetic landscape of CD8+ T cell fate and function, with a particular emphasis on cancer. Here, high EZH2 expression always leads to less CD8+ T cell infiltration. However, clear cell renal cell carcinoma (ccRCC) is reportedly a "hot" tumor, with contradictory high EZH2 expression. Our goal was to construct a EZH2-regulated immune risk score prognostic model to predict ccRCC outcomes, and provide a prospect of clinical EZH2 inhibitors in fine-tuning T cell responses with immune therapy. Methods: We downloaded and analyzed The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE), TISIDB database, and WebGestalt for ccRCC patients, EZH2-related tumor-infiltrating lymphocytes and immunomodulators. R packages "limma", "BiocManager", and "preprocessCore", etc. were downloaded to prepare CIBERSORT files, immune cells heatmap, multivariable Cox model and survival analysis. The EZH2-regulated immune risk model's prognostic ability was calculated by receiver operating characteristic (ROC) and area under the curve (AUC) analyses in R studio. Results: EZH2 was highly expressed and related to poor outcome in ccRCC. However, high-expression EZH2 was not related to a "cool" tumor. Of the 49 immunomodulators significantly regulated by EZH2, forest plot showed 26 immunomodulators signatures independently associated with overall survival. The EZH2-regulated immune-risk score prognostic model was an independent prognostic factor (AUC =0.816), especially combined with clinicopathologic parameters in ccRCC overall survival prediction. Conclusions: The EZH2-regulated immune-risk score prognostic model was an independent prognostic factor, with good accuracy and predictability, and could provide experimental data to the clinical area.

Design of stapled peptide-based PROTACs for MDM2/MDMX atypical degradation and tumor suppression.

Rationale: Although stapled peptides offer a powerful solution to overcome the susceptibility of linear peptides to proteolytic degradation and improve their ability to cross membranes, an efficient and durable disease treatment strategy has not yet been developed due to the inevitable elimination of peptide inhibitors and rapid accumulation of target proteins. Methods: Herein we developed stapled peptide-based proteolysis-targeting chimeras (SP-PROTACs), that simultaneously exhibited improved cellular uptake and proteolytic stability attributed to the stapled peptides, and efficient target protein degradation promoted by the PROTACs. Based on the PMI peptide with dual specificity for both MDM2 and MDMX, a series of SP-PROTACs were designed. Results: Among them, the optimized SPMI-HIF2-1 exhibited similar binding affinity with MDM2 and MDMX but obviously higher helical contents, improved proteolytic stability, better cellular permeability, and a better pharmacokinetic profile compared with its linear counterpart. Importantly, SPMI-HIF2-1 could effectively kill cancer cells and inhibit tumor progression in subcutaneous and orthotopic colorectal cancer xenograft models through simultaneously promoting the atypical degradation of both MDM2 and MDMX and durable p53 activation. An FP-based binding assay and structural modeling analysis of the ternary complex suggested that SPMI-HIF2-1 simultaneously bound with the target protein and E3 ligase. Conclusion: Our findings not only provide a new class of anticancer drug candidates, but also bridge the gap and reduce the physical distance between peptides and PROTACs.

Deubiquitinase OTUD5 modulates mTORC1 signaling to promote bladder cancer progression.

The mechanistic (formally "mammalian") target of rapamycin (mTOR) pathway serves as a crucial regulator of various biological processes such as cell growth and cancer progression. In bladder cancer, recent discoveries showing the cancer-promoting role of mTOR complex 1 have attracted wide attention. However, the regulation of mTOR signaling in bladder cancer is complicated and the underlying mechanism remains elusive. Here, we report that the deubiquitinating enzyme, ovarian tumor domain-containing protein 5 (OTUD5), can activate the mTOR signaling pathway, promote cancer progression, and show its oncogenic potential in bladder cancer. In our study, we found that OTUD5 deubiquitinated a RING-type E3 ligase, RNF186, and stabilized its function. In addition, the stabilization of RNF186 further led to the degradation of sestrin2, which is an inhibitor of the mTOR signaling pathway. Together, we provide novel insights into the pathogenesis of bladder cancer and first prove that OTUD5 can promote bladder cancer progression through the OTUD5-RNF186-sestrin2-mTOR axis, which may be exploited in the future for the diagnosis and treatment of this malignancy.

De Novo Design of an Androgen Receptor DNA Binding Domain-Targeted peptide PROTAC for Prostate Cancer Therapy.

Androgen receptor splice variant-7 (AR-V7), one of the major driving factors, is the most attractive drug target in castration-resistant prostate cancer (CRPC). Currently, no available drugs efficiently target AR-V7 in clinical practice. The DNA binding domain (DBD) is indispensable for the transcriptional activity of AR full length and AR splice variants, including AR-V7. Based on the homodimerization structure of the AR DBD, a novel peptide-based proteolysis-targeting chimera (PROTAC) drug is designed to induce AR and AR-V7 degradation in a DBD and MDM2-dependent manner, without showing any activity on other hormone receptors. To overcome the short half-life and poor cell penetrability of peptide PROTAC drugs, an ultrasmall gold (Au)-peptide complex platform to deliver the AR DBD PROTAC in vivo is developed. The obtained Au-AR pep-PROTAC effectively degrades AR and AR-V7 in prostate cancer cell lines, particularly in CWR22Rv1 cells with DC50 values 48.8 and 79.2 nM, respectively. Au-AR pep-PROTAC results in suppression of AR levels and induces tumor regression in both enzalutamide sensitive and resistant prostate cancer animal models. Further optimization of the Au-AR pep-PROTAC can ultimately lead to a new therapy for AR-V7-positive CRPC.

Kill Two Birds with One Stone: A Multifunctional Dual-Targeting Protein Drug to Overcome Imatinib Resistance in Philadelphia Chromosome-Positive Leukemia.

The Bcr/Abl plays a central role in Philadelphia chromosome-positive (Ph+) leukemia because of the constitutively activated Abl tyrosine kinase and its downstream pathways. Currently, the clinical treatment of imatinib-resistant patients with tyrosine kinase inhibitors is severely limited by drug resistance and adverse effects. Herein, a dual-targeting proteolysis-targeting chimera (PROTAC) protein drug, termed PMI Bcr/Abl-R6, is designed by engrafting an MDM2/p53 inhibition peptide sequence onto the Bcr/Abl tetramerization domain. PMI Bcr/Abl-R6, harboring a Bcr/Abl targeting sequence and an MDM2 binding sequence, acts as a PROTAC drug in Ph+ leukemia cells. Its dual-targeting constitution suggests that PMI Bcr/Abl-R6 designs to target the tetramerization domain instead of the Abl kinase domain, therefore has the potential to overcome drug resistance mutations in the kinase domain. The efficient ability of PMI Bcr/Abl-R6 is demonstrated to simultaneously induce Bcr/Abl degradation and activate the p53 pathway. PMI Bcr/Abl-R6 has the potential to overcome drug resistance in Ph+ leukemias by multiple mechanisms.

Ultrasound-assisted extraction of polysaccharide from spent Lentinus edodes substrate: Process optimization, precipitation, structural characterization and antioxidant activity.

Lentinus edodes is the second-most popular and cultivated mushroom worldwide due to its nutritional and health-promoting benefit. However, the mushroom production generates vast amounts of spent L. edodes substrate (SLS) that is generally discharged into the environment, posing a great challenge within mushroom by-product valorization. In this work, SLS polysaccharide (SP) was ultrasonically extracted by optimizing the process conditions with response surface methodology. Using gradient ethanol precipitation, SP was separated into SP40, SP60 and SP80, and their monosaccharide composition, structural properties, and antioxidant potential were further characterized and compared. The results showed the total polysaccharide content reached up to 37.05 ± 0.31 mg/g under the optimal conditions including an extraction temperature of 50 °C, a liquid-solid ratio of 30 mL/g and an ultrasonic power of 120 W. SP and its fractional precipitations were heteropolysaccharides sharing a similar monosaccharide composition including L-rhamnose, D-glucuronic acid, D-galacturonic acid, d-glucose and D-xylose, and a typical infrared spectrum for polysaccharide. These fractions also varied in the surface morphology, where SP80 was looser and more porous than SP40 and SP60. Furthermore, SP and SP80 displayed the strongest antioxidant activities in vitro. This study identified a novel and practical strategy to valorize SLS for valuable polysaccharide.

Circular RNAs acting as ceRNAs mediated by miRNAs may be involved in the synthesis of soybean fatty acids.

Soybean oil is composed of fatty acids and glycerol. The content and composition of fatty acids partly determine the quality of soybean seeds. Circular RNAs (circRNAs) are endogenous non-coding RNAs that competitively bind to microRNAs (miRNAs) through miRNA recognition elements, thereby acting as sponges to regulate the expression of target genes. Although circRNAs have been identified previously in soybean, only their expression has been investigated without exploration of the competitive endogenous RNAs (ceRNAs) network of circRNAs-miRNAs-mRNAs. In this study, circRNAs in immature pods of a low linolenic acid soybean Mutant 72' (MT72) and the wild-type control 'Jinong 18' (JN18) were systematically identified and analyzed at 30 and 40 days after flowering using high-throughput sequencing technology. We identified 6377 circRNAs, of which 114 were differentially expressed. Gene ontology and KEGG pathway analyses of targeted mRNAs in the ceRNAs network indicated that the differentially expressed circRNAs may be involved in fatty acid transport, suggesting that circRNAs may play a post-transcriptional regulatory role in soybean oil synthesis. This study provides a foundation for future exploration of the function of circRNAs in soybean and presents novel insights to guide further studies of plant circRNAs.

Genome-wide identification and analysis of long non-coding RNAs involved in fatty acid biosynthesis in young soybean pods.

Long non-coding RNAs (lncRNAs) are non-coding RNAs of more than 200 nucleotides. To date, the roles of lncRNAs in soybean fatty acid synthesis have not been fully studied. Here, the low-linolenic acid mutant 'MT72' and the wild-type control 'JN18' were used as materials. The lncRNAs in young pods at 30 and 40 days (d) after flowering were systematically identified and analyzed using transcriptome sequencing technology combined with bioinformatics tools. A total of 39,324 lncRNAs and 561 differentially expressed lncRNAs were identified. A lncRNAs-miRNAs-protein-coding genes (mRNAs) network was constructed, and 46 lncRNAs, 46 miRNAs and 137 mRNAs were found to be correlated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of 12 targeted mRNAs in the competing endogenous RNA network showed that these lncRNAs may be involved in the biological processes of fatty acid transport, lipid synthesis and cell division. Finally, the expression levels of differentially expressed lncRNAs, miRNAs and mRNAs were verified using qRT-PCR. The expression patterns of most genes were consistent with the sequencing results. In conclusion, new information was provided for the study of fatty acid synthesis by lncRNAs in young soybean pods.

Trimeric structure of the mouse Kupffer cell C-type lectin receptor Clec4f.

The C-type lectin receptor Clec4f has been identified as a specific surface marker for Kupffer cells, although its ortholog is absent in humans and its biological function remains elusive. Here, we report the crystal structure of a truncated mouse trimeric Clec4f. The orientation between the carbohydrate-recognition domain of Clec4f and its neck region differs from other C-type lectins, resulting in an observed distance of 45 Å between the glycan-binding sites within the Clec4f trimer. Interestingly, the trimeric coiled-coil interface within its heptad neck region contains multiple polyglutamine interactions instead of the predominantly hydrophobic leucine zipper found in other C-type lectin receptors. The Clec4f trimeric structure displays unique features regarding its assembly and ligand recognition, shedding light on the evolution and diversity of the C-type lectin family.

Tanshinones: First-in-Class Inhibitors of the Biogenesis of the Type 3 Secretion System Needle of Pseudomonas aeruginosa for Antibiotic Therapy.

The type 3 secretion system (T3SS) found as cell-surface appendages of many pathogenic Gram-negative bacteria, although nonessential for bacterial survival, is an important therapeutic target for drug discovery and development aimed at inhibiting bacterial virulence without inducing antibiotic resistance. We designed a fluorescence-polarization-based assay for high-throughput screening as a mechanistically well-defined general strategy for antibiotic discovery targeting the T3SS and made a serendipitous discovery of a subset of tanshinones-natural herbal compounds in traditional Chinese medicine widely used for the treatment of cardiovascular and cerebrovascular diseases-as effective inhibitors of the biogenesis of the T3SS needle of multi-drug-resistant Pseudomonas aeruginosa. By inhibiting the T3SS needle assembly and, thus, cytotoxicity and pathogenicity, selected tanshinones reduced the secretion of bacterial virulence factors toxic to macrophages in vitro, and rescued experimental animals challenged with lethal doses of Pseudomonas aeruginosa in a murine model of acute pneumonia. As first-in-class inhibitors with a demonstrable safety profile in humans, tanshinones may be used directly to alleviate Pseudomonas-aeruginosa-associated pulmonary infections without inducing antibiotic resistance. Since the T3SS is highly conserved among Gram-negative bacteria, this antivirulence strategy may be applicable to the discovery and development of novel classes of antibiotics refractory to existing resistance mechanisms for the treatment of many bacterial infections.

A tetrameric protein scaffold as a nano-carrier of antitumor peptides for cancer therapy.

A major pharmacological barrier to peptide therapeutics is their susceptibility to proteolytic degradation and poor membrane permeability, which, in principle, can be overcome by nanoparticle-based delivery technologies. Proteins, by definition, are nano materials and have been clinically proven as an efficient delivery vehicle for small molecule drugs. Here we describe the design of a protein-based peptide drug carrier derived from the tetramerization domain of the chimeric oncogenic protein Bcr/Abl of chronic myeloid leukemia. A dodecameric peptide inhibitor of the p53-MDM2/MDMX interaction, termed PMI, was grafted to the N-terminal helical region of Bcr/Abl tetramer. To antagonize intracellular MDM2/MDMX for p53 activation, we extended this protein, PMIBcr/Abl, by a C-terminal Arg-repeating hexapeptide to facilitate its cellular uptake. The resultant tetrameric protein PMIBcr/Abl-R6 adopted an alpha-helical conformation in solution and bound to MDM2 at an affinity of 32 nM. PMIBcr/Abl-R6 effectively induced apoptosis of HCT116 p53+/+ cells in vitro in a p53-dependent manner and potently inhibited tumor growth in a nude mouse xenograft model by stabilizing p53 in vivo. Our protein-based delivery strategy thus provides a clinically viable solution to p53-inspired anticancer therapy and is likely applicable to the development of many other peptide therapeutics to target a great variety of intracellular protein-protein interactions responsible for disease initiation and progression.

Systematic mutational analysis of human neutrophil α-defensin HNP4.

Defensins are a family of cationic antimicrobial peptides of innate immunity with immunomodulatory properties. The prototypic human α-defensins, also known as human neutrophil peptides 1-3 or HNP1-3, are extensively studied for their structure, function and mechanisms of action, yet little is known about HNP4 - the much less abundant "distant cousin" of HNP1-3. Here we report a systematic mutational analysis of HNP4 with respect to its antibacterial activity against E. coli and S. aureus, inhibitory activity against anthrax lethal factor (LF), and binding activity for LF and HIV-1 gp120. Except for nine conserved and structurally important residues (6xCys, 1xArg, 1xGlu and 1xGly), the remaining 24 residues of HNP4 were each individually mutated to Ala. The crystal structures of G23A-HNP4 and T27A-HNP4 were determined, both exhibiting a disulfide-stabilized canonical α-defensin dimer identical to wild-type HNP4. Unlike HNP1-3, HNP4 preferentially killed the Gram-negative bacterium, a property largely attributable to three clustered cationic residues Arg10, Arg11 and Arg15. The cationic cluster was also important for HNP4 killing of S. aureus, inhibition of LF and binding to LF and gp120. However, F26A, while functionally inconsequential for E. coli killing, was far more deleterious than any other mutations. Similarly, N-methylation of Leu20 to destabilize the HNP4 dimer had little effect on E. coli killing, but significantly reduced the ability of HNP4 to kill S. aureus, inhibit LF, and bind to LF and gp120. Our findings unveil the molecular determinants of HNP4 function, completing the atlas of structure and function relationships for all human neutrophil α-defensins.