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Cell Oct 2022The G protein-coupled receptor cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable proteins, with the exception of...
The G protein-coupled receptor cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable proteins, with the exception of the Gα subunit, Gαs. GTPases remain largely undruggable given the difficulty of displacing high-affinity guanine nucleotides and the lack of other drug binding sites. We explored a chemical library of 10 cyclic peptides to expand the chemical search for inhibitors of this enzyme class. We identified two macrocyclic peptides, GN13 and GD20, that antagonize the active and inactive states of Gαs, respectively. Both macrocyclic peptides fine-tune Gαs activity with high nucleotide-binding-state selectivity and G protein class-specificity. Co-crystal structures reveal that GN13 and GD20 distinguish the conformational differences within the switch II/α3 pocket. Cell-permeable analogs of GN13 and GD20 modulate Gαs/Gβγ signaling in cells through binding to crystallographically defined pockets. The discovery of cyclic peptide inhibitors targeting Gαs provides a path for further development of state-dependent GTPase inhibitors.
Topics: GTP Phosphohydrolases; Guanine Nucleotides; Nucleotides; Peptides; Peptides, Cyclic; Receptors, G-Protein-Coupled
PubMed: 36170854
DOI: 10.1016/j.cell.2022.09.019 -
Microbiology and Molecular Biology... Jun 2023Clinical management of Staphylococcus aureus infections presents a challenge due to the high incidence, considerable virulence, and emergence of drug resistance... (Review)
Review
Clinical management of Staphylococcus aureus infections presents a challenge due to the high incidence, considerable virulence, and emergence of drug resistance mechanisms. The treatment of drug-resistant strains, such as methicillin-resistant S. aureus (MRSA), is further complicated by the development of tolerance and persistence to antimicrobial agents in clinical use. To address these challenges, membrane disruptors, that are not generally considered during drug discovery for agents against S. aureus, should be explored. The cell membrane protects S. aureus from external stresses and antimicrobial agents, but membrane-targeting antimicrobial agents are probably less likely to promote bacterial resistance. Nontypical linear cationic antimicrobial peptides (AMPs), highly modified AMPs such as daptomycin (lipopeptide), bacitracin (cyclic peptide), and gramicidin S (cyclic peptide), are currently in clinical use. Recent studies have demonstrated that AMPs and small molecules can penetrate the cell membrane of S. aureus, inhibit phospholipid biosynthesis, or block the passage of solutes between the periplasm and the exterior of the cell. In addition to their primary mechanism of action (MOA) that targets the bacterial membrane, AMPs and small molecules may also impact bacteria through secondary mechanisms such as targeting the biofilm, and downregulating virulence genes of S. aureus. In this review, we discuss the current state of research into cell membrane-targeting AMPs and small molecules and their potential mechanisms of action against drug-resistant physiological forms of S. aureus, including persister cells and biofilms.
Topics: Humans; Staphylococcus aureus; Anti-Bacterial Agents; Methicillin-Resistant Staphylococcus aureus; Antimicrobial Peptides; Anti-Infective Agents; Peptides, Cyclic; Cell Membrane; Biofilms; Staphylococcal Infections
PubMed: 37129495
DOI: 10.1128/mmbr.00037-22 -
Redox Biology Aug 2023Necroptosis and pyroptosis, two types of proinflammatory programmed cell death, were recently found to play important roles in spinal cord injury (SCI). Moreover, cyclic...
BACKGROUND
Necroptosis and pyroptosis, two types of proinflammatory programmed cell death, were recently found to play important roles in spinal cord injury (SCI). Moreover, cyclic helix B peptide (CHBP) was designed to maintain erythropoietin (EPO) activity and protect tissue against the adverse effects of EPO. However, the protective mechanism of CHBP following SCI is still unknown. This research explored the necroptosis- and pyroptosis-related mechanism underlying the neuroprotective effect of CHBP after SCI.
METHODS
Gene Expression Omnibus (GEO) datasets and RNA sequencing were used to identify the molecular mechanisms of CHBP for SCI. A mouse model of contusion SCI was constructed, and HE staining, Nissl staining, Masson staining, footprint analysis and the Basso Mouse Scale (BMS) were applied for histological and behavioural analyses. qPCR, Western blot analysis, immunoprecipitation and immunofluorescence were utilized to analyse the levels of necroptosis, pyroptosis, autophagy and molecules associated with the AMPK signalling pathway.
RESULTS
The results revealed that CHBP significantly improved functional restoration, elevated autophagy, suppressed pyroptosis, and mitigated necroptosis after SCI. 3-Methyladenine (3-MA), an autophagy inhibitor, attenuated these beneficial effects of CHBP. Furthermore, CHBP-triggered elevation of autophagy was mediated by the dephosphorylation and nuclear translocation of TFEB, and this effect was due to stimulation of the AMPK-FOXO3a-SPK2-CARM1 and AMPK-mTOR signalling pathways.
CONCLUSION
CHBP acts as a powerful regulator of autophagy that improves functional recovery by alleviating proinflammatory cell death after SCI and thus might be a prospective therapeutic agent for clinical application.
Topics: Mice; Animals; Peptides, Cyclic; AMP-Activated Protein Kinases; Spinal Cord Injuries; Apoptosis; Signal Transduction; Autophagy
PubMed: 37290302
DOI: 10.1016/j.redox.2023.102767 -
Theranostics 2022To enhance tumor uptake and retention, we designed and developed bi-specific heterodimeric radiotracers targeting both FAP and αvβ3, [Ga]Ga-FAPI-RGD. The present study...
To enhance tumor uptake and retention, we designed and developed bi-specific heterodimeric radiotracers targeting both FAP and αvβ3, [Ga]Ga-FAPI-RGD. The present study aimed to evaluate the specificity, pharmacokinetics, and dosimetry of [Ga]Ga-FAPI-RGD by preclinical and preliminary clinical studies. FAPI-RGD was designed and synthesized with the quinoline-based FAPI-02 and the cyclic RGDfK peptide. Preclinical pharmacokinetics were determined in Panc02 xenograft model using microPET and biodistribution experiments. The safety and effective dosimetry of [Ga]Ga-FAPI-RGD was evaluated in 6 cancer patients, and compared with 2-[F]FDG imaging. The [Ga]Ga-FAPI-RGD had good stability in saline for at least 4 h, and showed favorable binding affinity and specificity and . Compared to [Ga]Ga-FAPI-02 and [Ga]Ga-RGDfK, the tumor uptake and retention of [Ga]Ga-FAPI-RGD were very much enhanced than its monomeric counterparts at all the time points examined by microPET imaging. A total of 6 patients with various malignant tumors were prospectively enrolled. The effective dose of [Ga]Ga-FAPI-RGD was 1.94E-02 mSv/MBq. The biodistribution of [Ga]Ga-FAPI-RGD from 0 to 2 h after injection demonstrated rapid and high tumor uptake, prolonged tumor retention, and high tumor-to-background ratios (TBRs) which further increased over time. No significant difference in mean SUVmax of [Ga]Ga-FAPI-RGD and 2-[F]FDG was present in primary tumors (8.9±3.2 10.3 ± 6.9; p = 0.459). The dual targeting PET tracer [Ga]Ga-FAPI-RGD showed significantly improved tumor uptake and retention, as well as cleaner background over Ga-labeled FAPI and RGD monospecific tracers. The first-in-human biodistribution study showed high TBRs over time, suggesting high diagnostic performance and favorable tracer kinetics for potential therapeutic applications.
Topics: Humans; Gallium Radioisotopes; Tissue Distribution; Fluorodeoxyglucose F18; Radiometry; Neoplasms; Peptides, Cyclic; Quinolines; Positron-Emission Tomography; Oligopeptides
PubMed: 36276644
DOI: 10.7150/thno.79144 -
International Journal of Molecular... Jun 2019In recent decades, the incidence of neuroendocrine tumors (NETs) has steadily increased. Due to the slow-growing nature of these tumors and the lack of early symptoms,... (Review)
Review
In recent decades, the incidence of neuroendocrine tumors (NETs) has steadily increased. Due to the slow-growing nature of these tumors and the lack of early symptoms, most cases are diagnosed at advanced stages, when curative treatment options are no longer available. Prognosis and survival of patients with NETs are determined by the location of the primary lesion, biochemical functional status, differentiation, initial staging, and response to treatment. Somatostatin analogue (SSA) therapy has been a mainstay of antisecretory therapy in functioning neuroendocrine tumors, which cause various clinical symptoms depending on hormonal hypersecretion. Beyond symptomatic management, recent research demonstrates that SSAs exert antiproliferative effects and inhibit tumor growth via the somatostatin receptor 2 (SSTR2). Both the PROMID (placebo-controlled, prospective, randomized study in patients with metastatic neuroendocrine midgut tumors) and the CLARINET (controlled study of lanreotide antiproliferative response in neuroendocrine tumors) trial showed a statistically significant prolongation of time to progression/progression-free survival (TTP/PFS) upon SSA treatment, compared to placebo. Moreover, the combination of SSA with peptide receptor radionuclide therapy (PRRT) in small intestinal NETs has proven efficacy in the phase 3 neuroendocrine tumours therapy (NETTER 1) trial. PRRT is currently being tested for enteropancreatic NETs versus everolimus in the COMPETE trial, and the potential of SSTR-antagonists in PRRT is now being evaluated in early phase I/II clinical trials. This review provides a synopsis on the pharmacological development of SSAs and their use as antisecretory drugs. Moreover, this review highlights the clinical evidence of SSAs in monotherapy, and in combination with other treatment modalities, as applied to the antiproliferative management of neuroendocrine tumors with special attention to recent high-quality phase III trials.
Topics: Animals; Antineoplastic Agents, Hormonal; Clinical Trials as Topic; Disease-Free Survival; Humans; Neuroendocrine Tumors; Octreotide; Peptides, Cyclic; Receptors, Somatostatin; Signal Transduction; Somatostatin
PubMed: 31234481
DOI: 10.3390/ijms20123049 -
Nature Biotechnology Apr 2021Molecules that covalently bind macromolecular targets have found widespread applications as activity-based probes and as irreversibly binding drugs. However, the general...
Molecules that covalently bind macromolecular targets have found widespread applications as activity-based probes and as irreversibly binding drugs. However, the general reactivity of the electrophiles needed for covalent bond formation makes control of selectivity difficult. There is currently no rapid, unbiased screening method to identify new classes of covalent inhibitors from highly diverse pools of candidate molecules. Here we describe a phage display method to directly screen for ligands that bind to protein targets through covalent bond formation. This approach makes use of a reactive linker to form cyclic peptides on the phage surface while simultaneously introducing an electrophilic 'warhead' to covalently react with a nucleophile on the target. Using this approach, we identified cyclic peptides that irreversibly inhibited a cysteine protease and a serine hydrolase with nanomolar potency and exceptional specificity. This approach should enable rapid, unbiased screening to identify new classes of highly selective covalent inhibitors for diverse molecular targets.
Topics: Cell Surface Display Techniques; Cysteine Proteinase Inhibitors; Hydrolases; Models, Molecular; Molecular Dynamics Simulation; Molecular Structure; Peptides, Cyclic; Proteins
PubMed: 33199876
DOI: 10.1038/s41587-020-0733-7 -
European Journal of Nuclear Medicine... Aug 2023Fibroblast activation protein-α (FAP)-targeting radioligands have recently demonstrated high diagnostic potential. However, their therapeutic value is impaired by the...
Head-to-head comparison of different classes of FAP radioligands designed to increase tumor residence time: monomer, dimer, albumin binders, and small molecules vs peptides.
PURPOSE
Fibroblast activation protein-α (FAP)-targeting radioligands have recently demonstrated high diagnostic potential. However, their therapeutic value is impaired by the short tumor residence time. Several strategies have been tested to overcome this limitation, but a head-to-head comparison has never been done. With the aim to identify strengths and limitations of the suggested strategies, we compared the monomer FAPI-46 versus (a) its dimer (FAPI-46-F1D), (b) two albumin binders conjugates (FAPI-46-Ibu (ibuprofen) and FAPI-46-EB (Evans Blue)), and (c) cyclic peptide FAP-2286.
METHODS
Lu-labeled ligands were evaluated in vitro in cell lines with low (HT-1080.hFAP) and high (HEK-293.hFAP) humanFAP expression. SPECT/CT imaging and biodistribution studies were conducted in HT-1080.hFAP and HEK-293.hFAP xenografts. The areas under the curve (AUC) of the tumor uptake and tumor-to-critical-organs ratios and the absorbed doses were estimated.
RESULTS
Radioligands showed IC in the picomolar range. Striking differences were observed in vivo regarding tumor uptake, residence, specificity, and total body distribution. All [Lu]Lu-FAPI-46-based radioligands showed similar uptake between the two tumor models. [Lu]Lu-FAP-2286 showed higher uptake in HEK-293.hFAP and the least background. The AUC of the tumor uptake and absorbed dose was higher for [Lu]Lu-FAPI-46-F1D and the two albumin binder conjugates, [Lu]Lu-FAPI-46-Ibu and [Lu]Lu-FAPI-46-EB, in HT1080.hFAP xenografts and for [Lu]Lu-FAPI-46-EB and [Lu]Lu-FAP-2286 in HEK293.hFAP xenografts. The tumor-to-critical-organs AUC values and the absorbed doses were in favor of [Lu]Lu-FAP-2286, but tumor-to-kidneys.
CONCLUSION
The study indicated dimerization and cyclic peptide structures as promising strategies for prolonging tumor residence time, sparing healthy tissues. Albumin binding strategy outcome depended on the albumin binding moiety. The peptide showed advantages in terms of tumor-to-background ratios, besides tumor-to-kidneys, but its tumor uptake was FAP expression-dependent.
Topics: Humans; HEK293 Cells; Tissue Distribution; Cell Line, Tumor; Albumins; Peptides; Peptides, Cyclic; Positron Emission Tomography Computed Tomography; Gallium Radioisotopes
PubMed: 37261473
DOI: 10.1007/s00259-023-06272-7 -
International Journal of Molecular... Oct 2020Using a random non-standard peptide integrated discovery system, we obtained cyclic peptides that bind to hepatocyte growth factor (HGF) or mesenchymal-epithelial... (Review)
Review
Using a random non-standard peptide integrated discovery system, we obtained cyclic peptides that bind to hepatocyte growth factor (HGF) or mesenchymal-epithelial transition factor. (MET) HGF-inhibitory peptide-8 (HiP-8) selectively bound to two-chain active HGF, but not to single-chain precursor HGF. HGF showed a dynamic change in its molecular shape in atomic force microscopy, but HiP-8 inhibited dynamic change in the molecular shape into a static status. The inhibition of the molecular dynamics of HGF by HiP-8 was associated with the loss of the ability to bind MET. HiP-8 could selectively detect active HGF in cancer tissues, and active HGF probed by HiP-8 showed co-localization with activated MET. Using HiP-8, cancer tissues with active HGF could be detected by positron emission tomography. HiP-8 seems to be applicable for the diagnosis and treatment of cancers. In contrast, based on the receptor dimerization as an essential process for activation, the cross-linking of the cyclic peptides that bind to the extracellular region of MET successfully generated an artificial ligand to MET. The synthetic MET agonists activated MET and exhibited biological activities which were indistinguishable from the effects of HGF. MET agonists composed of cyclic peptides can be manufactured by chemical synthesis but not recombinant protein expression, and thus are expected to be new biologics that are applicable to therapeutics and regenerative medicine.
Topics: Animals; Binding Sites; Biological Products; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Neoplasms; Peptides, Cyclic; Protein Binding; Proto-Oncogene Proteins c-met; Signal Transduction
PubMed: 33121208
DOI: 10.3390/ijms21217977 -
Molecules (Basel, Switzerland) Jun 2021Macrocyclic peptides are predominantly peptide structures bearing one or more rings and spanning multiple amino acid residues. Macrocyclization has become a common... (Review)
Review
Macrocyclic peptides are predominantly peptide structures bearing one or more rings and spanning multiple amino acid residues. Macrocyclization has become a common approach for improving the pharmacological properties and bioactivity of peptides. A variety of ribosomal-derived and non-ribosomal synthesized cyclization approaches have been established. The biosynthesis of backbone macrocyclic peptides using seven new emerging methodologies will be discussed with regard to the features and strengths of each platform rather than medicinal chemistry tools. The mRNA display variant, known as the random nonstandard peptide integrated discovery (RaPID) platform, utilizes flexible in vitro translation (FIT) to access macrocyclic peptides containing nonproteinogenic amino acids (NAAs). As a new discovery approach, the ribosomally synthesized and post-translationally modified peptides (RiPPs) method involves the combination of ribosomal synthesis and the phage screening platform together with macrocyclization chemistries to generate libraries of macrocyclic peptides. Meanwhile, the split-intein circular ligation of peptides and proteins (SICLOPPS) approach relies on the in vivo production of macrocyclic peptides. In vitro and in vivo peptide library screening is discussed as an advanced strategy for cyclic peptide selection. Specifically, biosynthetic bicyclic peptides are highlighted as versatile and attractive modalities. Bicyclic peptides represent another type of promising therapeutics that allow for building blocks with a heterotrimeric conjugate to address intractable challenges and enable multimer complexes via linkers. Additionally, we discuss the cell-free chemoenzymatic synthesis of macrocyclic peptides with a non-ribosomal catalase known as the non-ribosomal synthetase (NRPS) and chemo-enzymatic approach, with recombinant thioesterase (TE) domains. Novel insights into the use of peptide library tools, activity-based two-hybrid screening, structure diversification, inclusion of NAAs, combinatorial libraries, expanding the toolbox for macrocyclic peptides, bicyclic peptides, chemoenzymatic strategies, and future perspectives are presented. This review highlights the broad spectrum of strategy classes, novel platforms, structure diversity, chemical space, and functionalities of macrocyclic peptides enabled by emerging biosynthetic platforms to achieve bioactivity and for therapeutic purposes.
Topics: Cyclization; Humans; Peptide Library; Peptide Synthases; Peptides, Cyclic; Two-Hybrid System Techniques
PubMed: 34206124
DOI: 10.3390/molecules26113338 -
Nature Communications Dec 2023Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of...
Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-β-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-β-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-β-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.
Topics: Male; Mice; Animals; Receptors, Opioid, kappa; Ligands; Analgesics, Opioid; Receptors, Opioid, mu; Peptides, Cyclic
PubMed: 38052802
DOI: 10.1038/s41467-023-43718-w