bims-cepepe 2024-06-30 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2024–06–30
thirty-six papers selected by
Henry Lamb, Queensland University of Technology

Employing unnatural promiscuity of sortase to construct peptide macrocycle libraries for ligand discovery.
Phage-encoded bismuth bicycles enable instant access to targeted bioactive peptides.
Intranasal Delivery of Cell-Penetrating Therapeutic Peptide Enhances Brain Delivery, Reduces Inflammation, and Improves Neurologic Function in Moderate Traumatic Brain Injury.
Arginine-Rich Cell-Penetrating Peptides Induce Lipid Rearrangements for Their Active Translocation across Laterally Heterogeneous Membranes.
Exploring Macroscopic Dipoles of Designed Cyclic Peptide Ordered Assemblies to Harvest Piezoelectric Properties.
Multielectrode array characterization of human induced pluripotent stem cell derived neurons in co-culture with primary human astrocytes.
Subtle Structural Differences Affect the Inhibitory Potency of RGD-Containing Cyclic Peptide Inhibitors Targeting SPSB Proteins.
Expression and Subcellular Localization of Lanthipeptides in Human Cells.
Comparative Study of the Potential Cell-Penetrating Peptide ∆M4 on Apoptosis Cell Signaling in A375 and A431 Cancer Cell Lines.
Study on the design, synthesis, and activity of anti-tumor staple peptides targeting MDM2/MDMX.
Tuning Peptide-Based Nanofibers for Achieving Selective Doxorubicin Delivery in Triple-Negative Breast Cancer.
Design of a Lead-Like Cysteine-Targeting Covalent Library and the Identification of Hits to Cys55 of Bfl-1.
[64Cu]Cu-FAP-NOX, a N-oxalyl modified cyclic peptide for FAP PET imaging with a flexible imaging time window.
Peptide macrocyclisation via intramolecular interception of visible-light-mediated desulfurisation.
The importance of tyrosines in multimers of cyclic RGD nonapeptides: towards αvβ6-integrin targeted radiotherapeutics.
FZ-AD005, A Novel DLL3-Targeted Antibody-drug Conjugate with Topoisomerase I Inhibitor, Shows Potent Antitumor Activity in Preclinical Models.
In Vivo Evaluation of 68Ga-Labeled NOTA-EGFRvIII Aptamer in EGFRvIII-Positive Glioblastoma Xenografted Model.
Design of an Acidic pH-Activated NIR Fluorescent Convertible Rhodamine-Hemicyanine Probe-Peptide Conjugate for Living Cancer Cell Active Targeted Selective Tracking of Lysosomes.
PyAMPA: a high-throughput prediction and optimization tool for antimicrobial peptides.
Discovery of MK-1084: An Orally Bioavailable and Low-Dose KRASG12C Inhibitor.
Production of constrained L-cyclo-tetrapeptides by epimerization-resistant direct aminolysis.
Head-to-Head Comparison of Caco-2 Transwell and Gut-on-a-Chip Models for Assessing Oral Peptide Formulations.
Real-time label-free three-dimensional invasion assay for anti-metastatic drug screening using impedance sensing.
DiPPI: A Curated Data Set for Drug-like Molecules in Protein-Protein Interfaces.
Protein-Protein Interaction Prediction via Structure-Based Deep Learning.
Comparative Analysis of the Effect of the BRAF Inhibitor Dabrafenib in 2D and 3D Cell Culture Models of Human Metastatic Melanoma Cells.
Interaction of DisBa01 peptide from Bothrops alternatus venom with BRAF melanoma receptors: Modeling and molecular docking.
The Disulfide Bond-Mediated Cyclization of Oral Peptides.
Design of a covalent protein-protein interaction inhibitor of SRPKs to suppress angiogenesis and invasion of cancer cells.
Pembrolizumab plus Abiraterone Acetate and Prednisone in Patients with Chemotherapy-naïve Metastatic Castration-resistant Prostate Cancer: Results from KEYNOTE-365 Cohort D.
Preclinical proof of principle for orally delivered Th17 antagonist miniproteins.
Poptosis or Peptide-Induced Transmembrane Pore Formation: A Novel Way to Kill Cancer Cells without Affecting Normal Cells.
Structural insights into somatostatin receptor 5 bound with cyclic peptides.
Automated Analysis of Extracellular Matrix Invasion of Cancer Cells from Tumor Spheroids.
A real-time antibody-dependent cellular phagocytosis assay by live cell imaging.
Design of Proteolytic-Resistant Antifungal Peptides by Utilizing Minimum d-Amino Acid Ratios.

Chem Sci. 2024 Jun 26. 15(25): 9649-9656

Employing unnatural promiscuity of sortase to construct peptide macrocycle libraries for ligand discovery.

Yan-Ni Zhang, Xiao-Cui Wan, Yang Tang, Ying Chen, Feng-Hao Zheng, Zhi-Hui Cui, Hua Zhang, Zhaocai Zhou, Ge-Min Fang.

With the increasing attention paid to macrocyclic scaffolds in peptide drug development, genetically encoded peptide macrocycle libraries have become invaluable sources for the discovery of high-affinity peptide ligands targeting disease-associated proteins. The traditional phage display technique of constructing disulfide-tethered macrocycles by cysteine oxidation has the inherent drawback of reduction instability of the disulfide bond. Chemical macrocyclization solves the problem of disulfide bond instability, but the involved highly electrophilic reagents are usually toxic to phages and may bring undesirable side reactions. Here, we report a unique Sortase-mediated Peptide Ligation and One-pot Cyclization strategy (SPLOC) to generate peptide macrocycle libraries, avoiding the undesired reactions of electrophiles with phages. The key to this platform is to mine the unnatural promiscuity of sortase on the X residue of the pentapeptide recognition sequence (LPXTG). Low reactive electrophiles are incorporated into the X-residue side chain, enabling intramolecular cyclization with the cysteine residue of the phage-displayed peptide library. Utilizing the genetically encoded peptide macrocycle library constructed by the SPLOC platform, we found a high-affinity bicyclic peptide binding TEAD4 with a nanomolar KD value (63.9 nM). Importantly, the binding affinity of the bicyclic peptide ligand is 102-fold lower than that of the acyclic analogue. To our knowledge, this is the first time to mine the unnatural promiscuity of ligases to generate peptide macrocycles, providing a new avenue for the construction of genetically encoded cyclic peptide libraries.

DOI: https://doi.org/10.1039/d4sc01992j
Commun Chem. 2024 Jun 27. 7(1): 143

Phage-encoded bismuth bicycles enable instant access to targeted bioactive peptides.

Sven Ullrich, Upamali Somathilake, Minghao Shang, Christoph Nitsche.

Genetically encoded libraries play a crucial role in discovering structurally rigid, high-affinity macrocyclic peptide ligands for therapeutic applications. Bicyclic peptides with metal centres like bismuth were recently developed as a new type of constrained peptide with notable affinity, stability and membrane permeability. This study represents the genetic encoding of peptide-bismuth and peptide-arsenic bicycles in phage display. We introduce bismuth tripotassium dicitrate (gastrodenol) as a water-soluble bismuth(III) reagent for phage library modification and in situ bicyclic peptide preparation, eliminating the need for organic co-solvents. Additionally, we explore arsenic(III) as an alternative thiophilic element that is used analogously to our previously introduced bicyclic peptides with a bismuth core. The modification of phage libraries and peptides with these elements is instantaneous and entirely biocompatible, offering an advantage over conventional alkylation-based methods. In a pilot display screening campaign aimed at identifying ligands for the biotin-binding protein streptavidin, we demonstrate the enrichment of bicyclic peptides with dissociation constants two orders of magnitude lower than those of their linear counterparts, underscoring the impact of structural constraint on binding affinity.

DOI: https://doi.org/10.1038/s42004-024-01232-0
Pharmaceutics. 2024 Jun 07. pii: 774. [Epub ahead of print]16(6):

Intranasal Delivery of Cell-Penetrating Therapeutic Peptide Enhances Brain Delivery, Reduces Inflammation, and Improves Neurologic Function in Moderate Traumatic Brain Injury.

Yaswanthi Yanamadala, Ritika Roy, Afrika Alake Williams, Navya Uppu, Audrey Yoonsun Kim, Mark A DeCoster, Paul Kim, Teresa Ann Murray.

  Following traumatic brain injury (TBI), secondary brain damage due to chronic inflammation is the most predominant cause of the delayed onset of mood and memory disorders. Currently no therapeutic approach is available to effectively mitigate secondary brain injury after TBI. One reason is the blood-brain barrier (BBB), which prevents the passage of most therapeutic agents into the brain. Peptides have been among the leading candidates for CNS therapy due to their low immunogenicity and toxicity, bioavailability, and ease of modification. In this study, we demonstrated that non-invasive intranasal (IN) administration of KAFAK, a cell penetrating anti-inflammatory peptide, traversed the BBB in a murine model of diffuse, moderate TBI. Notably, KAFAK treatment reduced the production of proinflammatory cytokines that contribute to secondary injury. Furthermore, behavioral tests showed improved or restored neurological, memory, and locomotor performance after TBI in KAFAK-treated mice. This study demonstrates KAFAK's ability to cross the blood-brain barrier, to lower proinflammatory cytokines in vivo, and to restore function after a moderate TBI.

Keywords:  KAFAK; TBI; blood–brain barrier; cell penetrating peptide; intranasal drug delivery; peptide therapeutics; proinflammatory cytokines; secondary injury; traumatic brain injury

DOI:  https://doi.org/10.3390/pharmaceutics16060774
Adv Sci (Weinh). 2024 Jun 26. e2404563

Arginine-Rich Cell-Penetrating Peptides Induce Lipid Rearrangements for Their Active Translocation across Laterally Heterogeneous Membranes.

Sujin Park, Jinmin Kim, Seung Soo Oh, Siyoung Q Choi.

  Arginine-rich cell-penetrating peptides (CPPs) have emerged as valuable tools for the intracellular delivery of bioactive molecules, but their membrane perturbation during cell penetration is not fully understood. Here, nona-arginine (R9)-mediated membrane reorganization that facilitates the translocation of peptides across laterally heterogeneous membranes is directly visualized. The electrostatic binding of cationic R9 to anionic phosphatidylserine (PS)-enriched domains on a freestanding lipid bilayer induces lateral lipid rearrangements; in particular, in real-time it is observed that R9 fluidizes PS-rich liquid-ordered (Lo) domains into liquid-disordered (Ld) domains, resulting in the membrane permeabilization. The experiments with giant unilamellar vesicles (GUVs) confirm the preferential translocation of R9 through Ld domains without pore formation, even when Lo domains are more negatively charged. Indeed, whenever R9 comes into contact with negatively charged Lo domains, it dissolves the Lo domains first, promoting translocation across phase-separated membranes. Collectively, the findings imply that arginine-rich CPPs modulate lateral membrane heterogeneity, including membrane fluidization, as one of the fundamental processes for their effective cell penetration across densely packed lipid bilayers.

Keywords:  cell‐penetrating peptide; freestanding lipid bilayer; giant unilamellar vesicle; membrane remodeling; real‐time visualization

DOI:  https://doi.org/10.1002/advs.202404563
Angew Chem Int Ed Engl. 2024 Jun 26. e202409969

Exploring Macroscopic Dipoles of Designed Cyclic Peptide Ordered Assemblies to Harvest Piezoelectric Properties.

Souvik Panda Mahapatra, Saikat Pahan, Abhijit Chatterjee, Souvik Roy, DRGKoppalu R Puneeth Kumar, Hosahudya Gopi.

  Crystalline materials exhibiting non-centrosymmetry and possessing substantial surface dipole moments play a critical role in piezoelectricity. Designing biocompatible self-assembled materials with these attributes is particularly challenging when compared to inorganic materials and ceramics. In this study, we elucidate the crystal conformations of novel cyclic peptides that exhibit self-assembly into tubular structures characterized by unidirectional hydrogen bonding and piezoelectric properties. Unlike cyclic peptides derived from alternating L- and D-amino acids, those derived from new δ-amino acids demonstrate the formation of self-assembled tubes with unidirectional hydrogen bonds. Further, the tightly packed tubular assemblies and higher macrodipole moments result in superior piezoelectric coefficients compared to peptides with lower macrodipole moments. Our findings underscore the potential for designing cyclic peptides with unidirectional hydrogen bonds, thereby paving the way for their application in design of biocompatible piezo- and ferroelectric materials.

Keywords:  Cyclic Peptide; Dipole moment; Tubular Assembly *. Piezoelectric Property; δ-Amino Acid

DOI:  https://doi.org/10.1002/anie.202409969
PLoS One. 2024 ;19(6): e0303901

Multielectrode array characterization of human induced pluripotent stem cell derived neurons in co-culture with primary human astrocytes.

Maddie R Lemieux, Bernhard Freigassner, Jessica L Hanson, Zahra Thathey, Mark R Opp, Charles A Hoeffer, Christopher D Link.

Human induced pluripotent stem cells (hiPSCs) derived into neurons offer a powerful in vitro model to study cellular processes. One method to characterize functional network properties of these cells is using multielectrode arrays (MEAs). MEAs can measure the electrophysiological activity of cellular cultures for extended periods of time without disruption. Here we used WTC11 hiPSCs with a doxycycline-inducible neurogenin 2 (NGN2) transgene differentiated into neurons co-cultured with primary human astrocytes. We achieved a synchrony index ∼0.9 in as little as six-weeks with a mean firing rate of ∼13 Hz. Previous reports show that derived 3D brain organoids can take several months to achieve similar strong network burst synchrony. We also used this co-culture to model aspects of blood-brain barrier breakdown by using human serum. Our fully human co-culture achieved strong network burst synchrony in a fraction of the time of previous reports, making it an excellent first pass, high-throughput method for studying network properties and neurodegenerative diseases.

DOI: https://doi.org/10.1371/journal.pone.0303901
Int J Mol Sci. 2024 Jun 20. pii: 6764. [Epub ahead of print]25(12):

Subtle Structural Differences Affect the Inhibitory Potency of RGD-Containing Cyclic Peptide Inhibitors Targeting SPSB Proteins.

Kefa Li, Yanhong Luo, Weiwei Hu, Jinjin Yang, Danting Zhang, Huan Wei, Tingting You, Hai-Shu Lin, Zhihe Kuang.

  The SPRY domain-containing SOCS box proteins SPSB1, SPSB2, and SPSB4 utilize their SPRY/B30.2 domain to interact with a short region in the N-terminus of inducible nitric oxide synthase (iNOS), and recruit an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in the proteasomal degradation of iNOS. Inhibitors that can disrupt the endogenous SPSB-iNOS interactions could be used to augment cellular NO production, and may have antimicrobial and anticancer activities. We previously reported the rational design of a cyclic peptide inhibitor, cR8, cyclo(RGDINNNV), which bound to SPSB2 with moderate affinity. We, therefore, sought to develop SPSB inhibitors with higher affinity. Here, we show that cyclic peptides cR7, cyclo(RGDINNN), and cR9, cyclo(RGDINNNVE), have ~6.5-fold and ~2-fold, respectively, higher SPSB2-bindng affinities than cR8. We determined high-resolution crystal structures of the SPSB2-cR7 and SPSB2-cR9 complexes, which enabled a good understanding of the structure-activity relationships for these cyclic peptide inhibitors. Moreover, we show that these cyclic peptides displace full-length iNOS from SPSB2, SPSB1, and SPSB4, and that their inhibitory potencies correlate well with their SPSB2-binding affinities. The strongest inhibition was observed for cR7 against all three iNOS-binding SPSB proteins.

Keywords:  SPRY domain-containing SOCS box protein; crystal structure; cyclic peptide; inducible nitric oxide synthase; inhibitor

DOI:  https://doi.org/10.3390/ijms25126764
ACS Synth Biol. 2024 Jun 26.

Expression and Subcellular Localization of Lanthipeptides in Human Cells.

Sara M Eslami, Chandrashekhar Padhi, Imran R Rahman, Wilfred A van der Donk.

  Cyclic peptides, such as most ribosomally synthesized and post-translationally modified peptides (RiPPs), represent a burgeoning area of interest in therapeutic and biotechnological research because of their conformational constraints and reduced susceptibility to proteolytic degradation compared to their linear counterparts. Herein, an expression system is reported that enables the production of structurally diverse lanthipeptides and derivatives in mammalian cells. Successful targeting of lanthipeptides to the nucleus, the endoplasmic reticulum, and the plasma membrane is demonstrated. In vivo expression and targeting of such peptides in mammalian cells may allow for screening of lanthipeptide-based cyclic peptide inhibitors of native, organelle-specific protein-protein interactions in mammalian systems.

Keywords:  RiPPs; cyclic peptides; cytolysin; haloduracin; organelle localization; protein−protein interactions

DOI:  https://doi.org/10.1021/acssynbio.4c00178
Pharmaceutics. 2024 Jun 07. pii: 775. [Epub ahead of print]16(6):

Comparative Study of the Potential Cell-Penetrating Peptide ∆M4 on Apoptosis Cell Signaling in A375 and A431 Cancer Cell Lines.

Estefanía Fandiño-Devia, Aleksandra Brankiewicz, Gloria A Santa-González, Ibeth Guevara-Lora, Marcela Manrique-Moreno.

  In recent yearsjajajj, peptide-based therapeutics have attracted increasing interest as a potential approach to cancer treatment. Peptides are characterized by high specificity and low cytotoxicity, but they cannot be considered universal drugs for all types of cancer. Of the numerous anticancer-reported peptides, both natural and synthetic, only a few have reached clinical applications. However, in most cases, the mechanism behind the anticancer activity of the peptide is not fully understood. For this reason, in this work, we investigated the effect of the novel peptide ∆M4, which has documented anticancer activity, on two human skin cancer cell lines. A novel approach to studying the potential induction of apoptosis by anticancer peptides is the use of protein microarrays. The results of the apoptosis protein study demonstrated that both cell types, skin malignant melanoma (A375) and epidermoid carcinoma (A431), exhibited markers associated with apoptosis and cellular response to oxidative stress. Additionally, ∆M4 induced concentration- and time-dependent moderate ROS production, triggering a defensive response from the cells, which showed decreased activation of cytoplasmic superoxide dismutase. However, the studied cells exhibited a differential response in catalase activity, with A375 cells showing greater resistance to the peptide action, possibly mediated by the Nrf2 pathway. Nevertheless, both cell types showed moderate activity of caspases 3/7, suggesting that they may undergo partial apoptosis, although another pathway of programmed death cannot be excluded. Extended analysis of the mechanisms of action of anticancer peptides may help determine their effectiveness in overcoming chemoresistance in cancerous cells.

Keywords:  anticancer peptides; apoptosis; cell-penetrating peptides; epidermoid carcinoma; melanoma; oxidative stress

DOI:  https://doi.org/10.3390/pharmaceutics16060775
Front Chem. 2024 ;12 1403473

Study on the design, synthesis, and activity of anti-tumor staple peptides targeting MDM2/MDMX.

Jian Yang, Xiufei Liao, Damin Hu, Jinqiu Mo, Xiurong Gao, Hongli Liao.

  Staple peptides, which have a significantly enhanced pharmacological profile, are promising therapeutic molecules due to their remarkable resistance to proteolysis and cell-penetrating properties. In this study, we designed and synthesized a series of PMI-M3-based dual-targeting MDM2/MDMX staple peptides and compared them with straight-chain peptides. The staple peptide SM3-4 screened in the study induced apoptosis of tumor cells in vitro at low μM concentrations, and the helix was significantly increased. Studies have shown that the enhancement of staple activity is related to the increase in helicity, and SM3-4 provides an effective research basis for dual-targeted anti-tumor staple peptides.

Keywords:  MDM2; MDMX; anti-tumor; double-targeted; staple peptides

DOI:  https://doi.org/10.3389/fchem.2024.1403473
Int J Nanomedicine. 2024 ;19 6057-6084

Tuning Peptide-Based Nanofibers for Achieving Selective Doxorubicin Delivery in Triple-Negative Breast Cancer.

Rosa Bellavita, Marialuisa Piccolo, Linda Leone, Maria Grazia Ferraro, Principia Dardano, Luca De Stefano, Flavia Nastri, Carlo Irace, Annarita Falanga, Stefania Galdiero.

   Introduction: The design of delivery tools that efficiently transport drugs into cells remains a major challenge in drug development for most pathological conditions. Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer with poor prognosis and limited effective therapeutic options.
Purpose: In TNBC treatment, chemotherapy remains the milestone, and doxorubicin (Dox) represents the first-line systemic treatment; however, its non-selective distribution causes a cascade of side effects. To address these problems, we developed a delivery platform based on the self-assembly of amphiphilic peptides carrying several moieties on their surfaces, aimed at targeting, enhancing penetration, and therapy.
Methods: Through a single-step self-assembly process, we used amphiphilic peptides to obtain nanofibers decorated on their surfaces with the selected moieties. The surface of the nanofiber was decorated with a cell-penetrating peptide (gH625), an EGFR-targeting peptide (P22), and Dox bound to the cleavage sequence selectively recognized and cleaved by MMP-9 to obtain on-demand drug release. Detailed physicochemical and cellular analyses were performed.
Results: The obtained nanofiber (NF-Dox) had a length of 250 nm and a diameter of 10 nm, and it was stable under dilution, ionic strength, and different pH environments. The biological results showed that the presence of gH625 favored the complete internalization of NF-Dox after 1h in MDA-MB 231 cells, mainly through a translocation mechanism. Interestingly, we observed the absence of toxicity of the carrier (NF) on both healthy cells such as HaCaT and TNBC cancer lines, while a similar antiproliferative effect was observed on TNBC cells after the treatment with the free-Dox at 50 µM and NF-Dox carrying 7.5 µM of Dox.
Discussion: We envision that this platform is extremely versatile and can be used to efficiently carry and deliver diverse moieties. The knowledge acquired from this study will provide important guidelines for applications in basic research and biomedicine.

Keywords:  doxorubicin; nanofiber; on-demand strategy; self-assembling peptides; triple negative breast cancer

DOI:  https://doi.org/10.2147/IJN.S453958
J Med Chem. 2024 Jun 25.

Design of a Lead-Like Cysteine-Targeting Covalent Library and the Identification of Hits to Cys55 of Bfl-1.

Simon C C Lucas, Alexander G Milbradt, J Henry Blackwell, Silvia Bonomo, Andrew Brierley, Doyle J Cassar, Jared Freeman, Thomas E Hadfield, Lucas A Morrill, Rick Riemens, Sunil Sarda, Stefan Schiesser, Daniel Wiktelius, Samiyah Ahmed, Mark J Bostock, Ulf Börjesson, Claudia De Fusco, Carine Guerot, David Hargreaves, Sarah Hewitt, Michelle L Lamb, Nancy Su, Ryan Whatling, Matthew Wheeler, Jason G Kettle.

Covalent hit identification is a viable approach to identify chemical starting points against difficult-to-drug targets. While most researchers screen libraries of <2k electrophilic fragments, focusing on lead-like compounds can be advantageous in terms of finding hits with improved affinity and with a better chance of identifying cryptic pockets. However, due to the increased molecular complexity, larger numbers of compounds (>10k) are desirable to ensure adequate coverage of chemical space. Herein, the approach taken to build a library of 12k covalent lead-like compounds is reported, utilizing legacy compounds, robust library chemistry, and acquisitions. The lead-like covalent library was screened against the antiapoptotic protein Bfl-1, and six promising hits that displaced the BIM peptide from the PPI interface were identified. Intriguingly, X-ray crystallography of lead-like compound 8 showed that it binds to a previously unobserved conformation of the Bfl-1 protein and is an ideal starting point for the optimization of Bfl-1 inhibitors.

DOI: https://doi.org/10.1021/acs.jmedchem.4c00781
Eur J Nucl Med Mol Imaging. 2024 Jun 24.

[64Cu]Cu-FAP-NOX, a N-oxalyl modified cyclic peptide for FAP PET imaging with a flexible imaging time window.

Shaoyu Liu, Jiawei Zhong, Ziqi Zhang, Ruiyue Zhao, Qingsong Yan, Xinlu Wang.

   BACKGROUND: The aim of the present study was to develop a novel 64Cu-labeled cyclic peptide ([64Cu]Cu-FAP-NOX) that targets fibroblast activation protein (FAP) and may offer advantages in terms of image contrast, imaging time window, and low uptake in normal tissues.
METHODS: The novel cyclic peptide featuring with a N-oxalyl modified tail was constructed and conjugated to NOTA for 64Cu labeling. Biochemical and cellular assays were performed with A549.hFAP cells. The performance of [64Cu]Cu-FAP-NOX was compared to that of two established tracers ([64Cu]Cu-FAPI-04 and [68Ga]Ga-FAP-2286) and three different NOTA-conjugates in HEK-293T.hFAP xenograft mice using micro-PET imaging. Ex vivo biodistribution studies were performed to confirm the FAP specificity and to validate the PET data. Furthermore, a first-in-human study of this novel tracer was conducted on one patient with lung cancer.
RESULTS: Compared to [64Cu]Cu-FAPI-04, [64Cu]Cu-FAP-NOX demonstrated faster and higher rates of cellular uptake and internalization in A549.hFAP cells, but lower rates of cellular efflux. All six radiotracers were rapidly taken up by the tumor within the first 4 h post-injection. However, [64Cu]Cu-FAP-NOX had more intense tumor accumulation and slower washout from the target. The ratios of the tumor to normal tissue (including kidneys and muscles) increased significantly over time, with [64Cu]Cu-FAP-NOX reaching the highest ratio among all tracers. In the patient, [64Cu]Cu-FAP-NOX PET showed a comparable result to FDG PET in the primary malignant lesion while exhibiting higher uptake in pleural metastases, consistent with elevated FAP expression as confirmed by immunohistochemistry.
CONCLUSION: [64Cu]Cu-FAP-NOX is a promising FAP-targeted tracer with a highly flexible imaging time window, as evidenced by preclinical evaluation encompassing biodistribution and micro-PET studies, along with a successful patient application. Furthermore, [64Cu]Cu-FAP-NOX showed enhanced image contrast and favorable pharmacokinetic properties for FAP PET imaging, warranting translation into large cohort studies.

Keywords:  Copper-64; Cyclic peptide; Fibroblast activation protein; First-in-human

DOI:  https://doi.org/10.1007/s00259-024-06807-6
Chem Sci. 2024 Jun 26. 15(25): 9612-9619

Peptide macrocyclisation via intramolecular interception of visible-light-mediated desulfurisation.

Frances R Smith, Declan Meehan, Rhys C Griffiths, Harriet J Knowles, Peiyu Zhang, Huw E L Williams, Andrew J Wilson, Nicholas J Mitchell.

Synthetic methods that enable the macrocyclisation of peptides facilitate the development of effective therapeutic and diagnostic tools. Herein we report a peptide cyclisation strategy based on intramolecular interception of visible-light-mediated cysteine desulfurisation. This method allows cyclisation of unprotected peptides in an aqueous solution via the installation of a hydrocarbon linkage. We explore the limits of this chemistry using a range of model peptides of increasing length and complexity, including peptides of biological/therapeutic relevance. The method is applied to replace the native disulfide of the peptide hormone, oxytocin, with a proteolytically/redox-stable hydrocarbon, and internal macrocyclisation of an MCL-1-binding peptide.

DOI: https://doi.org/10.1039/d3sc05865d
RSC Med Chem. 2024 Jun 19. 15(6): 2018-2029

The importance of tyrosines in multimers of cyclic RGD nonapeptides: towards αvβ6-integrin targeted radiotherapeutics.

Neil Gerard Quigley, Maximilian Alexander Zierke, Beatrice Stefanie Ludwig, Frauke Richter, Nghia Trong Nguyen, Falco Reissig, Jakub Šimeček, Susanne Kossatz, Johannes Notni.

In a recent paper in this journal (RSC Med. Chem., 2023, 14, 2429), we described an unusually strong impact of regiospecific exchange of phenylalanines by tyrosines in 10 gallium-68-labeled trimers of certain cyclic RGD peptides, c[XRGDLAXp(NMe)K] (X = F or Y), on non-specific organ uptakes. We found that there was, in part, no correlation of liver uptake with established polarity proxies, such as the octanol-water distribution coefficient (log D). Since this observation could not be explained straightforwardly, we suggested that the symmetry of the compounds had resulted in a synergistic interaction of certain components of the macromolecules. In the present work, we investigated whether a comparable effect also occurred for a series of 5 tetramers labeled with lutetium-177. We found that in contrast to the trimers, liver uptake of the tetramers was well correlated to their polarity, indicating that the unusual observations along the trimer series indeed was a unique feature, probably related to their particular symmetry. Since the Lu-177 labeled tetramers are also potential agents for treatment of a variety of αvβ6-integrin expressing cancers, these were evaluated in mice bearing human lung adenocarcinoma xenografts. Due to their tumor-specific uptake and retention in biodistribution and SPECT imaging experiments, these compounds are considered a step forward on the way to αvβ6-integrin-targeted anticancer agents. Furthermore, we noticed that the presence of tyrosines in general had a positive impact on the in vivo performance of our peptide multimers. In view of the fact that a corresponding rule was already proposed in the context of protein engineering, we argue in favor of considering peptide multimers as a special class of small or medium-sized proteins. In summary, we contend that the performance of peptide multimers is less determined by the in vitro characteristics (particularly, affinity and selectivity) of monomers, but rather by the peptides' suitability for the overall macromolecular design concept, and peptides containing tyrosines are preferred.

DOI: https://doi.org/10.1039/d4md00073k
Mol Cancer Ther. 2024 Jun 28.

FZ-AD005, A Novel DLL3-Targeted Antibody-drug Conjugate with Topoisomerase I Inhibitor, Shows Potent Antitumor Activity in Preclinical Models.

Qingsong Guo, Bei Gao, Ruiwen Song, Weinan Li, Qian Xie, Sensen Lou, Jiafei Shen, Teng Zhao, Yifan Zhang, Jinsong Wu, Tong Yang.

Delta-like ligand 3 (DLL3) is overexpressed in small-cell lung cancer (SCLC) and has been considered an attractive target for SCLC therapy. Rovalpituzumab tesirine (Rova-T) was the first DLL3-targeted antibody-drug conjugate (ADC) to enter clinical studies. However, serious adverse events limited progress in the treatment of SCLC with Rova-T. In this study, we developed a novel DLL-3-targeted ADC, FZ-AD005, by using DXd with potent cytotoxicity and a relatively better safety profile to maximize the therapeutic index. FZ-AD005 was generated by a novel anti-DLL3 antibody FZ-A038 and a valine-alanine (Val-Ala) dipeptide linker to conjugate DXd. Moreover, Fc-silencing technology was introduced in FZ-AD005 to avoid off-target toxicity mediated by FcγRs and showed negligible Fc-mediated effector functions in vitro. In preclinical evaluation, FZ-AD005 exhibited DLL3-specific binding and demonstrated efficient internalization, bystander killing, and excellent in vivo antitumor activities in cell line-derived xenografts (CDX) and patient-derived xenograft (PDX) models. FZ-AD005 was stable in circulation with acceptable pharmacokinetic profiles in cynomolgus monkeys. FZ-AD005 was well tolerated in rats and monkeys. The safety profile of FZ-AD005 was favorable and the highest non-severely toxic dose was 30 mg/kg in cynomolgus monkeys. In conclusion, FZ-AD005 has the potential to be a superior DLL3-targeted ADC with a wide therapeutic window and is expected to provide clinical benefits for the treatment of SCLC patients.

DOI: https://doi.org/10.1158/1535-7163.MCT-23-0701
Pharmaceutics. 2024 Jun 16. pii: 814. [Epub ahead of print]16(6):

In Vivo Evaluation of 68Ga-Labeled NOTA-EGFRvIII Aptamer in EGFRvIII-Positive Glioblastoma Xenografted Model.

Jun Young Park, Ye Lim Cho, Tae Sup Lee, Daekyun Lee, Ju-Hyung Kang, Soryong Lim, Yujin Lee, Jae Hyun Lim, Won Jun Kang.

  EGFRvIII is expressed only in tumor cells and strongly in glioblastoma and is considered a promising target in cancer diagnosis and therapy. Aptamers are synthetic single-stranded oligonucleotides that bind to biochemical target molecules with high binding affinity and specificity. This study examined the potential of the 68Ga-NOTA-EGFRvIII aptamer as a nuclear imaging probe for visualizing EGFRvIII-expressing glioblastoma by positron emission tomography (PET). EGFRvIII aptamer was selected using the SELEX technology, and flow cytometry and fluorescence microscopy verified the high binding affinity to EGFRvIII positive U87MG vIII 4.12 glioma cells but not to EGFRvIII negative U87MG cells. The EGFRvIII aptamer was conjugated with a chelator (1,4,7-triazanonane-1,4,7-triyl)triacetic acid (NOTA) for 68Ga-labeling. The 68Ga-NOTA-EGFRvIII aptamer was prepared using the preconcentration-based labeling method with a high radiolabeling yield at room temperature. Ex vivo biodistribution analyses confirmed the significantly higher tumor uptake of the 68Ga-NOTA-EGFRvIII aptamer in EGFRvIII-expressing xenograft tumors than that in EGFRvIII negative tumors, confirming the specific tumor uptake of the 68Ga-NOTA-EGFRvIII aptamer in vivo. PET imaging studies revealed a high retention rate of the 68Ga-NOTA-EGFRvIII aptamer in U87MG vIII 4.12 tumors but only low uptake levels in U87-MG tumors, suggesting that the 68Ga-NOTA-EGFRvIII aptamer may be used as a PET imaging agent for EGFRvIII-expressing glioblastoma.

Keywords:  EGFRvIII; Ga-68; PET imaging; aptamer; glioblastoma

DOI:  https://doi.org/10.3390/pharmaceutics16060814
Chemistry. 2024 Jun 24. e202402146

Design of an Acidic pH-Activated NIR Fluorescent Convertible Rhodamine-Hemicyanine Probe-Peptide Conjugate for Living Cancer Cell Active Targeted Selective Tracking of Lysosomes.

Ayan Mukherjee, Samiran Kar, Shreya Das, Tapas Bera, Aniruddha Mondal, Arunima Sengupta, Samit Guha.

  We have synthesized an acidic pH-activatable dual targeting ratiometric fluorescent probe-peptide conjugate using the SPPS protocol on resin. Living carcinoma cell specific active targeting, successive cell penetration, and selective staining of lysosomes are accomplished. Real-time monitoring of lysosomes, 3D, and multicolor cancer cell imaging are attained. The de novo design consists of the integration of multifunctionality into a single molecular scaffold, e.g., RGDS peptide to target cancer cell overexpressed receptor αVβ3 integrin, live-cell penetrating rhodamine-hemicyanine chromophore comprising a lysosome targeting morpholine group, and an acidic pH openable spiro-lactam ring for a visible-to-NIR switchable ratiometric response. Water-soluble probe-peptide conjugate exhibits intramolecular spirolactamization at basic pH through Arg amide N. The visible spirolactam state predominantly exists at physiological and basic pH and can be switched to the highly conjugated NIR open amide state (λem=735 nm) through spiro-lactam ring opening triggered by acidic pH with a huge bathochromic shift (Δλabs=336 nm, ΔλFL=265 nm). pH-sensitive ratiometric switching is achieved. This in situ acidic cancer cell lysosome activatable multifunctional fluorophore-peptide conjugate shows augmented molar absorptivity, enhanced quantum yield, and improved fluorescence lifetime at acidic lysosomal pH; negligible cytotoxicity; and dual targeted ratiometric imaging capability of living cancer cell selective lysosomes with pKa value of 5.1.

Keywords:  Acidic pH-activated; Lysosomes; NIR fluorescent; Ratiometric imaging; Rhodamine−hemicyanine

DOI:  https://doi.org/10.1002/chem.202402146
mSystems. 2024 Jun 27. e0135823

PyAMPA: a high-throughput prediction and optimization tool for antimicrobial peptides.

Marc Ramos-Llorens, Roberto Bello-Madruga, Javier Valle, David Andreu, Marc Torrent.

  The alarming rise of antibiotic-resistant bacterial infections is driving efforts to develop alternatives to conventional antibiotics. In this context, antimicrobial peptides (AMPs) have emerged as promising candidates for their ability to target a broad range of microorganisms. However, the development of AMPs with optimal potency, selectivity, and/or stability profiles remains a challenge. To address it, computational tools for predicting AMP properties and designing novel peptides have gained increasing attention. PyAMPA is a novel platform for AMP discovery. It consists of five modules, namely AMPScreen, AMPValidate, AMPSolve, AMPMutate, and AMPOptimize, that allow high-throughput proteome inspection, candidate screening, and optimization through point-mutation and genetic algorithms. The platform also offers additional tools for predicting and evaluating AMP properties, including antimicrobial and cytotoxic activity, and peptide half-life. By providing innovative and accessible inroads into AMP motifs in proteomes, PyAMPA will enable advances in AMP development and potential translation into clinically useful molecules. PyAMPA is available at: https://github.com/SysBioUAB/PyAMPA.
IMPORTANCE: This paper introduces PyAMPA, a new bioinformatics platform designed for the discovery and optimization of antimicrobial peptides (AMPs). It addresses the urgent need for new antimicrobials due to the rise of antibiotic-resistant infections. PyAMPA, with its five predictive modules -AMPScreen, AMPValidate, AMPSolve, AMPMutate and AMPOptimize, enables high-throughput screening of proteomes to identify potential AMP motifs and optimize them for clinical use. Its unique approach, combining prediction, design, and optimization tools, makes PyAMPA a robust solution for developing new AMP-based therapies, offering a significant advance in combatting antibiotic resistance.

Keywords:  antimicrobial peptide

DOI:  https://doi.org/10.1128/msystems.01358-23
J Med Chem. 2024 Jun 26.

Discovery of MK-1084: An Orally Bioavailable and Low-Dose KRASG12C Inhibitor.

Xiaoshen Ma, David L Sloman, Ruchia Duggal, Kenneth D Anderson, Jeanine E Ballard, Indu Bharathan, Christopher Brynczka, Symon Gathiaka, Timothy J Henderson, Thomas W Lyons, Richard Miller, Erik V Munsell, Peter Orth, Ryan D Otte, Anandan Palani, Danica A Rankic, Michelle R Robinson, Aaron C Sather, Nicolas Solban, Xuelei Sherry Song, Xin Wen, Zangwei Xu, Yi Yang, Ruojing Yang, Phil J Day, Alexander Stoeck, David Jonathan Bennett, Yongxin Han.

Oncogenic mutations in the RAS gene account for 30% of all human tumors; more than 60% of which present as KRAS mutations at the hotspot codon 12. After decades of intense pursuit, a covalent inhibition strategy has enabled selective targeting of this previously "undruggable" target. Herein, we disclose our journey toward the discovery of MK-1084, an orally bioavailable and low-dose KRASG12C covalent inhibitor currently in phase I clinical trials (NCT05067283). We leveraged structure-based drug design to identify a macrocyclic core structure, and hypothesis-driven optimization of biopharmaceutical properties to further improve metabolic stability and tolerability.

DOI: https://doi.org/10.1021/acs.jmedchem.4c00572
Nat Commun. 2024 Jun 25. 15(1): 5372

Production of constrained L-cyclo-tetrapeptides by epimerization-resistant direct aminolysis.

Huan Chen, Yuchen Zhang, Yuming Wen, Xinhao Fan, Nicholas Sciolino, Yanyun Lin, Leonard Breindel, Yuanwei Dai, Alexander Shekhtman, Xiao-Song Xue, Qiang Zhang.

The synthesis of constrained 12-membered rings is notably difficult. The main challenges result from constraints during the linear peptide cyclization. Attempts to overcome constraints through excessive activation frequently cause peptidyl epimerization, while insufficient activation of the C-terminus hampers cyclization and promotes intermolecular oligomer formation. We present a β-thiolactone framework that enables the synthesis of cyclo-tetrapeptides via direct aminolysis. This tactic utilizes a mechanism that restricts C-terminal carbonyl rotation while maintaining high reactivity, thereby enabling efficient head-to-tail amidation, reducing oligomerization, and preventing epimerization. A broad range of challenging cyclo-tetrapeptides ( > 20 examples) are synthesized in buffer and exhibits excellent tolerance toward nearly all proteinogenic amino acids. Previously unattainable macrocycles, such as cyclo-L-(Pro-Tyr-Pro-Val), have been produced and identified as μ-opioid receptor (MOR) agonists, with an EC50 value of 2.5 nM. Non-epimerizable direct aminolysis offers a practical solution for constrained peptide cyclization, and the discovery of MOR agonist activity highlights the importance of overcoming synthetic challenges for therapeutic development.

DOI: https://doi.org/10.1038/s41467-024-49329-3
Mol Pharm. 2024 Jun 28.

Head-to-Head Comparison of Caco-2 Transwell and Gut-on-a-Chip Models for Assessing Oral Peptide Formulations.

John P Gleeson, Stephanie Y Zhang, Natalia Subelzu, Jing Ling, Becky Nissley, Whitney Ong, Rebecca Nofsinger, Filippos Kesisoglou.

  Oral delivery of potent peptide drugs provides key formulation challenges in the pharmaceutical industry: stability, solubility, and permeability. Intestinal permeation enhancers (PEs) can overcome the low oral bioavailability by improving the drug permeability. Conventional in vitro and ex vivo models for assessing PEs fail to predict efficacy in vivo. Here, we compared Caco-2 cells cultured in the conventional static Transwell model to a commercially available continuous flow microfluidic Gut-on-a-Chip model. We determined baseline permeability of FITC-Dextan 3 kDa (FD3) in Transwell (5.3 ± 0.8 × 10-8 cm/s) vs Chip (3.2 ± 1.8 × 10-7 cm/s). We screened the concentration impact of two established PEs sodium caprate and sucrose monolaurate and indicated a requirement for higher enhancer concentration in the Chip model to elicit equivalent efficacy e.g., 10 mM sodium caprate in Transwells vs 25 mM in Chips. Fasted and fed state simulated intestinal fluids (FaSSIF/FeSSIF) were introduced into the Chip and increased basal FD3 permeability by 3-fold and 20-fold, respectively, compared to 4-fold and 4000-fold in Transwells. We assessed the utility of this model to peptides (Insulin and Octreotide) with PEs and observed much more modest permeability enhancement in the Chip model in line with observations in ex vivo and in vivo preclinical models. These data indicate that microfluidic Chip models are well suited to bridge the gap between conventional in vitro and in vivo models.

Keywords:  Gut-on-a-Chip; IVIVC; intestinal permeation enhancers; microfluidics; oral peptide

DOI:  https://doi.org/10.1021/acs.molpharmaceut.4c00210
Front Pharmacol. 2024 ;15 1387949

Real-time label-free three-dimensional invasion assay for anti-metastatic drug screening using impedance sensing.

Kai Ding, Hailong Li, Qian Xu, Yongmei Zhao, Kaikai Wang, Tianqing Liu.

  Tumor metastasis presents a formidable challenge in cancer treatment, necessitating effective tools for anti-cancer drug development. Conventional 2D cell culture methods, while considered the "gold standard" for invasive studies, exhibit limitations in representing cancer hallmarks and phenotypes. This study proposes an innovative approach that combines the advantages of 3D tumor spheroid culture with impedance-based biosensing technologies to establish a high-throughput 3D cell invasion assay for anti-metastasis drug screening through multicellular tumor spheroids. In addition, the xCELLigence device is employed to monitor the time-dependent kinetics of cell behavior, including attachment and invasion out of the 3D matrix. Moreover, an iron chelator (deferoxamine) is employed to monitor the inhibition of epithelial-mesenchymal transition in 3D spheroids across different tumor cell types. The above results indicate that our integrated 3D cell invasion assay with impedance-based sensing could be a promising tool for enhancing the quality of the drug development pipeline by providing a robust platform for predicting the efficacy and safety of anti-metastatic drugs before advancing into preclinical or clinical trials.

Keywords:  biosensing; drug development; drug screening; tumor metastasis; tumor spheroid

DOI:  https://doi.org/10.3389/fphar.2024.1387949
J Chem Inf Model. 2024 Jun 22.

DiPPI: A Curated Data Set for Drug-like Molecules in Protein-Protein Interfaces.

Fatma Cankara, Simge Senyuz, Ahenk Zeynep Sayin, Attila Gursoy, Ozlem Keskin.

Proteins interact through their interfaces, and dysfunction of protein-protein interactions (PPIs) has been associated with various diseases. Therefore, investigating the properties of the drug-modulated PPIs and interface-targeting drugs is critical. Here, we present a curated large data set for drug-like molecules in protein interfaces. We further introduce DiPPI (Drugs in Protein-Protein Interfaces), a two-module web site to facilitate the search for such molecules and their properties by exploiting our data set in drug repurposing studies. In the interface module of the web site, we present several properties, of interfaces, such as amino acid properties, hotspots, evolutionary conservation of drug-binding amino acids, and post-translational modifications of these residues. On the drug-like molecule side, we list drug-like small molecules and FDA-approved drugs from various databases and highlight those that bind to the interfaces. We further clustered the drugs based on their molecular fingerprints to confine the search for an alternative drug to a smaller space. Drug properties, including Lipinski's rules and various molecular descriptors, are also calculated and made available on the web site to guide the selection of drug molecules. Our data set contains 534,203 interfaces for 98,632 protein structures, of which 55,135 are detected to bind to a drug-like molecule. 2214 drug-like molecules are deposited on our web site, among which 335 are FDA-approved. DiPPI provides users with an easy-to-follow scheme for drug repurposing studies through its well-curated and clustered interface and drug data and is freely available at http://interactome.ku.edu.tr:8501.

DOI: https://doi.org/10.1021/acs.jcim.3c01905
Proteins. 2024 Jun 23.

Protein-Protein Interaction Prediction via Structure-Based Deep Learning.

Yucong Liu, Yijun Liu, Zhenhai Li.

  Protein-protein interactions (PPIs) play an essential role in life activities. Many artificial intelligence algorithms based on protein sequence information have been developed to predict PPIs. However, these models have difficulty dealing with various sequence lengths and suffer from low generalization and prediction accuracy. In this study, we proposed a novel end-to-end deep learning framework, RSPPI, combining residual neural network (ResNet) and spatial pyramid pooling (SPP), to predict PPIs based on the protein sequence physicochemistry properties and spatial structural information. In the RSPPI model, ResNet was employed to extract the structural and physicochemical information from the protein three-dimensional structure and primary sequence; the SPP layer was used to transform feature maps to a single vector and avoid the fixed-length requirement. The RSPPI model possessed excellent cross-species performance and outperformed several state-of-the-art methods based either on protein sequence or gene ontology in most evaluation metrics. The RSPPI model provides a novel strategy to develop an AI PPI prediction algorithm.

Keywords:  cross‐species prediction; protein–protein interactions; residual neural network; spatial pyramid pooling; structural features

DOI:  https://doi.org/10.1002/prot.26721
In Vivo. 2024 Jul-Aug;38(4):38(4): 1579-1593

Comparative Analysis of the Effect of the BRAF Inhibitor Dabrafenib in 2D and 3D Cell Culture Models of Human Metastatic Melanoma Cells.

David Tovar-Parra, Marion Zammit-Mangion.

   BACKGROUND/AIM: Melanoma, a variant of skin cancer, presents the highest mortality rates among all skin cancers. Despite advancements in targeted therapies, immunotherapies, and tissue culture techniques, the absence of an effective early treatment model remains a challenge. This study investigated the impact of dabrafenib on both 2D and 3D cell culture models with distinct molecular profiles.
MATERIALS AND METHODS: We developed a high-throughput workflow enabling drug screening on spheroids. Our approach involved cultivating 2D and 3D cultures derived from normal melanocytes and metastatic melanoma cells, treating them with dabrafenib and conducting viability, aggregation, migration, cell cycle, and apoptosis assays.
RESULTS: Dabrafenib exerted multifaceted influences, particularly on migration at concentrations of 10 and 25 μM. It induced a decrease in cell viability, impeded cellular adhesion to the matrix, inhibited cellular aggregation and spheroid formation, arrested the cell cycle in the G1 phase, and induced apoptosis.
CONCLUSION: These results confirm the therapeutic potential of dabrafenib in treating melanoma with the BRAF V600E mutation and that 3D models are validated models to study the potential of new molecules for therapeutic purposes. Furthermore, our study underscores the relevance of 3D models in simulating physiological in vivo microenvironments, providing insights into varied treatment responses between normal and tumor cells.

Keywords:  BRAF; Melanoma; dabrafenib; spheroid cellular

DOI:  https://doi.org/10.21873/invivo.13608
Int J Biol Macromol. 2024 Jun 21. pii: S0141-8130(24)04088-1. [Epub ahead of print]274(Pt 1): 133283

Interaction of DisBa01 peptide from Bothrops alternatus venom with BRAF melanoma receptors: Modeling and molecular docking.

Tatiane Senna Bialves, Luana Luiza Bastos, John Alexanders Amaya Parra, Maurício Nogueira Moysés, Edleusa Marques, Adriano Monteiro de Castro Pimenta, Fernando Marques Quintela, Diego César Batista Mariano, Frederico Chaves Carvalho, Raquel C de Melo-Minardi, Robert Tew Boyle.

  Metastatic melanoma is highly aggressive and challenging, often leading to a grim prognosis. Its progression is swift, especially when mutations like BRAFV600E continuously activate pathways vital for cell growth and survival. Although several treatments target this mutation, resistance typically emerges over time. In recent decades, research has underscored the potential of snake venoms and peptides as bioactive substances for innovative drugs, including anti-coagulants, anti-microbial, and anti-cancer agents. Leveraging this knowledge, we propose employing a bioinformatics simulation approach to: a) Predict how well a peptide (DisBa01) from Bothrops alternatus snake venom binds to the melanoma receptor BRAFV600E via Molecular Docking. b) Identify the specific peptide binding sites on receptors and analyze their proximity to active receptor sites. c) Evaluate the behavior of resulting complexes through molecular dynamics simulations. d) Assess whether this peptide qualifies as a candidate for anti-melanoma therapy. Our findings reveal that DisBa01 enhances stability in the BRAFV600E melanoma receptor structure by binding to its RGD motif, an interaction absent in the BRAF WT model. Consequently, both docking and molecular dynamics simulations suggest that DisBa01 shows promise as a BRAFV600E inhibitor.

Keywords:  BRAF; DisBa01; Inhibitor; Melanoma; Snake venom - disintegrin

DOI:  https://doi.org/10.1016/j.ijbiomac.2024.133283
Curr Protein Pept Sci. 2024 ;25(6): 438-442

The Disulfide Bond-Mediated Cyclization of Oral Peptides.

Chenguang Yao, Guoguo Ye, Qin Yang, Zhenwang Chen, Minghui Yang.

  'Structure determines function' is a consensus in the current biological community, but the structural characteristics corresponding to a certain function have always been a hot field of scientific exploration. A peptide is a bio-active molecule that is between the size of an antibody and a small molecule. Still, the gastrointestinal barrier and the physicochemical properties of peptides have always limited the oral administration of peptides. Therefore, we analyze the main ways oral peptide conversion strategies of peptide modification and permeation enhancers. Based on our analysis of the structure of natural oral peptides, which can be absorbed through the gastrointestinal tract, we believe that the design strategy of natural stapled peptides based on disulfide bonds is good for oral peptide design. This cannot only be used to identify anti-gastrointestinal digestive structural proteins in nature but also provide a solid structural foundation for the construction of new oral peptide drugs.

Keywords:  Oral peptides; aglycin; cell-penetration; cyclization; proteins.; vglycin

DOI:  https://doi.org/10.2174/0113892037280719231214095428
Commun Chem. 2024 Jun 27. 7(1): 144

Design of a covalent protein-protein interaction inhibitor of SRPKs to suppress angiogenesis and invasion of cancer cells.

Gongli Cai, Yishu Bao, Qingyun Li, Pang-Hung Hsu, Jiang Xia, Jacky Chi Ki Ngo.

Serine-arginine (SR) proteins are splicing factors that play essential roles in both constitutive and alternative pre-mRNA splicing. Phosphorylation of their C-terminal RS domains by SR protein kinases (SRPKs) regulates their localization and diverse cellular activities. Dysregulation of phosphorylation has been implicated in many human diseases, including cancers. Here, we report the development of a covalent protein-protein interaction inhibitor, C-DBS, that targets a lysine residue within the SRPK-specific docking groove to block the interaction and phosphorylation of the prototypic SR protein SRSF1. C-DBS exhibits high specificity and conjugation efficiency both in vitro and in cellulo. This self-cell-penetrating inhibitor attenuates the phosphorylation of endogenous SR proteins and subsequently inhibits the angiogenesis, migration, and invasion of cancer cells. These findings provide a new foundation for the development of covalent SRPK inhibitors for combatting diseases such as cancer and viral infections and overcoming the resistance encountered by ATP-competitive inhibitors.

DOI: https://doi.org/10.1038/s42004-024-01230-2
Eur Urol Oncol. 2024 Jun 25. pii: S2588-9311(24)00145-7. [Epub ahead of print]

Pembrolizumab plus Abiraterone Acetate and Prednisone in Patients with Chemotherapy-naïve Metastatic Castration-resistant Prostate Cancer: Results from KEYNOTE-365 Cohort D.

Evan Y Yu, Cristiano Ferrario, Mark D Linch, Michael Stoeckle, Brigitte Laguerre, Jose A Arranz, Tilman Todenhöfer, Peter C Fong, Josep M Piulats, William Berry, Urban Emmenegger, Loic Mourey, Anthony M Joshua, Nataliya Mar, Leonard J Appleman, Henry J Conter, Gwenaelle Gravis, Xin Tong Li, Charles Schloss, Christian Poehlein, Johann S de Bono.

   BACKGROUND AND OBJECTIVE: Abiraterone acetate (abiraterone) plus prednisone is approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Our aim was to evaluate the efficacy and safety of pembrolizumab plus abiraterone in mCRPC.
METHODS: In cohort D of the phase 1b/2 KEYNOTE-365 study (NCT02861573), patients were chemotherapy-naïve, had disease progression ≤6 mo before screening, and had either not received prior next-generation hormonal agents for mCRPC or had received prior enzalutamide for mCRPC and had disease progression or became intolerant to enzalutamide. Patients received pembrolizumab 200 mg intravenously every 3 wk plus abiraterone 1000 mg orally once daily and prednisone 5 mg orally twice daily. The primary endpoints were safety, prostate-specific antigen (PSA) response rate, and objective response rate (ORR) according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) by blinded independent central review (BICR). Secondary endpoints included radiographic progression-free survival (rPFS) according to Prostate Cancer Clinical Trials Working Group 3-modified RECIST v1.1 by BICR and overall survival (OS).
KEY FINDINGS AND LIMITATIONS: For the 103 patients who were treated, median follow-up was 28 mo (interquartile range 26-31). The confirmed PSA response rate was 56% (58/103 patients). The ORR for patients with RECIST v1.1-measurable disease was 16% (6/37 patients). Median rPFS was 15 mo (95% confidence interval 9.2-22) and median OS was 30 mo (95% confidence interval 23-not reached); the estimated 24-mo OS rate was 58%. In total, 91% of patients experienced treatment-related adverse events, and 39% experienced grade 3-5 events. Grade 3/4 elevation of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) was observed in 12% and 6.8% of patients, respectively. One patient died due to treatment-related myasthenic syndrome. Study limitations include the single-arm design.
CONCLUSIONS: Pembrolizumab plus abiraterone and prednisone demonstrated antitumor activity and acceptable safety in patients with chemotherapy-naïve mCRPC. Higher incidence of grade 3/4 elevated ALT/AST occurred than was reported for the individual agents.
PATIENT SUMMARY: For patients with metastatic castratation-resistant prostate cancer, the drug combination of pembrolizumab plus abiraterone and prednisone showed antitumor activity and acceptable safety.

Keywords:  Abiraterone acetate; Metastatic castration-resistant prostate cancer; Pembrolizumab

DOI:  https://doi.org/10.1016/j.euo.2024.05.013
Cell. 2024 Jun 23. pii: S0092-8674(24)00631-7. [Epub ahead of print]

Preclinical proof of principle for orally delivered Th17 antagonist miniproteins.

Stephanie Berger, Franziska Seeger, Ta-Yi Yu, Merve Aydin, Huilin Yang, Daniel Rosenblum, Laure Guenin-Macé, Caleb Glassman, Lauren Arguinchona, Catherine Sniezek, Alyssa Blackstone, Lauren Carter, Rashmi Ravichandran, Maggie Ahlrichs, Michael Murphy, Ingrid Swanson Pultz, Alex Kang, Asim K Bera, Lance Stewart, K Christopher Garcia, Shruti Naik, Jamie B Spangler, Florian Beigel, Matthias Siebeck, Roswitha Gropp, David Baker.

  Interleukin (IL)-23 and IL-17 are well-validated therapeutic targets in autoinflammatory diseases. Antibodies targeting IL-23 and IL-17 have shown clinical efficacy but are limited by high costs, safety risks, lack of sustained efficacy, and poor patient convenience as they require parenteral administration. Here, we present designed miniproteins inhibiting IL-23R and IL-17 with antibody-like, low picomolar affinities at a fraction of the molecular size. The minibinders potently block cell signaling in vitro and are extremely stable, enabling oral administration and low-cost manufacturing. The orally administered IL-23R minibinder shows efficacy better than a clinical anti-IL-23 antibody in mouse colitis and has a favorable pharmacokinetics (PK) and biodistribution profile in rats. This work demonstrates that orally administered de novo-designed minibinders can reach a therapeutic target past the gut epithelial barrier. With high potency, gut stability, and straightforward manufacturability, de novo-designed minibinders are a promising modality for oral biologics.

Keywords:  IL-17; IL-23R; Th17; autoinflammation; computational protein design; inflammatory bowel disease; oral biologics; protein engineering

DOI:  https://doi.org/10.1016/j.cell.2024.05.052
Biomedicines. 2024 May 22. pii: 1144. [Epub ahead of print]12(6):

Poptosis or Peptide-Induced Transmembrane Pore Formation: A Novel Way to Kill Cancer Cells without Affecting Normal Cells.

Matthew R Pincus, Miriam Silberstein, Nitzan Zohar, Ehsan Sarafraz-Yazdi, Wilbur B Bowne.

  Recent advances in cancer treatment like personalized chemotherapy and immunotherapy are aimed at tumors that meet certain specifications. In this review, we describe a new approach to general cancer treatment, termed peptide-induced poptosis, in which specific peptides, e.g., PNC-27 and its shorter analogue, PNC-28, that contain the segment of the p53 transactivating 12-26 domain that bind to HDM-2 in its 1-109 domain, bind to HDM-2 in the membranes of cancer cells, resulting in transmembrane pore formation and the rapid extrusion of cancer cell contents, i.e., tumor cell necrosis. These peptides cause tumor cell necrosis of a wide variety of solid tissue and hematopoietic tumors but have no effect on the viability and growth of normal cells since they express at most low levels of membrane-bound HDM-2. They have been found to successfully treat a highly metastatic pancreatic tumor as well as stem-cell-enriched human acute myelogenous leukemias in nude mice, with no evidence of off-target effects. These peptides also are cytotoxic to chemotherapy-resistant cancers and to primary tumors. We performed high-resolution scanning immuno-electron microscopy and visualized the pores in cancer cells induced by PNC-27. This peptide forms 1:1 complexes with HDM-2 in a temperature-independent step, followed by dimerization of these complexes to form transmembrane channels in a highly temperature-dependent step parallel to the mode of action of other membranolytic but less specific agents like streptolysin. These peptides therefore may be effective as general anti-cancer agents.

Keywords:  PNC-27; PNC-27–HDM-2 membrane complexes; transmembrane pore formation; tumor cell necrosis

DOI:  https://doi.org/10.3390/biomedicines12061144
Acta Pharmacol Sin. 2024 Jun 26.

Structural insights into somatostatin receptor 5 bound with cyclic peptides.

Ying-Ge Li, Xian-Yu Meng, Xiru Yang, Sheng-Long Ling, Pan Shi, Chang-Lin Tian, Fan Yang.

  Somatostatin receptor 5 (SSTR5) is highly expressed in ACTH-secreting pituitary adenomas and is an important drug target for the treatment of Cushing's disease. Two cyclic SST analog peptides (pasireotide and octreotide) both can activate SSTR5 and SSTR2. Pasireotide is preferential binding to SSTR5 than octreotide, while octreotide is biased to SSTR2 than SSTR5. The lack of selectivity of both pasireotide and octreotide causes side effects, such as hyperglycemia, gastrointestinal disturbance, and abnormal glucose homeostasis. However, little is known about the binding and selectivity mechanisms of pasireotide and octreotide with SSTR5, limiting the development of subtype-selective SST analog drugs specifically targeting SSTR5. Here, we report two cryo-electron microscopy (cryo-EM) structures of SSTR5-Gi complexes activated by pasireotide and octreoitde at resolutions of 3.09 Å and 3.24 Å, respectively. In combination with structural analysis and functional experiments, our results reveal the molecular mechanisms of ligand recognition and receptor activation. We also demonstrate that pasireotide preferentially binds to SSTR5 through the interactions between Tyr(Bzl)/DTrp of pasireotide and SSTR5. Moreover, we find that the Q2.63, N6.55, F7.35 and ECL2 of SSTR2 play a crucial role in octreotide biased binding of SSTR2. Our results will provide structural insights and offer new opportunities for the drug discovery of better selective pharmaceuticals targeting specific SSTR subtypes.

Keywords:  G protein-coupled receptors; Somatostatin receptor 5; cryo-EM; octreotide; pasireotide

DOI:  https://doi.org/10.1038/s41401-024-01314-8
ACS Meas Sci Au. 2024 Jun 19. 4(3): 260-266

Automated Analysis of Extracellular Matrix Invasion of Cancer Cells from Tumor Spheroids.

Jacob Heiss, Hossein Tavana.

The main cause of mortality among cancer patients is metastatic disease. Metastasis develops from cancer cells that invade the stromal tissue and intravasate the circulatory or lymphatic systems to eventually form new tumors in other organs. Blocking cancer cell invasion can potentially prevent or reduce the metastatic progression of cancers. Testing different chemical compounds against cell invasion in three-dimensional cultures is a common laboratory technique. The efficacy of the treatments is often evaluated from confocal microscopic images of the cells using image processing. However, the analysis approaches are often subject to variations and inconsistencies due to user decisions that must be made while processing each image. To overcome this limitation, we developed a fully automated method to quantify the invasion of cancer cells from a 3D tumor spheroid into the surrounding extracellular matrix. We demonstrated that this method resolves cell invasion from spheroids of different shapes and sizes and from cells that invade as a cluster or individually. We also showed that this approach can help quantify the dose-dependent anti-invasive effects of a commonly used chemotherapy drug. Our automated method significantly reduces the time and increases the consistency and accuracy of cancer cell invasion analysis in three-dimensional cultures.

DOI: https://doi.org/10.1021/acsmeasuresciau.3c00064
J Immunol Methods. 2024 Jun 25. pii: S0022-1759(24)00100-5. [Epub ahead of print] 113715

A real-time antibody-dependent cellular phagocytosis assay by live cell imaging.

Yongchang Shi, Yonglian Sun, Akiko Seki, Sascha Rutz, James T Koerber, Jianyong Wang.

  Antibody-dependent cellular phagocytosis (ADCP) is a cellular process by which antibody-opsonized targets (pathogens or cells) activate the Fc receptors on the surface of phagocytes to induce phagocytosis, resulting in internalization and degradation of pathogens or target cells through phagosome acidification. Besides NK cells-mediated antibody-dependent cellular cytotoxicity (ADCC), tumor-infiltrated monocytes and macrophages can directly kill tumor cells in the presence of tumor antigen-specific antibodies through ADCP, representing another attractive strategy for cancer immunotherapy. Even though several methods have been developed to measure ADCP, an automated and high-throughput quantitative assay should offer highly desirable advantages for drug discovery. In this study we established a new ADCP assay to identify therapeutical monoclonal antibodies (mAbs) that facilitate macrophages phagocytosis of live target cells. We used Incucyte, an imaging system for live cell analysis. By labeling the live target cells with a pH sensitive dye (pHrodo), we successfully monitored the ADCP in real time. We demonstrated that our image-based assay is robust and quantitative, suitable for screening and characterization of therapeutical mAbs that directly kill target cells through ADCP. Furthermore, we found different subtypes of macrophages have distinct ADCP activities using both mouse and human primary macrophages differentiated in vitro. By studying various mAbs with mutations in their Fc regions using our assay, we showed that the variants with increased binding to Fc gamma receptors (FcγRs) have enhanced ADCP activities.

Keywords:  Antibody-dependent cellular phagocytosis (ADCP); Cancer immunotherapy; Macrophage; Phagocytosis

DOI:  https://doi.org/10.1016/j.jim.2024.113715
J Med Chem. 2024 Jun 27.

Design of Proteolytic-Resistant Antifungal Peptides by Utilizing Minimum d-Amino Acid Ratios.

Zhenheng Lai, Xiaojie Yuan, Wenwen Chen, Hongyu Chen, Bowen Li, Zhongpeng Bi, Yinfeng Lyu, Anshan Shan.

Antifungal peptides are an appealing alternative to standard antifungal medicines due to their unique mechanism of action and low-level resistance. However, their susceptibility to protease degradation keeps hindering their future development. Herein, a library was established to design peptides with protease resistance and high antifungal activity. The peptides were incorporated with minimal D-amino acids to further improve the protease stability. The most active peptide, IR3, demonstrated good antifungal activity and low toxicity, and its molecular integrity was maintained after protease hydrolysis for 8 h at 2 mg/mL. Furthermore, IR3 could permeate the fungal cell wall, disrupt the cell membrane, produce reactive oxygen species, and induce apoptosis in fungal cells. In vivo experiments confirmed that IR3 could effectively treat fungal keratitis. Collectively, these findings suggest that IR3 is a promising antifungal agent and may be beneficial in the design and development of protease-resistant antifungal peptides.

DOI: https://doi.org/10.1021/acs.jmedchem.4c00394