bims-cepepe 2024-10-06 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2024‒10‒06
sixteen papers selected by
Henry Lamb, Queensland University of Technology

Design and Biophysical Characterization of Second-Generation cyclic peptide LAG-3 inhibitors for cancer immunotherapy.
Computational Design and Optimization of Peptide Inhibitors for SIRT2.
Histidine-bridged cyclic peptide natural products: isolation, biosynthesis and synthetic studies.
Protocol for Designing De Novo Noncanonical Peptide Binders in OSPREY.
Structural insights into molecular-targeting helix-loop-helix peptide against vascular endothelial growth factor-A.
Fluorescent Labeling Can Significantly Perturb Measured Binding Affinity and Selectivity of Peptide-Protein Interactions.
Designer pseudopeptides: autofluorescent polygonal tubes via Phe-zipper and triple helix.
HemoFuse: multi-feature fusion based on multi-head cross-attention for identification of hemolytic peptides.
A CYCLIC PEPTIDE TARGETS GLIOBLASTOMA BY BINDING TO ABERRANTLY EXPOSED SNAP25.
Mammalian Esterase Activity: Implications for Peptide Prodrugs.
VDAC1-Based Peptides as Potential Modulators of VDAC1 Interactions with Its Partners and as a Therapeutic for Cancer, NASH, and Diabetes.
Preclinical Evaluation of 226Ac as a Theranostic Agent: Imaging, Dosimetry, and Therapy.
Structure and functional studies of Avt1, a novel peptide from the sea anemone Aulactinia veratra.
Optimization of Potent, Efficacious, Selective and Blood-Brain Barrier Penetrating Inhibitors Targeting EGFR Exon20 Insertion Mutations.
Probing Deuteration-Induced Phase Separation in Supported Lipid Monolayers using Hyperspectral TERS Imaging.
Exploration of the tunability of BRD4 degradation by DCAF16 trans-labelling covalent glues.

Bioorg Med Chem Lett. 2024 Sep 26. pii: S0960-894X(24)00381-0. [Epub ahead of print]113 129979

Design and Biophysical Characterization of Second-Generation cyclic peptide LAG-3 inhibitors for cancer immunotherapy.

Laura Calvo-Barreiro, Longfei Zhang, Yasir Ali, Ashfaq Ur Rehman, Moustafa Gabr.

  Lymphocyte activation gene 3 (LAG-3) is an inhibitory immune checkpoint crucial for suppressing the immune response against cancer. Blocking LAG-3 interactions enables T cells to recover their cytotoxic capabilities and diminishes the immunosuppressive effects of regulatory T cells. A cyclic peptide (Cys-Val-Pro-Met-Thr-Tyr-Arg-Ala-Cys, disulfide bridge: 1-9) was recently reported as a LAG-3 inhibitor. Based on this peptide, we designed 19 derivatives by substituting tyrosine residue to maximize LAG-3 inhibition. Screening via TR-FRET assay identified 8 outperforming derivatives, with cyclic peptides 12 [Tyr6(L-3-CN-Phe)], 13 [Tyr6(L-4-NH2-Phe)], and 17 [Tyr6(L-3,5-DiF-Phe)] as top candidates. Cyclic peptide 12 exhibited the highest inhibition (IC50 = 4.45 ± 1.36 µM). MST analysis showed cyclic peptides 12 and 13 bound LAG-3 with KD values of 2.66 ± 2.06 µM and 1.81 ± 1.42 µM, respectively, surpassing the original peptide (9.94 ± 4.13 µM). Docking simulations revealed that cyclic peptide 12 exhibited significantly enhanced binding, with a docking score of -7.236 kcal/mol, outperforming the original peptide (-5.236 kcal/mol) and cyclic peptide 5 (L-4-CN-Phe) (-5.131 kcal/mol). A per-residue decomposition of the interaction energy indicated that the 3-cyano group in cyclic peptide 12 contributes to a more favorable conformation, yielding an interaction energy of -9.22 kcal/mol with Phe443 of MHC-II, compared to -6.03 kcal/mol and -5.619 kcal/mol for cyclic peptides 0 and 5, respectively. Despite promising in vitro results, cyclic peptide 12 failed to inhibit tumor growth in vivo, underscoring the importance of dual immunotherapies targeting several immune checkpoints to achieve anti-tumor efficacy.

Keywords:  Cancer immunotherapy; Computational chemistry; Cyclic peptides; Drug discovery; Lymphocyte-activation gene 3

DOI:  https://doi.org/10.1016/j.bmcl.2024.129979
Pharmaceuticals (Basel). 2024 Aug 24. pii: 1120. [Epub ahead of print]17(9):

Computational Design and Optimization of Peptide Inhibitors for SIRT2.

Heba A Alkhatabi, Fatmah M A Naemi, Reem Alsolami, Hisham N Alatyb.

  Sirtuin 2 (SIRT2), an NAD+-dependent deacetylase, is crucial for regulating vital physiological processes, including aging, DNA repair, and cell cycle progression. Its abnormal activity is linked to diseases such as Parkinson's disease, cancer, and metabolic disorders, making it a potential target for therapeutic intervention. While small molecule inhibitors have been studied, peptide-based inhibitors offer a promising alternative due to their selectivity and bioavailability. This study explores the effects of converting the naturally occurring cyclic inhibitor peptide of SIRT2 (S2iL5) into a non-cyclic form by replacing a residue with FAK (LYS + CF3CO-). The new peptide sequence, Tyr-His-Thr-Tyr-His-Val-FAK (LYS)-Arg-Arg-Thr-Asn-Tyr-Tyr-Cys, was modeled to confirm its stable conformation. Docking studies and MM/GBSA calculations showed that the non-cyclic peptide had a better binding free energy (-50.66 kcal/mol) compared to the cyclic S2iL5 (-49.44 kcal/mol). Further mutations generated 160,000 unique peptides, screened using a machine learning-based QSAR model. Three promising peptides (Peptide 1: YGGNNVKRRTNYYC, Peptide 2: YMGEWVKRRTNYYC, and Peptide 3: YGGNGVKRRTNYYC) were selected and further modeled. Molecular dynamics (MD) analyses demonstrated that Peptide 1 and Peptide 2 had significant potential as SIRT2 inhibitors, showing moderate stability and some structural flexibility. Their best binding free energies were -59.07 kcal/mol and -46.01 kcal/mol, respectively. The study aimed to enhance peptide flexibility and binding affinity, suggesting that optimized peptide-based inhibitors can interact effectively with SIRT2. However, further experimental validation is necessary to confirm these computational predictions and evaluate the therapeutic potential of the identified peptides.

Keywords:  SIRT2 inhibitors; binding free energy calculations; computational biology; cyclic peptides; molecular dynamics simulation

DOI:  https://doi.org/10.3390/ph17091120
Org Biomol Chem. 2024 Oct 01.

Histidine-bridged cyclic peptide natural products: isolation, biosynthesis and synthetic studies.

Pascal M Engelhardt, Robert Keyzers, Margaret A Brimble.

The histidine bridge is a rare and often overlooked structural motif in macrocyclic peptide natural products, yet there are several examples in nature of cyclic peptides bearing this moiety that exhibit potent biological activity. These interesting compounds have been the focus of several studies reporting their isolation, biosynthesis and chemical synthesis over the last four decades. This review summarises the findings on the structure, biological activity and, where possible, proposed biosynthesis and progress towards the synthesis of histidine-bridged cyclic peptides.

DOI: https://doi.org/10.1039/d4ob01259c
J Comput Biol. 2024 Oct 04.

Protocol for Designing De Novo Noncanonical Peptide Binders in OSPREY.

Henry Childs, Nathan Guerin, Pei Zhou, Bruce R Donald.

  D-peptides, the mirror image of canonical L-peptides, offer numerous biological advantages that make them effective therapeutics. This article details how to use DexDesign, the newest OSPREY-based algorithm, for designing these D-peptides de novo. OSPREY physics-based models precisely mimic energy-equivariant reflection operations, enabling the generation of D-peptide scaffolds from L-peptide templates. Due to the scarcity of D-peptide:L-protein structural data, DexDesign calls a geometric hashing algorithm, Method of Accelerated Search for Tertiary Ensemble Representatives, as a subroutine to produce a synthetic structural dataset. DexDesign enables mixed-chirality designs with a new user interface and also reduces the conformation and sequence search space using three new design techniques: Minimum Flexible Set, Inverse Alanine Scanning, and K*-based Mutational Scanning.

Keywords:  D-peptides; OSPREY; de novo peptide design; protein:ligand binding; protocol

DOI:  https://doi.org/10.1089/cmb.2024.0669
Biochem Biophys Res Commun. 2024 Sep 27. pii: S0006-291X(24)01285-3. [Epub ahead of print]734 150749

Structural insights into molecular-targeting helix-loop-helix peptide against vascular endothelial growth factor-A.

Masataka Michigami, Kunpei Notsu, Masayuki Kamo, Takatsugu Hirokawa, Takayoshi Kinoshita, Koji Inaka, Ikuhiko Nakase, Ikuo Fujii.

  Mid-sized binding peptides have recently emerged as a new therapeutic modality. A helix-loop-helix (HLH) peptide was designed as a scaffold for combinatorial peptide libraries. We screened the HLH peptide libraries against human vascular endothelial growth factor-A (VEGF) to generate a peptide, VS42-LR3, which inhibited VEGF/receptor interaction and suppressed tumor growth in a murine xenograft model of human colorectal cancer. Here, we report the first crystal structure of the HLH peptide in a complex with VEGF at high resolution using space-grown protein crystals. The X-ray structural analysis revealed that the monomeric VS42-LR3 adopted an HLH structure and bound to VEGF at the VEGF receptor-binding site. Interestingly, from the site-directed mutagenesis, thermodynamic analysis, and molecular dynamic simulations, it turned out that the loop region in the non-interacting surface to VEGF affected the structural rigidity of the whole HLH to increase the binding affinity. These findings provide valuable insights for the design of more structurally stable and higher affinity mid-sized binding peptides as well as HLH peptides, that could play a crucial role in advancing molecular-targeting therapies.

Keywords:  Helix–loop–helix; VEGF; X-ray crystal structure

DOI:  https://doi.org/10.1016/j.bbrc.2024.150749
J Phys Chem Lett. 2024 Oct 03. 10252-10257

Fluorescent Labeling Can Significantly Perturb Measured Binding Affinity and Selectivity of Peptide-Protein Interactions.

Sara Bobone, Claudia Storti, Chiara Fulci, Alessia Damiani, Chiara Innamorati, Daniela Roversi, Paolo Calligari, Luca Pannone, Simone Martinelli, Marco Tartaglia, Gianfranco Bocchinfuso, Fernando Formaggio, Cristina Peggion, Barbara Biondi, Lorenzo Stella.

Peptide-based drugs are powerful inhibitors of therapeutically relevant protein-protein interactions. Their affinity and selectivity for target proteins are commonly assessed using fluorescence-based assays such as anisotropy/polarization or quantitative microarrays. This study reveals that labeling can perturb peptide/protein binding by more than 1 order of magnitude. We have recently developed inhibitors targeted to the N-terminal Src homology 2 (SH2) domain of oncogenic phosphatase SHP2. Despite their high activity and selectivity, these molecules demonstrated an undesired interaction with the SH2 domain of another protein, known as APS, in a fluorescence microarray assay. Fluorescence anisotropy measurement in solution showed that the dissociation constant was significantly influenced by labeling (∼10 times), and the effect depended on the specific fluorophore and SH2 domain. Notably, displacement assays performed with unlabeled peptides were successfully used to eliminate these artifacts, demonstrating that the inhibitors' affinity for their target is over 1,000 times higher than for APS.

DOI: https://doi.org/10.1021/acs.jpclett.4c01767
Chem Sci. 2024 Sep 18.

Designer pseudopeptides: autofluorescent polygonal tubes via Phe-zipper and triple helix.

V Haridas, Govind P Maurya, Souvik Dutta.

Chemists are increasingly turning to biology for inspiration to develop novel and superior synthetic materials. Here, we present an innovative peptide design strategy for tubular assembly. In this simple design, a phenylene urea unit is introduced as an aglet at the N-terminus of the peptide. When α-amino isobutyric acid (Aib) is the first residue and phenylalanine (Phe) is the second residue from the phenylene urea entity, it induces an edge-to-face π-π interaction resulting in a turn conformation. The peptides with a unique reverse turn conformation associate to form polygonal peptide tubes via a Phe-zipper arrangement, as evidenced by microscopic and single crystal X-ray studies. Ultra-microscopic imaging revealed that the tubular assembly is hexagonal, square, and triangular in shape. This hierarchical assembly reveals the interplay between π-π interactions and hydrogen bonding. In another design, pseudopeptide 5, wherein a Phe-Phe (FF) unit is linked to phenylene urea, formed polygonal tubes via a triple helical arrangement. Interestingly, the extension of this design to the bis-urea core resulted in vesicular assembly. These supramolecular polygonal tubes and vesicles showed autofluorescence, which allowed confocal imaging. The observed fluorescence is an additional advantage for applications in biological and medical sciences.

DOI: https://doi.org/10.1039/d4sc04023f
Sci Rep. 2024 09 28. 14(1): 22518

HemoFuse: multi-feature fusion based on multi-head cross-attention for identification of hemolytic peptides.

Ya Zhao, Shengli Zhang, Yunyun Liang.

  Hemolytic peptides are therapeutic peptides that damage red blood cells. However, therapeutic peptides used in medical treatment must exhibit low toxicity to red blood cells to achieve the desired therapeutic effect. Therefore, accurate prediction of the hemolytic activity of therapeutic peptides is essential for the development of peptide therapies. In this study, a multi-feature cross-fusion model, HemoFuse, for hemolytic peptide identification is proposed. The feature vectors of peptide sequences are transformed by word embedding technique and four hand-crafted feature extraction methods. We apply multi-head cross-attention mechanism to hemolytic peptide identification for the first time. It captures the interaction between word embedding features and hand-crafted features by calculating the attention of all positions in them, so that multiple features can be deeply fused. Moreover, we visualize the features obtained by this module to enhance its interpretability. On the comprehensive integrated dataset, HemoFuse achieves ideal results, with ACC, SP, SN, MCC, F1, AUC, and AP of 0.7575, 0.8814, 0.5793, 0.4909, 0.6620, 0.8387, and 0.7118, respectively. Compared with HemoDL proposed by Yang et al., it is 3.32%, 3.89%, 5.93%, 10.6%, 8.17%, 5.88%, and 2.72% higher. Other ablation experiments also prove that our model is reasonable and efficient. The codes and datasets are accessible at https://github.com/z11code/Hemo .

Keywords:  Feature fusion; Hemolytic peptides; Multi-head cross-attention mechanism; Transformer

DOI:  https://doi.org/10.1038/s41598-024-74326-3
bioRxiv. 2024 Sep 21. pii: 2024.09.18.613627. [Epub ahead of print]

A CYCLIC PEPTIDE TARGETS GLIOBLASTOMA BY BINDING TO ABERRANTLY EXPOSED SNAP25.

Alberto G Arias, Laura Tovar-Martinez, Eliana K Asciutto, Aman Mann, Kristina Posnograjeva, Lorena Simon Gracia, Miriam Royo, Maarja Haugas, Tambet Teesalu, Cristian Smulski, Erkki Ruoslahti, Pablo Scodeller.

Disease-specific changes in tumors and other diseased tissues are an important target of research because they provide clues on the pathophysiology of the disease as well as uncovering potentially useful markers for diagnosis and treatment. Here, we report a new cyclic peptide, CESPLLSEC (CES), that specifically accumulated (homed) in intracranial U87MG and the WT-GBM model of glioblastoma from intravenous (IV) injection, associating with the vasculature. Affinity chromatography of U87MG tumor extracts on insolubilized CES peptide identified Synaptosomal Associated Protein 25 (SNAP25) as a candidate target molecule (receptor) for CES. Several results supported the identification of SNAP25 as the CES receptor. IV-injected FAM-CES colocalized with SNAP25 in the tumors, and direct binding studies showed specific CES peptide binding to recombinant human SNAP25. A CES peptide-drug conjugate designed for photodynamic therapy showed selective cytotoxicity to SNAP25+ glioblastoma cell lines. Specific accumulation of systemically injected anti-SNAP25 antibody in U87MG glioblastoma, and labeling of intact U87MG cells with anti-SNAP in flow cytometry showed that SNAP25 is available from the circulation but not in normal tissues and that it is present at the cell surface. Using an array of ECM proteins and surface plasmon resonance revealed that SNAP25 binds moderately to collagen V and strongly to collagen VI. Modeling studies suggested that CES and collagen VI compete for the same binding site on SNAP25. Our results introduce CES as a valuable targeting peptide for drug delivery, and its receptor SNAP25 as a possible molecular marker of interest for glioblastoma.

DOI: https://doi.org/10.1101/2024.09.18.613627
Biochemistry. 2024 Oct 03.

Mammalian Esterase Activity: Implications for Peptide Prodrugs.

Yana D Petri, Ruben Verresen, Clair S Gutierrez, Volga Kojasoy, Erika Zhang, Nile S Abularrage, Evans C Wralstad, Kaya R Weiser, Ronald T Raines.

As a traceless, bioreversible modification, the esterification of carboxyl groups in peptides and proteins has the potential to increase their clinical utility. An impediment is the lack of strategies to quantify esterase-catalyzed hydrolysis rates for esters in esterified biologics. We have developed a continuous Förster resonance energy transfer (FRET) assay for esterase activity based on a peptidic substrate and a protease, Glu-C, that cleaves a glutamyl peptide bond only if the glutamyl side chain is a free acid. Using pig liver esterase (PLE) and human carboxylesterases, we validated the assay with substrates containing simple esters (e.g., ethyl) and esters designed to be released by self-immolation upon quinone methide elimination. We found that simple esters were not cleaved by esterases, likely for steric reasons. To account for the relatively low rate of quinone methide elimination, we extended the mathematics of the traditional Michaelis-Menten model to conclude with a first-order intermediate decay step. By exploring two regimes of our substrate → intermediate → product (SIP) model, we evaluated the rate constants for the PLE-catalyzed cleavage of an ester on a glutamyl side chain (kcat/KM = 1.63 × 103 M-1 s-1) and subsequent spontaneous quinone methide elimination to regenerate the unmodified peptide (kI = 0.00325 s-1; t1/2 = 3.55 min). The detection of esterase activity was also feasible in the human intestinal S9 fraction. Our assay and SIP model increase the understanding of the release kinetics of esterified biologics and facilitate the rational design of efficacious peptide prodrugs.

DOI: https://doi.org/10.1021/acs.biochem.4c00446
Biomolecules. 2024 Sep 09. pii: 1139. [Epub ahead of print]14(9):

VDAC1-Based Peptides as Potential Modulators of VDAC1 Interactions with Its Partners and as a Therapeutic for Cancer, NASH, and Diabetes.

Anna Shteinfer-Kuzmine, Manikandan Santhanam, Varda Shoshan-Barmatz.

  This review presents current knowledge related to the voltage-dependent anion channel-1 (VDAC1) as a multi-functional mitochondrial protein that acts in regulating both cell life and death. The location of VDAC1 at the outer mitochondrial membrane (OMM) allows control of metabolic cross-talk between the mitochondria and the rest of the cell, and also enables its interaction with proteins that are involved in metabolic, cell death, and survival pathways. VDAC1's interactions with over 150 proteins can mediate and regulate the integration of mitochondrial functions with cellular activities. To target these protein-protein interactions, VDAC1-derived peptides have been developed. This review focuses specifically on cell-penetrating VDAC1-based peptides that were developed and used as a "decoy" to compete with VDAC1 for its VDAC1-interacting proteins. These peptides interfere with VDAC1 interactions, for example, with metabolism-associated proteins such as hexokinase (HK), or with anti-apoptotic proteins such as Bcl-2 and Bcl-xL. These and other VDAC1-interacting proteins are highly expressed in many cancers. The VDAC1-based peptides in cells in culture selectively affect cancerous, but not non-cancerous cells, inducing cell death in a variety of cancers, regardless of the cancer origin or genetics. They inhibit cell energy production, eliminate cancer stem cells, and act very rapidly and at low micro-molar concentrations. The activity of these peptides has been validated in several mouse cancer models of glioblastoma, lung, and breast cancers. Their anti-cancer activity involves a multi-pronged attack targeting the hallmarks of cancer. They were also found to be effective in treating non-alcoholic fatty liver disease and diabetes mellitus. Thus, VDAC1-based peptides, by targeting VDAC1-interacting proteins, offer an affordable and innovative new conceptual therapeutic paradigm that can potentially overcome heterogeneity, chemoresistance, and invasive metastatic formation.

Keywords:  VDAC1; apoptosis; cancer; mitochondria; peptide

DOI:  https://doi.org/10.3390/biom14091139
J Nucl Med. 2024 Oct 03. pii: jnumed.124.267999. [Epub ahead of print]

Preclinical Evaluation of 226Ac as a Theranostic Agent: Imaging, Dosimetry, and Therapy.

Helena Koniar, Luke Wharton, Aidan Ingham, Ana Paulina Morales Oliver, Helen Merkens, Cristina Rodríguez-Rodríguez, Peter Kunz, Valery Radchenko, Hua Yang, Arman Rahmim, Carlos Uribe, Paul Schaffer.

  226Ac (t½ = 29.37 h) has been proposed as a theranostic radioisotope leveraging both its diagnostic γ-emissions and therapeutic α-emissions. 226Ac emits 158 and 230 keV γ-photons ideal for quantitative SPECT imaging and acts as an in vivo generator of 4 high-energy α-particles. Because of these nuclear decay properties, 226Ac has potential to act as a standalone theranostic isotope. In this proof-of-concept study, we evaluated a preclinical 226Ac-radiopharmaceutical for its theranostic efficacy and present the first 226Ac-targeted α-therapy study. Methods: 226Ac was produced at TRIUMF and labeled with the chelator-peptide bioconjugate crown-TATE. [226Ac]Ac-crown-TATE was selected to target neuroendocrine tumors in male NRG mice bearing AR42J tumor xenografts for SPECT imaging, biodistribution, and therapy studies. A preclinical SPECT/CT scanner acquired quantitative images reconstructed from both the 158 and the 230 keV emissions. Mice in the biodistribution study were euthanized at 1, 3, 5, 24, and 48 h after injection, and internal radiation dosimetry was derived for the tumor and organs of interest to establish appropriate therapeutic activity levels. Mice in the therapy study were administered 125, 250, or 375 kBq treatments and were monitored for tumor size and body condition. Results: We present quantitative SPECT images of the in vivo biodistribution of [226Ac]Ac-crown-TATE, which showed agreement with ex vivo measurements. Biodistribution studies demonstrated high uptake (>30%IA/g at 5 h after injection) and retention in the tumor, with an estimated mean absorbed dose coefficient of 222 mGy/kBq. [226Ac]Ac-crown-TATE treatments significantly extended the median survival from 7 d in the control groups to 16, 24, and 27 d in the 125, 250, and 375 kBq treatment groups, respectively. Survival was prolonged by slowing tumor growth, and no weight loss or toxicities were observed. Conclusion: This study highlights the theranostic potential of 226Ac as a standalone therapeutic isotope in addition to its demonstrated diagnostic capabilities to assess dosimetry in matched 225Ac-radiopharmaceuticals. Future studies will investigate maximum dose and toxicity to further explore the therapeutic potential of 226Ac-radiopharmaceuticals.

Keywords:  226Ac; SPECT imaging; preclinical radiopharmaceutical; targeted α-therapy; theranostics

DOI:  https://doi.org/10.2967/jnumed.124.267999
Biochim Biophys Acta Proteins Proteom. 2024 Sep 30. pii: S1570-9639(24)00057-8. [Epub ahead of print] 141050

Structure and functional studies of Avt1, a novel peptide from the sea anemone Aulactinia veratra.

Renad A Albar, Hayden L Smith, Karoline Sanches, Dorothy C C Wai, Muhammad Umair Naseem, Tibor G Szanto, Gyorgy Panyi, Peter J Prentis, Raymond S Norton.

  Sea anemones are a rich source of peptide toxins spanning a diverse range of biological activities, typically targeting proteins such as ion channels, receptors and transporters. These peptide toxins and their analogues are usually highly stable and selective for their molecular targets, rendering them of interest as molecular tools, insecticides and therapeutics. Recent transcriptomic and proteomic analyses of the sea anemone Aulactinia veratra identified a novel 28-residue peptide, named Avt1. Avt1 was produced using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The liquid chromatography-mass spectrometry profile of synthetic Avt1 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds. The solution structure determined by NMR revealed that Avt1 adopts an inhibitor cystine knot (ICK) fold, in which a ring is formed by two disulfide bonds with a third disulfide penetrating the ring to create the pseudo-knot. This structure provides ICK peptides with high structural, thermal and proteolytic stability. Consistent with its ICK structure, Avt1 was resistant to proteolysis by trypsin, chymotrypsin and pepsin, although it was not a trypsin inhibitor. Avt1 at 100 nM showed no activity in patch-clamp electrophysiological assays against several mammalian voltage-gated ion channels, but has structural features similar to toxins targeting insect sodium ion channels. Although sequence homologues of Avt1 are found in a number of sea anemones, this is the first representative of this family to be characterised structurally and functionally.

Keywords:  Cysteine-rich peptide; ICK fold; Ion channels; NMR spectroscopy; Sea anemone

DOI:  https://doi.org/10.1016/j.bbapap.2024.141050
J Med Chem. 2024 Sep 28.

Optimization of Potent, Efficacious, Selective and Blood-Brain Barrier Penetrating Inhibitors Targeting EGFR Exon20 Insertion Mutations.

Clare Thomson, Erin Braybrooke, Nicola Colclough, Nichola L Davies, Nicolas Floc'h, Ryan Greenwood, Carine Guérot, David Hargreaves, Peter Johnstrom, Puneet Khurana, Demetrios H Kostomiris, Songlei Li, Andrew Lister, Olivier Lorthioir, Scott Martin, William McCoull, Neville J McLean, Lisa McWilliams, Jonathan P Orme, Martin J Packer, Stuart Pearson, Aisha M Swaih, Sharon Tentarelli, Michael J Tucker, Richard A Ward, Stephen Wilkinson, Poppy Winlow, Isabel L Wood.

Herein, we report the optimization of a series of epidermal growth factor receptor (EGFR) Exon20 insertion (Ex20Ins) inhibitors using structure-based drug design (SBDD), leading to the discovery of compound 28, a potent and wild type selective molecule, which demonstrates efficacy in multiple EGFR Ex20Ins xenograft models and blood-brain barrier penetration in preclinical species. Building on our earlier discovery of an in vivo probe, SBDD was used to design a novel bicyclic core with a lower molecular weight to facilitate blood-brain barrier penetration. Further optimization including strategic linker replacement and diversification of the ring system interacting with the c-helix enabled photolytic and metabolic stability improvements. Together with refinement of molecular properties important for achieving high brain exposure, including molecular weight, H-bonding, and polarity, 28 was identified.

DOI: https://doi.org/10.1021/acs.jmedchem.4c01792
J Phys Chem Lett. 2024 Oct 02. 10237-10243

Probing Deuteration-Induced Phase Separation in Supported Lipid Monolayers using Hyperspectral TERS Imaging.

Chengcheng Xu, Timon Käser, Yuanzhi Xia, Naresh Kumar, Renato Zenobi.

In this study, we investigate the impact of deuteration on the formation of phase-separated domains in supported lipid monolayers using hyperspectral Tip-Enhanced Raman Spectroscopy (TERS) imaging. The intricate organization of biological membranes plays a crucial role in cellular functions. Various factors that influence domain formation have been identified in previous studies such as lipid tail length and cholesterol concentration. Deuterium labeling of lipids has proven useful for probing cellular structures and dynamics, but its impact on lipid phase separation remains underexplored. By examining 1:1 mixed monolayers of dipalmitoylphosphatidylcholine (DPPC) and deuterated DPPC on Au(111) surfaces, we reveal partial segregation of domains rich in deuterated and nondeuterated lipids. This study addresses a gap in knowledge by examining the impact of deuteration on lipid tail behavior, offering new insights into how even subtle structural modifications can influence phase behavior. Furthermore, it demonstrates that TERS can be a powerful, nondestructive, and label-free nanoanalytical tool for analyzing lipid membranes and advance the field of membrane biophysics.

DOI: https://doi.org/10.1021/acs.jpclett.4c01994
Eur J Med Chem. 2024 Sep 24. pii: S0223-5234(24)00785-2. [Epub ahead of print]279 116904

Exploration of the tunability of BRD4 degradation by DCAF16 trans-labelling covalent glues.

Muhammad Murtaza Hassan, Yen-Der Li, Michelle W Ma, Mingxing Teng, Woong Sub Byun, Kedar Puvar, Ryan Lumpkin, Brittany Sandoval, Justine C Rutter, Cyrus Y Jin, Michelle Y Wang, Shawn Xu, Anna M Schmoker, Hakyung Cheong, Brian J Groendyke, Jun Qi, Eric S Fischer, Benjamin L Ebert, Nathanael S Gray.

Chemically induced proximity modalities such as targeted protein degradation (TPD) hold promise for expanding the number of proteins that can be manipulated pharmacologically. However, current TPD strategies are often limited to proteins with preexisting ligands. Molecular glues (e.g. glutarimide ligands for CUL4CRBN), offer the potential to target undruggable proteins. Yet, their rational design is largely unattainable due to the unpredictability of the 'gain-of-function' nature of the glue interaction upon chemical modification of ligands. We recently reported a covalent trans-labelling glue mechanism which we named 'Template-assisted covalent modification', where an electrophile decorated BRD4 inhibitor was effectively delivered to a cysteine residue on DCAF16 due to an electrophile-induced BRD4-DCAF16 interaction. Herein, we report our efforts to evaluate how various electrophilic modifications to the BRD4 binder, JQ1, affect DCAF16 recruitment and subsequent BRD4 degradation efficiency. We discovered a moderate correlation between the electrophile-induced BRD4-DCAF16 ternary complex formation and BRD4 degradation. Moreover, we show that a more solvent-exposed warhead presentation optimally recruits DCAF16 and promotes BRD4 degradation. The diversity of covalent attachments in this class of BRD4 degraders suggests a high tolerance and tunability for the BRD4-DCAF16 interaction. This offers a new avenue for rational glue design by introducing covalent warheads to known binders.

DOI: https://doi.org/10.1016/j.ejmech.2024.116904