Rachita Dash

Front Immunol. 2023 Oct 2;14:1237964. doi: 10.3389/fimmu.2023.1237964. eCollection 2023.

ABSTRACT

INTRODUCTION: Our previous research has found that degradation of palmitoyltransferase in tumor cells using a linear peptide PROTAC leads to a significant decrease in PD-L1 expression in tumors. However, this degradation is not a sustained and efficient process. Therefore, we designed a cyclic peptide PROTAC to achieve this efficient anti-PD-L1 effect.

METHODS: We designed and synthesized an improvement in linear peptide PROTAC targeting palmitoyltransferase DHHC3, and used disulfide bonds to stabilize the continuous N- and C-termini of the peptides to maintain their structure. Cellular and molecular biology techniques were used to test the effect of this cyclic peptide on PD-L1.

RESULTS: In human cervical cancer cells, our cyclic peptide PROTAC can significantly downregulate palmitoyl transferase DHHC3 and PD-L1 expressions. This targeted degradation effect is enhanced with increasing doses and treatment duration, with a DC50 value much lower than that of linear peptides. Additionally, flow cytometry analysis of fluorescence intensity shows an increase in the amount of cyclic peptide entering the cell membrane with prolonged treatment time and higher concentrations. The Cellular Thermal Shift Assay (CETSA) method used in this study indicates effective binding between our novel cyclic peptide and DHHC3 protein, leading to a change in the thermal stability of the latter. The degradation of PD-L1 can be effectively blocked by the proteasome inhibitor MG132. Results from clone formation experiments illustrate that our cyclic peptide can enhance the proliferative inhibition effect of cisplatin on the C33A cell line. Furthermore, in the T cell-C33A co-culture system, cyclic peptides target the degradation of PD-L1, thereby blocking the interaction between PD-L1 and PD-1, and promoting the secretion of IFN-γ and TNF-α in the co-culture system supernatant.

CONCLUSION: Our results demonstrate that a disulfide-bridged cyclic peptide PROTAC targeting palmitoyltransferase can provide a stable and improved anti-PD-L1 activity in human tumor cells.

PMID:37849747 | PMC:PMC10577221 | DOI:10.3389/fimmu.2023.1237964

Innate Immun. 2023 Nov;29(8):186-200. doi: 10.1177/17534259231207198. Epub 2023 Oct 13.

ABSTRACT

NOD1 and NOD2 sense small bacterial peptidoglycan fragments, often called muropeptides, that access the cytosol. These muropeptides include iE-DAP and MDP, the minimal agonists for NOD1 and NOD2, respectively. Here, we synthesized and validated alkyne-modified muropeptides, iE-DAP-Alk and MDP-Alk, for use in click-chemistry reactions. While it has long been known that many cell types respond to extracellular exposure to muropeptides, it is unclear how these innate immune activators access their cytosolic innate immune receptors, NOD1 and NOD2. The subcellular trafficking and transport mechanisms by which muropeptides access these cytosolic innate immune receptors are a major gap in our understanding of these critical host responses. The click-chemistry-enabled agonists developed here will be particularly powerful to decipher the underlying cell biology and biochemistry of NOD1 and NOD2 innate immune sensing.

PMID:37828863 | PMC:PMC10621468 | DOI:10.1177/17534259231207198

Methods Mol Biol. 2024;2727:193-204. doi: 10.1007/978-1-0716-3491-2_15.

ABSTRACT

Cell wall-anchored surface proteins are integral components of Gram-positive bacterial cell envelope and vital for bacterial survival in different environmental niches. To fulfill their functions, surface protein precursors translocate from cytoplasm to bacterial cell surface in three sequential steps: secretion across the cytoplasmic membrane, covalently anchoring to the cell wall precursor lipid II by sortase A, and incorporation of the lipid II-linked precursors into mature cell wall peptidoglycan. Here, we describe a series of immunofluorescence microscopy methods to track the subcellular localization of cell wall-anchored proteins along the sorting pathway. While the protocols are tailored to Staphylococcus aureus, they can be readily adapted to localize cell wall-anchored proteins as well as membrane proteins in other Gram-positive bacteria.

PMID:37815718 | DOI:10.1007/978-1-0716-3491-2_15

ACS Omega. 2023 Aug 25;8(36):32258-32270. doi: 10.1021/acsomega.3c03557. eCollection 2023 Sep 12.

ABSTRACT

M. tuberculosis, an etiological agent of tuberculosis, requires a long treatment regimen due to its ability to respond to stress and persist inside the host. The second messenger (p)ppGpp-mediated stress response plays a critical role in such long-term survival, persistence, and antibiotic tolerance which may also lead to the emergence of multiple drug resistance. In mycobacteria, (pp)pGpp molecules are synthesized predominantly by two bifunctional enzymes-long RSH-Rel and short SAS-RelZ. The long RSH-Rel is a major (p)ppGpp synthetase and hydrolase. How it switches its activity from synthesis to hydrolysis remains unclear. Rel_Mtb mutant has been reported to be defective in biofilm formation, cell wall function, and persister cell formation. The survival of such mutants has also been observed to be compromised in infection models. In M. smegmatis, short SAS-RelZ has RNase HII activity in addition to (pp)Gpp synthesis activity. The RNase HII function of RelZ has been implicated in resolving replication-transcription conflicts by degrading R-loops. However, the mechanism and regulatory aspects of such a regulation remain elusive. In this article, we have discussed (p)ppGpp metabolism and its role in managing the stress response network of mycobacteria, which is responsible for long-term survival inside the host, making it an important therapeutic target.

PMID:37720788 | PMC:PMC10500699 | DOI:10.1021/acsomega.3c03557

Angew Chem Int Ed Engl. 2023 Nov 13;62(46):e202308408. doi: 10.1002/anie.202308408. Epub 2023 Oct 13.

ABSTRACT

Expanding the chemical diversity of peptide macrocycle libraries for display selection is desirable to improve their potential to bind biomolecular targets. We now have implemented a considerable expansion through a large aromatic helical foldamer inclusion. A foldamer was first identified that undergoes flexizyme-mediated tRNA acylation and that is capable of initiating ribosomal translation with yields sufficiently high to perform an mRNA display selection of macrocyclic foldamer-peptide hybrids. A hybrid macrocyclic nanomolar binder to the C-lobe of the E6AP HECT domain was selected that showed a highly converged peptide sequence. A crystal structure and molecular dynamics simulations revealed that both the peptide and foldamer are helical in an intriguing reciprocal stapling fashion. The strong residue convergence could be rationalized based on their involvement in specific interactions with the target protein. The foldamer stabilizes the peptide helix through stapling and through contacts with key residues. These results altogether represent a significant extension of the chemical space amenable to display selection and highlight possible benefits of inserting an aromatic foldamer into a peptide macrocycle for the purpose of protein recognition.

PMID:37707879 | DOI:10.1002/anie.202308408

Bioeng Transl Med. 2023 May 13;8(5):e10542. doi: 10.1002/btm2.10542. eCollection 2023 Sep.

ABSTRACT

Cyclic peptides are poised to target historically difficult to drug intracellular protein-protein interactions, however, their general cell impermeability poses a challenge for characterizing function. Recent advances in microfluidics have enabled permeabilization of the cytoplasmic membrane by physical cell deformation (i.e., mechanoporation), resulting in intracellular delivery of impermeable macromolecules in vector- and electrophoretic-free approaches. However, the number of payloads (e.g., peptides) and/or concentrations delivered via microfluidic mechanoporation is limited by having to pre-mix cells and payloads, a manually intensive process. In this work, we show that cells are momentarily permeable (t _1/2 = 1.1-2.8 min) after microfluidic vortex shedding (μVS) and that lower molecular weight macromolecules can be cytosolically delivered upon immediate exposure after cells are processed/permeabilized. To increase the ability to screen peptides, we built a system, dispensing-microfluidic vortex shedding (DμVS), that integrates a μVS chip with inline microplate-based dispensing. To do so, we synced an electronic pressure regulator, flow sensor, on/off dispense valve, and an x-y motion platform in a software-driven feedback loop. Using this system, we were able to deliver low microliter-scale volumes of transiently mechanoporated cells to hundreds of wells on microtiter plates in just several minutes (e.g., 96-well plate filled in <2.5 min). We validated the delivery of an impermeable peptide directed at MDM2, a negative regulator of the tumor suppressor p53, using a click chemistry- and NanoBRET-based cell permeability assay in 96-well format, with robust delivery across the full plate. Furthermore, we demonstrated that DμVS could be used to identify functional, low micromolar, cellular activity of otherwise cell-inactive MDM2-binding peptides using a p53 reporter cell assay in 96- and 384-well format. Overall, DμVS can be combined with downstream cell assays to investigate intracellular target engagement in a high-throughput manner, both for improving structure-activity relationship efforts and for early proof-of-biology of non-optimized peptide (or potentially other macromolecular) tools.

PMID:37693049 | PMC:PMC10487316 | DOI:10.1002/btm2.10542

Chem Biol Drug Des. 2023 Sep 1. doi: 10.1111/cbdd.14331. Online ahead of print.

ABSTRACT

Medium sized molecules such as peptides and macrocycles have recently drawn much attention as potent sources of medicinal lead compounds, whereas the possibility of obtaining a practical drug from them remains limited. The present paper describes a concept of discovering novel medicinal targets or binding modes as well as lead compounds by the one-peptide-on-one-bead (OPOB) technology for comprehensive screening. The difficulty and problems in conventional drug discovery methods that generally deal with one predetermined target are considered. The building blocks used for the present libraries were selected based on previous results in development of peptidic drugs. Each constituent has the common structure of cyclic form prepared by disulfide of cysteinyl residues or thioether linkages, additionally a methionine residue was inserted for the site-specific rapid cleavage by cyanogen bromide to liberate the immobilized peptides allowing reliable characterization by MALDI-TOF-MS/MS without LC-purification. Thus, a high throughput construction method for cyclic peptide libraries as well as characterization of single bead are proposed for drug discovery.

PMID:37658589 | DOI:10.1111/cbdd.14331

Mol Divers. 2023 Sep 2. doi: 10.1007/s11030-023-10722-7. Online ahead of print.

ABSTRACT

Listeria monocytogenes is an important human and animal pathogen able to cause an infection named listeriosis and is mainly transmitted through contaminated food. Among its virulence traits, the ability to form biofilms and to survive in harsh environments stand out and lead to the persistence of L. monocytogenes for long periods in food processing environments. Virulence and biofilm formation are phenotypes regulated by quorum sensing (QS) and, therefore, the control of L. monocytogenes through an anti-QS strategy is promising. This study aimed to identify, by in silico approaches, proteins secreted by lactic acid bacteria (LAB) potentially able to interfere with the agr QS system of L. monocytogenes. The genome mining of Lacticaseibacillus rhamnosus GG and Lactobacillus acidophilus NCFM revealed 151 predicted secreted proteins. Concomitantly, the three-dimensional (3D) structures of AgrB and AgrC proteins of L. monocytogenes were modeled and validated, and their active sites were predicted. Through protein-protein docking and molecular dynamic, Serine-type D-Ala-D-Ala carboxypeptidase and L,D-transpeptidase, potentially secreted by L. rhamnosus GG and L. acidophilus NCFM, respectively, were identified with high affinity to AgrB and AgrC proteins, respectively. By inhibiting the translocation of the cyclic autoinducer peptide (cyclic AIP) via AgrB, and its recognition in the active site of AgrC, these LAB proteins could disrupt L. monocytogenes communication by impairing the agr QS system. The application of the QS inhibitors predicted in this study can emerge as a promising strategy in controlling L. monocytogenes in food processing environment and as an adjunct to antibiotic therapy for the treatment of listeriosis.

PMID:37658910 | DOI:10.1007/s11030-023-10722-7

Elife. 2023 Sep 4;12:e81924. doi: 10.7554/eLife.81924.

ABSTRACT

Lateral partitioning of proteins and lipids shapes membrane function. In model membranes, partitioning can be influenced both by bilayer-intrinsic factors like molecular composition and by bilayer-extrinsic factors such as interactions with other membranes and solid supports. While cellular membranes can departition in response to bilayer-intrinsic or -extrinsic disruptions, the mechanisms by which they partition de novo are largely unknown. The plasma membrane of Mycobacterium smegmatis spatially and biochemically departitions in response to the fluidizing agent benzyl alcohol, then repartitions upon fluidizer washout. By screening for mutants that are sensitive to benzyl alcohol, we show that the bifunctional cell wall synthase PonA2 promotes membrane partitioning and cell growth during recovery from benzyl alcohol exposure. PonA2's role in membrane repartitioning and regrowth depends solely on its conserved transglycosylase domain. Active cell wall polymerization promotes de novo membrane partitioning and the completed cell wall polymer helps to maintain membrane partitioning. Our work highlights the complexity of membrane-cell wall interactions and establishes a facile model system for departitioning and repartitioning cellular membranes.

PMID:37665120 | PMC:PMC10547480 | DOI:10.7554/eLife.81924

PNAS Nexus. 2023 Aug 17;2(8):pgad270. doi: 10.1093/pnasnexus/pgad270. eCollection 2023 Aug.

ABSTRACT

The lack of available treatments for many antimicrobial-resistant infections highlights the critical need for antibiotic discovery innovation. Peptides are an underappreciated antibiotic scaffold because they often suffer from proteolytic instability and toxicity toward human cells, making in vivo use challenging. To investigate sequence factors related to serum activity, we adapt an antibacterial display technology to screen a library of peptide macrocycles for antibacterial potential directly in human serum. We identify dozens of new macrocyclic peptide antibiotic sequences and find that serum activity within our library is influenced by peptide length, cationic charge, and the number of disulfide bonds present. Interestingly, an optimized version of our most active lead peptide permeates the outer membrane of Gram-negative bacteria without strong inner-membrane disruption and kills bacteria slowly while causing cell elongation. This contrasts with traditional cationic antimicrobial peptides, which kill rapidly via lysis of both bacterial membranes. Notably, this optimized variant is not toxic to mammalian cells and retains its function in vivo, suggesting therapeutic promise. Our results support the use of more physiologically relevant conditions when screening peptides for antimicrobial activity which retain in vivo functionality.

PMID:37637199 | PMC:PMC10449418 | DOI:10.1093/pnasnexus/pgad270

PNAS Nexus. 2023 Aug 17;2(8):pgad270. doi: 10.1093/pnasnexus/pgad270. eCollection 2023 Aug.

ABSTRACT

The lack of available treatments for many antimicrobial-resistant infections highlights the critical need for antibiotic discovery innovation. Peptides are an underappreciated antibiotic scaffold because they often suffer from proteolytic instability and toxicity toward human cells, making in vivo use challenging. To investigate sequence factors related to serum activity, we adapt an antibacterial display technology to screen a library of peptide macrocycles for antibacterial potential directly in human serum. We identify dozens of new macrocyclic peptide antibiotic sequences and find that serum activity within our library is influenced by peptide length, cationic charge, and the number of disulfide bonds present. Interestingly, an optimized version of our most active lead peptide permeates the outer membrane of Gram-negative bacteria without strong inner-membrane disruption and kills bacteria slowly while causing cell elongation. This contrasts with traditional cationic antimicrobial peptides, which kill rapidly via lysis of both bacterial membranes. Notably, this optimized variant is not toxic to mammalian cells and retains its function in vivo, suggesting therapeutic promise. Our results support the use of more physiologically relevant conditions when screening peptides for antimicrobial activity which retain in vivo functionality.

PMID:37637199 | PMC:PMC10449418 | DOI:10.1093/pnasnexus/pgad270

J Med Chem. 2023 Sep 14;66(17):11843-11854. doi: 10.1021/acs.jmedchem.3c00426. Epub 2023 Aug 26.

ABSTRACT

The κ-opioid receptor (KOR) is an attractive target for the development of novel drugs. KOR agonists are potentially safer pain medications, whereas KOR antagonists are promising drug candidates for the treatment of neuropsychiatric disorders. Hitherto, the vast majority of selective drug leads that have been developed for KOR are small molecules. In this study, novel peptide probes were designed by using an endogenous dynorphin A_1-13 sequence as a template for peptide stapling via late-stage cysteine functionalization. Leveraging this strategy, we developed a stable and potent KOR antagonist, CSD-CH_2(1,8)-NH₂, with approximately 1000-fold improved selectivity for KOR over μ- and δ-opioid receptors. Its potent competitive KOR antagonism was verified in KOR-expressing cells, peripheral dorsal root ganglion neurons, and using the tail-flick and rotarod tests in mice. This work highlights the value of cysteine stapling to develop selective peptide probes to modulate central KOR function, as innovative peptide drug candidates for the treatment of KOR-related illnesses.

PMID:37632447 | PMC:PMC10510397 | DOI:10.1021/acs.jmedchem.3c00426

ACS Symp Ser Am Chem Soc. 2022;1417:179-197. doi: 10.1021/bk-2022-1417.ch007. Epub 2022 Aug 4.

ABSTRACT

Intracellular protein-protein interactions (PPIs) represent a large class of exciting as well as challenging drug targets for traditional drug modalities (i.e., small molecules and biologics). Peptides (especially cyclic peptides) have proven highly effective as PPI inhibitors in vitro but are generally impermeable to the cell membrane. The recent discovery of a family of highly active cyclic cell-penetrating peptides (CPPs) has enabled the delivery of peptides into the cytosol of mammalian cells at therapeutically relevant levels. This chapter describes the various strategies that have been developed to conjugate or integrate different types of peptidyl cargoes (e.g., linear, cyclic, and stapled peptides) with cyclic CPPs to generate cell-permeable, metabolically stable, and biologically active macrocyclic peptides against intracellular targets including PPIs.

PMID:37621949 | PMC:PMC10448808 | DOI:10.1021/bk-2022-1417.ch007

Protein Sci. 2023 Aug 26:e4769. doi: 10.1002/pro.4769. Online ahead of print.

ABSTRACT

Targeted intracellular delivery via receptor-mediated endocytosis requires the delivered cargo to escape the endosome to prevent lysosomal degradation. This can in principle be achieved by membrane lysis tightly restricted to endosomal membranes upon internalization to avoid general membrane insertion and lysis. Here we describe the design of small monomeric proteins with buried histidine containing pH-responsive hydrogen bond networks and membrane permeating amphipathic helices. Of 30 designs that were experimentally tested, all expressed in E. coli, 13 were monomeric with the expected secondary structure, and 4 designs disrupted artificial liposomes in a pH-dependent manner. Mutational analysis showed that the buried histidine hydrogen bond networks mediate pH-responsiveness and control lysis of model membranes within a very narrow range of pH (6.0 - 5.5) with almost no lysis occurring at neutral pH. These tightly controlled lytic monomers could help mediate endosomal escape in designed targeted delivery platforms. This article is protected by copyright. All rights reserved.

PMID:37632837 | DOI:10.1002/pro.4769