bims-cepepe 2023-10-22 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2023–10–22
eleven papers selected by
Henry Lamb, Queensland University of Technology

Deciphering variations in the endocytic uptake of a cell-penetrating peptide: the crucial role of cell culture protocols.
Development of Melanocortin 4 Receptor Agonists by Exploiting Animal-Derived Macrocyclic, Disulfide-Rich Peptide Scaffolds.
PEGylation of a Peptide-Based Amphiphilic Delivery Agent and Influence on Protein Delivery to Cells.
A cyclic peptide-based PROTAC induces intracellular degradation of palmitoyltransferase and potently decreases PD-L1 expression in human cervical cancer cells.
Screening and optimization of phage display cyclic peptides against the WDR5 WBM site.
Streamlined Protein-Protein Interface Loop Mimicry.
AS602801 treatment suppresses breast cancer metastasis to the brain by interfering with gap-junction communication by regulating Cx43 expression.
Distinct membrane perturbation effects of Catestatin and its CST-364 S variant: Insights from molecular simulations and anisotropy measurements.
Design of Synthetic Surrogates for the Macrolactone Linker Motif in Coibamide A.
Warburg effect-related risk scoring model to assess clinical significance and immunity characteristics of glioblastoma.
Design, synthesis, and biological evaluation of novel 4,4'-bipyridine derivatives acting as CDK9-Cyclin T1 protein-protein interaction inhibitors against triple-negative breast cancer.

Cytotechnology. 2023 Dec;75(6): 473-490

Deciphering variations in the endocytic uptake of a cell-penetrating peptide: the crucial role of cell culture protocols.

Joshua Diaz, Jean-Philippe Pellois.

  Delivery tools, including cell-penetrating peptides (CPPs), are often inefficient due to a combination of poor endocytosis and endosomal escape. Aspects that impact the delivery of CPPs are typically characterized using tissue culture models. One problem of using cell culture is that cell culture protocols have the potential to contribute to endosomal uptake and endosomal release of CPPs. Hence, a systematic study to identify which aspects of cell culturing techniques impact the endocytic uptake of a typical CPP, the TMR-TAT peptide (peptide sequence derived from HIV1-TAT with the N-terminus labeled with tetramethylrhodamine), was conducted. Aspects of cell culturing protocols previously found to generally modulate endocytosis, such as cell density, washing steps, and cell aging, did not affect TMR-TAT endocytosis. In contrast, cell dissociation methods, media, temperature, serum starvation, and media composition all contributed to changes in uptake. To establish a range of endocytosis achievable by different cell culture protocols, TMR-TAT uptake was compared among protocols. These protocols led to changes in uptake of more than 13-fold, indicating that differences in cell culturing techniques have a cumulative effect on CPP uptake. Taken together this study highlights how different protocols can influence the amount of endocytic uptake of TMR-TAT. Additionally, parameters that can be exploited to improve CPP accumulation in endosomes were identified. The protocols identified herein have the potential to be paired with other delivery enhancing strategies to improve overall delivery efficiency of CPPs.
Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00591-1.

Keywords:  Cell culture protocols ; Cell culture variability ; Cell penetrating peptides ; Endocytosis ; HIV-TAT

DOI:  https://doi.org/10.1007/s10616-023-00591-1
ACS Pharmacol Transl Sci. 2023 Oct 13. 6(10): 1373-1381

Development of Melanocortin 4 Receptor Agonists by Exploiting Animal-Derived Macrocyclic, Disulfide-Rich Peptide Scaffolds.

Edin Muratspahić, Despoina Aslanoglou, Andrew M White, Claudia Draxler, Xaver Kozisek, Zara Farooq, David J Craik, Peter J McCormick, Thomas Durek, Christian W Gruber.

G protein-coupled receptors are among the most widely studied classes of drug targets. A major challenge in this field is to develop ligands that will selectively modulate a single receptor subtype to overcome the disadvantages of undesired "off target" effects caused by lack of target and thus signaling specificity. In the current study, we explored ligand design for the melanocortin 4 receptor (MC4R) since it is an attractive target for developing antiobesity drugs. Endogenously, the receptor is activated by peptide ligands, i.e., three melanocyte-stimulating hormones (α-MSH, β-MSH, and γ-MSH) and by adrenocorticotropic hormone. Therefore, we utilized a peptide drug design approach, utilizing "molecular grafting" of pharmacophore peptide sequence motifs onto a stable nature-derived peptide scaffold. Specifically, protegrin-4-like-peptide-1 (Pr4LP1) and arenicin-1-like-peptide-1 (Ar3LP1) fully activated MC4R in a functional cAMP assay with potencies of 3.7 and 1.0 nM, respectively. In a nanoluciferase complementation assay with less signal amplification, the designed peptides fully recruited mini-Gs with subnanomolar and nanomolar potencies. Interestingly, these novel peptide MC4R ligands recruited β-arrestin-2 with ∼2-fold greater efficacies and ∼20-fold increased potencies as compared to the endogenous α-MSH. The peptides were inactive at related MC1R and MC3R in a cAMP accumulation assay. These findings highlight the applicability of animal-derived disulfide-rich scaffolds to design pathway and subtype selective MC4R pharmacological probes. In the future, this approach could be exploited to develop functionally selective ligands that could offer safer and more effective obesity drugs.

DOI: https://doi.org/10.1021/acsptsci.3c00090
Biomacromolecules. 2023 Oct 20.

PEGylation of a Peptide-Based Amphiphilic Delivery Agent and Influence on Protein Delivery to Cells.

Andrea A Greschner, Nadine Brahiti, Maud Auger, Lei Hu, Hoda Soleymani Abyaneh, Xavier Barbeau, Victor Parent, Bruno Gaillet, David Guay, Al-Halifa Soultan, Marc A Gauthier.

The cell membrane is a restrictive biological barrier, especially for large, charged molecules, such as proteins. The use of cell-penetrating peptides (CPPs) can facilitate the delivery of proteins, protein complexes, and peptides across the membrane by a variety of mechanisms that are all limited by endosomal sequestration. To improve CPP-mediated delivery, we previously reported the rapid and effective cytosolic delivery of proteins in vitro and in vivo by their coadministration with the peptide S10, which combines a CPP and an endosomal leakage domain. Amphiphilic peptides with hydrophobic properties, such as S10, can interact with lipids to destabilize the cell membrane, thus promoting cargo internalization or escape from endosomal entrapment. However, acute membrane destabilization can result in a dose-limiting cytotoxicity. In this context, the partial or transient deactivation of S10 by modification with methoxy poly(ethylene glycol) (mPEG; i.e., PEGylation) may provide the means to alter membrane destabilization kinetics, thereby attenuating the impact of acute permeabilization on cell viability. This study investigates the influence of PEGylation parameters (molecular weight, architecture, and conjugation chemistry) on the delivery efficiency of a green fluorescent protein tagged with a nuclear localization signal (GFP-NLS) and cytotoxicity on cells in vitro. Results suggest that PEGylation mostly interferes with adsorption and secondary structure formation of S10 at the cell membrane, and this effect is exacerbated by the mPEG molecular weight. This effect can be compensated for by increasing the concentration of conjugates prepared with lower molecular weight mPEG (5 to ∼20 kDa) but not for conjugates prepared with higher molecular weight mPEG (40 kDa). For conjugates prepared with moderate-to-high molecular weight mPEG (10 to 20 kDa), partial compensation of inactivation could be achieved by the inclusion of a reducible disulfide bond, which provides a mechanism to liberate the S10 from the polymer. Grafting multiple copies of S10 to a high-molecular-weight multiarmed PEG (40 kDa) improved GFP-NLS delivery efficiency. However, these constructs were more cytotoxic than the native peptide. Considering that PEGylation could be harnessed for altering the pharmacokinetics and biodistribution profiles of peptide-based delivery agents in vivo, the trends observed herein provide new perspectives on how to manipulate the membrane permeabilization process, which is an important variable for achieving delivery.

DOI: https://doi.org/10.1021/acs.biomac.3c00603
Front Immunol. 2023 ;14 1237964

A cyclic peptide-based PROTAC induces intracellular degradation of palmitoyltransferase and potently decreases PD-L1 expression in human cervical cancer cells.

Yu-Ying Shi, Di-Rong Dong, Gang Fan, Meng-Yuan Dai, Miao Liu.

   Introduction: Our previous research has found that degradation of palmitoyltransferase in tumor cells using a linear peptide PROTAC leads to a significant decrease in PD-L1 expression in tumors. However, this degradation is not a sustained and efficient process. Therefore, we designed a cyclic peptide PROTAC to achieve this efficient anti-PD-L1 effect.
Methods: We designed and synthesized an improvement in linear peptide PROTAC targeting palmitoyltransferase DHHC3, and used disulfide bonds to stabilize the continuous N- and C-termini of the peptides to maintain their structure. Cellular and molecular biology techniques were used to test the effect of this cyclic peptide on PD-L1.
Results: In human cervical cancer cells, our cyclic peptide PROTAC can significantly downregulate palmitoyl transferase DHHC3 and PD-L1 expressions. This targeted degradation effect is enhanced with increasing doses and treatment duration, with a DC50 value much lower than that of linear peptides. Additionally, flow cytometry analysis of fluorescence intensity shows an increase in the amount of cyclic peptide entering the cell membrane with prolonged treatment time and higher concentrations. The Cellular Thermal Shift Assay (CETSA) method used in this study indicates effective binding between our novel cyclic peptide and DHHC3 protein, leading to a change in the thermal stability of the latter. The degradation of PD-L1 can be effectively blocked by the proteasome inhibitor MG132. Results from clone formation experiments illustrate that our cyclic peptide can enhance the proliferative inhibition effect of cisplatin on the C33A cell line. Furthermore, in the T cell-C33A co-culture system, cyclic peptides target the degradation of PD-L1, thereby blocking the interaction between PD-L1 and PD-1, and promoting the secretion of IFN-γ and TNF-α in the co-culture system supernatant.
Conclusion: Our results demonstrate that a disulfide-bridged cyclic peptide PROTAC targeting palmitoyltransferase can provide a stable and improved anti-PD-L1 activity in human tumor cells.

Keywords:  DHHC3; PD-L1; cervical cancer; cyclic peptide PROTAC; immunotherapy

DOI:  https://doi.org/10.3389/fimmu.2023.1237964
RSC Med Chem. 2023 Oct 18. 14(10): 2048-2057

Screening and optimization of phage display cyclic peptides against the WDR5 WBM site.

Lingyu Song, Jiawen Cao, Lin Chen, Zhiyan Du, Naixia Zhang, Danyan Cao, Bing Xiong.

Of the various WD40 family proteins, WDR5 is a particularly important multifunctional adaptor protein that can bind to several protein complexes to regulate gene activation, so it was considered as a promising epigenetic target in anti-cancer drug development. Despite many inhibitors having been discovered directing against the arginine-binding cavity in WDR5 called the WIN site, the side hydrophobic cavity called the WBM site receives rather scant attention. Herein, we aim to obtain novel WBM-targeted peptidic inhibitors with high potency and selectivity. We employed two improved biopanning approaches with a disulfide-constrained cyclic peptide phage library containing 7 randomized residues and identified several peptides with micromole binding activity by docking and binding assay. To further optimize the stability and activity, 9 thiol-reactive chemical linkers were utilized in the cyclization of the candidate peptide DH226027, which had good binding affinity. This study provides an effective method to discover potent peptides targeting protein-protein interactions and highlights a broader perspective of peptide-mimic drugs.

DOI: https://doi.org/10.1039/d3md00288h
Angew Chem Int Ed Engl. 2023 Oct 17. e202307092

Streamlined Protein-Protein Interface Loop Mimicry.

Tianxiong Mi, Siriwalee Siriwibool, Kevin Burgess.

  Cyclic peptides comprising endocyclic organic fragments, "cyclo-organopeptides", can be probes for perturbing protein-protein interactions (PPIs). Finding loop mimics is difficult because of high conformational variability amongst targets. Backbone Matching (BM), introduced here, helps solve this problem in the illustrative cases by facilitating efficient evaluation of virtual cyclo-organopeptide core-structure libraries. Thus, 86 rigid organic fragments were selected to build a library of 602 cyclo-organopeptides comprising Ala and organic parts: "cyclo-{-(Ala)n-organo-}". The central hypothesis is "hit" library members have accessible low energy conformers corresponding to backbone structures of target protein loops, while library members which cannot attain this conformation are probably unworthy of further evaluation. BM thereby prioritizes candidate loop mimics, so that less than 10 cyclo-organopeptides are needed to be prepared to find leads for two illustrative PPIs: iNOS•SPSB2, and uPA•uPAR.

Keywords:  Loop Mimicry * Protein-protein interactions * Macrocycles * Virtual screening

DOI:  https://doi.org/10.1002/anie.202307092
Drug Dev Res. 2023 Oct 19.

AS602801 treatment suppresses breast cancer metastasis to the brain by interfering with gap-junction communication by regulating Cx43 expression.

Zhigang Yang, Liguo Yang, Jun Zhang, Chenzeyue Qian, Yi Zhao.

  AS602801 has been reported as a potential drug candidate against brain metastasis by suppressing the gap-junction communication between lung cancer stem cells and astrocytes. In this study, we aimed to study the molecular mechanism underlying the role of AS602801 in the treatment of brain metastasis in breast cancer. We utilized female athymic BALB/c nude mice and MDA-MB-231/BT-474BR cells to establish experimental models. Polymerase chain reaction assays were performed to observe changes in the connexin 43 (Cx43) messenger RNA (mRNA) and c-Jun N-terminal kinase (JNK) mRNA levels. Dye transfer assay was used to observe the effect of AS602801 on cell-cell communication. An organotypic blood-brain barrier (BBB) model was utilized to observe the effect of AS602801 on transmigration through the BBB barrier. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and flow cytometry were performed to evaluate the proliferation and apoptosis of breast cancer cells co-cultivated with astrocytes. AS602801 inhibited the upregulation of Cx43 and JNK in brain metastasized breast cancer cells in a dose-dependent manner. Also, AS602801 significantly decreased the dye transfer rate from astrocytes to breast cancer cells, indicating the inhibitory effect of AS602801 on cell-cell communication. The transmigration ability of breast cancer cells co-cultured with astrocytes was decreased by AS602801. Furthermore, AS602801 reduced the elevated Cx43/JNK mRNA expression in the co-astrocyte group while suppressing the increased proliferation and promoting the decreased apoptosis of breast cancer cells co-cultivated with astrocytes. AS602801 also suppressed the brain metastasis of breast cancer cells and increased mouse survival. AS602801 downregulates the expressions of JNK and Cx43 to suppress the gap-junction activity. AS602801 also inhibits the communication between breast cancer cells and astrocytes, thus contributing to the treatment of brain metastasis in breast cancer.

Keywords:  AS602801; Cx43; JNK; breast cancer metastasis to brain; gap-junction communication

DOI:  https://doi.org/10.1002/ddr.22124
Biochim Biophys Acta Biomembr. 2023 Oct 14. pii: S0005-2736(23)00120-7. [Epub ahead of print]1866(1): 184238

Distinct membrane perturbation effects of Catestatin and its CST-364 S variant: Insights from molecular simulations and anisotropy measurements.

Garima Singh, Yuvaraju Venkataramaraju, Geetanjali Meher, Bhavani Shankar Sahu, Mohammed Saleem, Yusuf Akhter.

  Catestatin (CST), a versatile 21 amino acid long cationic peptide, is stored in chromaffin granules and exocytosed upon fusion with the plasma membrane. CST, produced by chromaffin cells and neutrophils, is derived from the processing of chromogranin A and induced in the skin after injury. It involves catecholamine inhibition, blood pressure control, inflammation, and innate immunity. It is thought that calcium influx is triggered by CST permeating within neutrophils. It is unknown whether CST can disturb the immediate environment enough to penetrate the cell membrane passively. We used molecular dynamics simulations to examine the behaviour of human CST in its wild-type state (CST WT) and one of its naturally occurring variants, CST-364 S, which has a high allelic frequency in the human population. Both peptides were incorporated into the model eukaryotic cell membrane known as the POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) lipid bilayer. The molecular modelling and simulations results show that CST WT and CST-364 S have different propensities for membrane disruption. It was shown that CST-364 S has higher membrane permeability than CST WT. In addition, we have used fluorescence anisotropy and leakage assay to study the interaction of peptides with PC membranes. Both peptides interacted with POPC and DOPC membranes, while CST-364 S penetrated the membrane more deeply via disorganizing the membrane interface, which supports our previous findings. According to this study, there is a good possibility that the peptides will passively permeate the cell membrane, distort, and pass through it.

Keywords:  Catestatin; DOPC; Exocytosis; Membrane deformation; POPC

DOI:  https://doi.org/10.1016/j.bbamem.2023.184238
ACS Med Chem Lett. 2023 Oct 12. 14(10): 1344-1350

Design of Synthetic Surrogates for the Macrolactone Linker Motif in Coibamide A.

Rikito Suzuki, Daphne R Mattos, Takashi Kitamura, Rina Tsujioka, Kazuya Kobayashi, Shinsuke Inuki, Hiroaki Ohno, Jane E Ishmael, Kerry L McPhail, Shinya Oishi.

A marine cyanobacterial cyclic depsipeptide, coibamide A (CbA), inhibits the mammalian protein secretory pathway by blocking the Sec61 translocon, which is an emerging drug target for cancer and other chronic diseases. In our previous structure-activity relationship study of CbA, the macrolactone ester linker was replaced with alkyl/alkenyl surrogates to provide synthetically accessible macrocyclic scaffolds. To optimize the cellular bioactivity profile of CbA analogues, novel lysine mimetics having β- and ε-methyl groups have now been designed and synthesized by a stereoselective route. A significant increase in cytotoxicity was observed upon introduction of these two methyl groups, corresponding to the d-MeAla α-methyl and MeThr β-methyl of CbA. All synthetic products retained the ability to inhibit secretion of a model Sec61 substrate. Tandem evaluation of secretory function inhibition in living cells and cytotoxicity was an effective strategy to assess the impact of structural modifications to the linker for ring closure.

DOI: https://doi.org/10.1021/acsmedchemlett.3c00232
Cancer Med. 2023 Oct 21.

Warburg effect-related risk scoring model to assess clinical significance and immunity characteristics of glioblastoma.

Rong Zhang, Can Wang, Xiaohong Zheng, Shenglan Li, Weichunbai Zhang, Zhuang Kang, Shuo Yin, Jinyi Chen, Feng Chen, Wenbin Li.

   BACKGROUND: Glioblastoma (GBM), the most common primary malignant brain tumor, has a poor prognosis, with a median survival of only 14.6 months. The Warburg effect is an abnormal energy metabolism, which is the main cause of the acidic tumor microenvironment. This study explored the role of the Warburg effect in the prognosis and immune microenvironment of GBM.
METHODS: A prognostic risk score model of Warburg effect-related genes (Warburg effect signature) was constructed using GBM cohort data from The Cancer Genome Atlas. Cox analysis was performed to identify independent prognostic factors. Next, the nomogram was built to predict the prognosis for GBM patients. Finally, the drug sensitivity analysis was utilized to find the drugs that specifically target Warburg effect-related genes.
RESULTS: Age, radiotherapy, chemotherapy, and WRGs score were confirmed as independent prognostic factors for GBM by Cox analyses. The C-index (0.633 for the training set and 0.696 for the validation set) and area under curve (>0.7) indicated that the nomogram exhibited excellent performance. The calibration curve also indicates excellent consistency of the nomogram between predictions and actual observations. In addition, immune microenvironment analysis revealed that patients with high WRGs scores had high immunosuppressive scores, a high abundance of immunosuppressive cells, and a low response to immunotherapy. The Cell Counting Kit-8 assays showed that the drugs targeting Warburg effect-related genes could inhibit the GBM cells growth in vitro.
CONCLUSION: Our research showed that the Warburg effect is connected with the prognosis and immune microenvironment of GBM. Therefore, targeting Warburg effect-related genes may provide novel therapeutic options.

Keywords:  Warburg effect; drug sensitivity; glioblastoma; immune microenvironment; prognostic model

DOI:  https://doi.org/10.1002/cam4.6627
Eur J Med Chem. 2023 Oct 06. pii: S0223-5234(23)00825-5. [Epub ahead of print]261 115858

Design, synthesis, and biological evaluation of novel 4,4'-bipyridine derivatives acting as CDK9-Cyclin T1 protein-protein interaction inhibitors against triple-negative breast cancer.

Guiping Gao, Jiayi Li, Yin Cao, Xudan Li, Yuqing Qian, Xiumei Wang, Mengyu Li, Yingkun Qiu, Tong Wu, Liqiang Wang, Meijuan Fang.

  Cyclin-dependent kinase 9 (CDK9) is directly related to tumor development in triple-negative breast cancer (TNBC) patients. Increased CDK9 is significantly associated with poor patient prognosis, while inhibiting CDK9-Cyclin T1 protein-protein interaction has recently been demonstrated as a new approach to TNBC treatment. Herein, we synthesized a novel class of 4,4'-bipyridine derivatives as potential CDK9-Cyclin T1 PPI inhibitors against TNBC. The represented compound B19 was found to be an excellent and selective CDK9-Cyclin T1 PPI inhibitor with good potency against TNBC cell lines while exhibiting lower toxicity in normal human cell lines than the positive compound I-CDK9. Notably, compound B19 showed good pharmacokinetic properties and excellent antitumor activity against TNBC (4T1) allografts in mice with a therapeutic index of more than 42 (TGI4T1(12.5 mg/kg,i.p.) = 63.1% vs. LD50 = 537 mg/kg). Moreover, the administration of B19 in combination with the PARP inhibitor Olaparib results in a significant increase of the antitumor activity in MDA-MB-231 cells relative to that of either single agent. To our knowledge, B19 is the first reported non-metal organic compound that acts as a selective CDK9-Cyclin T1 PPI inhibitor with in vivo antitumor activity, and it may be alone and in combination with PARP inhibitor Olaparib for TNBC therapy.

Keywords:  4,4′-Bipyridine derivatives; Antitumor activity; CDK9-Cyclin T1; PPI inhibitor; Triple-negative breast cancer (TNBC)

DOI:  https://doi.org/10.1016/j.ejmech.2023.115858