ABSTRACT
The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics.
Subject(s)
Peptides , Plasmodium falciparum , Protozoan Proteins , Ubiquitin Thiolesterase , Plasmodium falciparum/enzymology , Plasmodium falciparum/metabolism , Plasmodium falciparum/drug effects , Ubiquitin Thiolesterase/metabolism , Ubiquitin Thiolesterase/antagonists & inhibitors , Ubiquitin Thiolesterase/genetics , Humans , Peptides/chemistry , Peptides/metabolism , Peptides/pharmacology , Protozoan Proteins/metabolism , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Protozoan Proteins/antagonists & inhibitors , Antimalarials/pharmacology , Antimalarials/chemistry , Ubiquitin/metabolism , Malaria, Falciparum/parasitology , Malaria, Falciparum/drug therapyABSTRACT
Ribosome-dependent protein biosynthesis is an essential cellular process mediated by transfer RNAs (tRNAs). Generally, ribosomally synthesized proteins are limited to the 22 proteinogenic amino acids (pAAs: 20 l-α-amino acids present in the standard genetic code, selenocysteine, and pyrrolysine). However, engineering tRNAs for the ribosomal incorporation of non-proteinogenic monomers (npMs) as building blocks has led to the creation of unique polypeptides with broad applications in cellular biology, material science, spectroscopy, and pharmaceuticals. Ribosomal polymerization of these engineered polypeptides presents a variety of challenges for biochemists, as translation efficiency and fidelity is often insufficient when employing npMs. In this Review, we will focus on the methodologies for engineering tRNAs to overcome these issues and explore recent advances both in vitro and in vivo. These efforts include increasing orthogonality, recruiting essential translation factors, and creation of expanded genetic codes. After our review on the biochemical optimizations of tRNAs, we provide examples of their use in genetic code manipulation, with a focus on the in vitro discovery of bioactive macrocyclic peptides containing npMs. Finally, an analysis of the current state of tRNA engineering is presented, along with existing challenges and future perspectives for the field.
Subject(s)
Protein Biosynthesis , RNA, Transfer , Ribosomes , RNA, Transfer/metabolism , RNA, Transfer/chemistry , RNA, Transfer/genetics , Ribosomes/metabolism , Ribosomes/genetics , Genetic Code , HumansABSTRACT
Ribosomal incorporation of ß-amino acids into nascent peptides is much less efficient than that of the canonical α-amino acids. To overcome this, we have engineered a tRNA chimera bearing T-stem of tRNAGlu and D-arm of tRNAPro1, referred to as tRNAPro1E2, which efficiently recruits EF-Tu and EF-P. Using tRNAPro1E2 indeed improved ß-amino acid incorporation. However, multiple/consecutive incorporations of ß-amino acids are still detrimentally poor. Here, we attempted fine-tuning of the anticodon arm of tRNAPro1E2 aiming at further enhancement of ß-amino acid incorporation. By screening various mutations introduced into tRNAPro1E2, C31G39/C28G42 mutation showed an approximately 3-fold enhancement of two consecutive incorporation of ß-homophenylglycine (ßPhg) at CCG codons. The use of this tRNA made it possible for the first time to elongate up to ten consecutive ßPhg's. Since the enhancement effect of anticodon arm mutations differs depending on the codon used for ß-amino acid incorporation, we optimized anticodon arm sequences for five codons (CCG, CAU, CAG, ACU and UGG). Combination of the five optimal tRNAs for these codons made it possible to introduce five different kinds of ß-amino acids and analogs simultaneously into model peptides, including a macrocyclic scaffold. This strategy would enable ribosomal synthesis of libraries of macrocyclic peptides containing multiple ß-amino acids.
Subject(s)
Amino Acids , Anticodon , Anticodon/genetics , Anticodon/chemistry , Amino Acids/chemistry , Amino Acids/genetics , RNA, Transfer/genetics , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Mutation , Codon/genetics , Ribosomes/metabolism , Ribosomes/genetics , Protein Biosynthesis , RNA, Transfer, Pro/genetics , RNA, Transfer, Pro/metabolism , RNA, Transfer, Pro/chemistry , Nucleic Acid Conformation , Peptide Elongation Factor Tu/genetics , Peptide Elongation Factor Tu/metabolism , Escherichia coli/genetics , Escherichia coli/metabolismABSTRACT
In the hypothetical RNA world, ribozymes could have acted as modern aminoacyl-tRNA synthetases (ARSs) to charge tRNAs, thus giving rise to the peptide synthesis along with the evolution of a primitive translation apparatus. We previously reported a T-boxzyme, Tx2.1, which selectively charges initiator tRNA with N-biotinyl-phenylalanine (BioPhe) in situ in a Flexible In-vitro Translation (FIT) system to produce BioPhe-initiating peptides. Here, we performed in vitro selection of elongation-capable T-boxzymes (elT-boxzymes), using para-azido-l-phenylalanine (PheAZ) as an acyl-donor. We implemented a new strategy to enrich elT-boxzyme-tRNA conjugates that self-aminoacylated on the 3'-terminus selectively. One of them, elT32, can charge PheAZ onto tRNA in trans in response to its cognate anticodon. Further evolution of elT32 resulted in elT49, with enhanced aminoacylation activity. We have demonstrated the translation of a PheAZ-containing peptide in an elT-boxzyme-integrated FIT system, revealing that elT-boxzymes are able to generate the PheAZ-tRNA in response to the cognate anticodon in situ of a custom-made translation system. This study, together with Tx2.1, illustrates a scenario where a series of ribozymes could have overseen aminoacylation and co-evolved with a primitive RNA-based translation system.
Subject(s)
Anticodon , Protein Biosynthesis , RNA, Catalytic , RNA, Transfer, Amino Acyl , RNA, Catalytic/metabolism , RNA, Catalytic/genetics , Anticodon/genetics , RNA, Transfer, Amino Acyl/metabolism , RNA, Transfer, Amino Acyl/genetics , Phenylalanine/metabolism , Phenylalanine/analogs & derivatives , Amino Acyl-tRNA Synthetases/metabolism , Amino Acyl-tRNA Synthetases/genetics , Transfer RNA Aminoacylation , Aminoacylation , Peptide Chain Elongation, TranslationalABSTRACT
Nonproteinogenic amino acids, including d-α-, ß-, and γ-amino acids, present in bioactive peptides play pivotal roles in their biochemical activities and proteolytic stabilities. d-α-Amino acids (dαAA) are widely used building blocks that can enhance the proteolytic stability. Cyclic ß2,3-amino acids (cßAA), for instance, can fold peptides into rigid secondary structures, improving the binding affinity and proteolytic stability. Cyclic γ2,4-amino acids (cγAA) are recently highlighted as rigid residues capable of preventing the proteolysis of flanking residues. Simultaneous incorporation of all dαAA, cßAA, and cγAA into a peptide is expected to yield l-α/d-α/ß/γ-hybrid peptides with improved stability and potency. Despite challenges in the ribosomal incorporation of multiple nonproteinogenic amino acids, our engineered tRNAPro1E2 successfully reaches such a difficulty. Here, we report the ribosomal synthesis of macrocyclic l-α/d-α/ß/γ-hybrid peptide libraries and their application to in vitro selection against interferon gamma receptor 1 (IFNGR1). One of the resulting l-α/d-α/ß/γ-hybrid peptides, IB1, exhibited remarkable inhibitory activity against the IFN-γ/IFNGR1 protein-protein interaction (PPI) (IC50 = 12 nM), primarily attributed to the presence of a cßAA in the sequence. Additionally, cγAAs and dαAAs in the resulting peptides contributed to their serum stability. Furthermore, our peptides effectively inhibit IFN-γ/IFNGR1 PPI at the cellular level (best IC50 = 0.75 µM). Altogether, our platform expands the chemical space available for exploring peptides with high activity and stability, thereby enhancing their potential for drug discovery.
Subject(s)
Interferon gamma Receptor , Interferon-gamma , Receptors, Interferon , Interferon-gamma/metabolism , Receptors, Interferon/metabolism , Receptors, Interferon/chemistry , Humans , Protein Binding , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/pharmacology , Peptides/chemistry , Peptides/pharmacology , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Peptides, Cyclic/metabolismABSTRACT
Thiopeptides make up a group of structurally complex peptidic natural products holding promise in bioengineering applications. The previously established thiopeptide/mRNA display platform enables de novo discovery of natural product-like thiopeptides with designed bioactivities. However, in contrast to natural thiopeptides, the discovered structures are composed predominantly of proteinogenic amino acids, which results in low metabolic stability in many cases. Here, we redevelop the platform and demonstrate that the utilization of compact reprogrammed genetic codes in mRNA display libraries can lead to the discovery of thiopeptides predominantly composed of nonproteinogenic structural elements. We demonstrate the feasibility of our designs by conducting affinity selections against Traf2- and NCK-interacting kinase (TNIK). The experiment identified a series of thiopeptides with high affinity to the target protein (the best KD = 2.1 nM) and kinase inhibitory activity (the best IC50 = 0.15 µM). The discovered compounds, which bore as many as 15 nonproteinogenic amino acids in an 18-residue macrocycle, demonstrated high metabolic stability in human serum with a half-life of up to 99 h. An X-ray cocrystal structure of TNIK in complex with a discovered thiopeptide revealed how nonproteinogenic building blocks facilitate the target engagement and orchestrate the folding of the thiopeptide into a noncanonical conformation. Altogether, the established platform takes a step toward the discovery of thiopeptides with high metabolic stability for early drug discovery applications.
Subject(s)
Amino Acids , Peptides , Humans , Peptides/chemistry , Amino Acids/chemistry , Genetic Code , RNA, MessengerABSTRACT
There have been significant advances in the flexibility and power of in vitro cell-free translation systems. The increasing ability to incorporate noncanonical amino acids and complement translation with recombinant enzymes has enabled cell-free production of peptide-based natural products (NPs) and NP-like molecules. We anticipate that many more such compounds and analogs might be accessed in this way. To assess the peptide NP space that is directly accessible to current cell-free technologies, we developed a peptide parsing algorithm that breaks down peptide NPs into building blocks based on ribosomal translation logic. Using the resultant data set, we broadly analyze the biophysical properties of these privileged compounds and perform a retrobiosynthetic analysis to predict which peptide NPs could be directly synthesized in augmented cell-free translation reactions. We then tested these predictions by preparing a library of highly modified peptide NPs. Two macrocyclases, PatG and PCY1, were used to effect the head-to-tail macrocyclization of candidate NPs. This retrobiosynthetic analysis identified a collection of high-priority building blocks that are enriched throughout peptide NPs, yet they had not previously been tested in cell-free translation. To expand the cell-free toolbox into this space, we established, optimized, and characterized the flexizyme-enabled ribosomal incorporation of piperazic acids. Overall, these results demonstrate the feasibility of cell-free translation for peptide NP total synthesis while expanding the limits of the technology. This work provides a novel computational tool for exploration of peptide NP chemical space, that could be expanded in the future to allow design of ribosomal biosynthetic pathways for NPs and NP-like molecules.
Subject(s)
Biological Products , Biological Products/chemistry , Cheminformatics , Peptides/chemistry , Peptide Biosynthesis , Amino AcidsABSTRACT
Ten-Eleven Translocation (TET) enzymes are Fe(II)/2OG-dependent oxygenases that play important roles in epigenetic regulation, but selective inhibition of the TETs is an unmet challenge. We describe the profiling of previously identified TET1-binding macrocyclic peptides. TiP1 is established as a potent TET1 inhibitor (IC50 = 0.26 µM) with excellent selectivity over other TETs and 2OG oxygenases. TiP1 alanine scanning reveals the critical roles of Trp10 and Glu11 residues for inhibition of TET isoenzymes. The results highlight the utility of the RaPID method to identify potent enzyme inhibitors with selectivity over closely related paralogues. The structure-activity relationship data generated herein may find utility in the development of chemical probes for the TETs.
Subject(s)
Dioxygenases , Peptides, Cyclic , Humans , Epigenesis, Genetic , DNA-Binding Proteins/metabolism , Mixed Function Oxygenases/metabolism , Dioxygenases/metabolism , DNA Methylation , Proto-Oncogene ProteinsABSTRACT
Despite the great advances in discovering cyclic peptides against protein targets, their reduced aqueous solubility, cell permeability, and activity of the cyclic peptide restrict its utilization in advanced biological research and therapeutic applications. Here we report on a novel approach of structural alternation of the exocyclic and linker parts that led to a new derivative with significantly improved cell activity allowing us to dissect its mode of action in detail. We have identified an effective cyclic peptide (CP7) that induces approximately a 9-fold increase in DNA damage accumulation and a remarkable increase in apoptotic cancer cell death compared to the reported molecule. Notably, treating cells with CP7 leads to a dramatic decrease in the efficiency of non-homologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs), which is accompanied by an increase in homologous recombination (HR) repair. Interestingly, treating BRCA1-deficient cells with CP7 restores HR integrity, which is accompanied by increased resistance to CP7. Additionally, CP7 treatment increases the sensitivity of cancer cells to ionizing radiation. Collectively, our findings demonstrate that CP7 is a selective inhibitor of NHEJ, offering a potential strategy to enhance the effectiveness of radiation therapy.
ABSTRACT
Prenylation of peptides is widely observed in the secondary metabolites of diverse organisms, granting peptides unique chemical properties distinct from proteinogenic amino acids. Discovery of prenylated peptide agents has largely relied on isolation or genome mining of naturally occurring molecules. To devise a platform technology for de novo discovery of artificial prenylated peptides targeting a protein of choice, here we have integrated the thioether-macrocyclic peptide (teMP) library construction/selection technology, so-called RaPID (Random nonstandard Peptides Integrated Discovery) system, with a Trp-C3-prenyltransferase KgpF involved in the biosynthesis of a prenylated natural product. This unique enzyme exhibited remarkably broad substrate tolerance, capable of modifying various Trp-containing teMPs to install a prenylated residue with tricyclic constrained structure. We constructed a vast library of prenylated teMPs and subjected it to in vitro selection against a phosphoglycerate mutase. This selection platform has led to the identification of a pseudo-natural prenylated teMP inhibiting the target enzyme with an IC50 of 30â nM. Importantly, the prenylation was essential for the inhibitory activity, enhanced serum stability, and cellular uptake of the peptide, highlighting the benefits of peptide prenylation. This work showcases the de novo discovery platform for pseudo-natural prenylated peptides, which is readily applicable to other drug targets.
ABSTRACT
Protein engineering techniques have vastly expanded their domain of impact, notably following the success of antibodies. Likewise, smaller peptide therapeutics have carved an increasingly significant niche for themselves in the pharmaceutical landscape. The concept of grafting such peptides onto larger protein scaffolds, thus harvesting the advantages of both, has given rise to a variety of protein engineering strategies that are reviewed herein. We also describe our own "Lasso-Grafting" approach, which combines traditional grafting concepts with mRNA display to streamline the production of multiple grafted drug candidates for virtually any target.
Subject(s)
Epitopes , Peptides , Protein Engineering , Peptides/chemistry , Epitopes/chemistry , Protein Engineering/methods , Humans , Proteins/chemistryABSTRACT
Oxidation of a thioether linkage in thioether-closed macrocyclic peptides led to collision-induced site-selective linearization of the peptides. This method has allowed for de novo sequencing of thioether macrocyclic peptides. The utility of the sequencing method was demonstrated by identifying the correct peptide sequences from a virtually randomized thioether macrocyclic peptide library.
ABSTRACT
Semaphorin-Plexin signaling plays a major role in the tumor microenvironment (TME). In particular, Semaphorin 4D (SEMA4D) has been shown to promote tumor growth and metastasis; however, the role of its high-affinity receptor Plexin-B1 (PLXNB1), which is expressed in the TME, is poorly understood. In this study, we directly targeted PLXNB1 in the TME of triple-negative murine breast carcinoma to elucidate its relevance in cancer progression. We found that primary tumor growth, and metastatic dissemination were strongly reduced in PLXNB1-deficient mice, which showed longer survival. PLXNB1-loss in the TME induced a switch in the polarization of tumor-associated macrophages (TAMs) towards a pro-inflammatory M1 phenotype and enhanced the infiltration of CD8+ T lymphocytes both in primary tumors and in distant metastases. Moreover, PLXNB1-deficiency promoted a shift in the Th1/Th2 balance of the T-cell population and an antitumor gene signature, with the up-regulation of Icos, Perforin-1, Stat3 and Ccl5 in tumor infiltrating lymphocytes (TILs). We thus tested the translational relevance of TME re-programming driven by PLXNB1 inactivation for responsiveness to immunotherapy. Indeed, in the absence of PLXNB1, the efficacy of anti-PD-1 blockade was strongly enhanced, efficiently reducing tumor growth and distant metastasis. Consistent with this, pharmacological PLXNB1 blockade by systemic treatment with a specific inhibitor significantly hampered breast cancer growth and enhanced the antitumor activity of the anti-PD1 treatment in a preclinical model. Altogether, these data indicate that PLXNB1 signaling controls the antitumor immune response in the TME and highlight this receptor as a promising immune therapeutic target for metastatic breast cancers.
ABSTRACT
The regenerative functions associated with cytokines and growth factors have immense therapeutic potential; however, their poor pharmacokinetics, resulting from structural features, hinder their effectiveness. In this study, we aimed to enhance the pharmacokinetics of growth factors by designing receptor-binding macrocyclic peptides through in vitro mRNA display and grafting them into loops of immunoglobulin's crystallizable region (Fc). As a model, we developed peptide-grafted Fc proteins with hepatocyte growth factor (HGF) functionality that exhibited a prolonged circulation half-life and could be administered subcutaneously. The Fc-based HGF mimetic alleviated liver fibrosis in a mouse model fed a choline-deficient high-fat diet, which induces hepatic features of non-alcoholic steatohepatitis, including fibrosis, showcasing its potential as a therapeutic intervention. This study provides a basis for developing growth factor and cytokine mimetics with improved pharmacokinetics, expanding their therapeutic applications.
ABSTRACT
Due to their constrained conformations, cyclic ß2,3-amino acids (cßAA) are key building blocks that can fold peptides into compact and rigid structures, improving peptidase resistance and binding affinity to target proteins, due to their constrained conformations. Although the translation efficiency of cßAAs is generally low, our engineered tRNA, referred to as tRNAPro1E2, enabled efficient incorporation of cßAAs into peptide libraries using the flexible in vitro translation (FIT) system. Here we report on the design and application of a macrocyclic peptide library incorporating 3 kinds of cßAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (ß1), (1S,2S)-2-aminocyclohexane carboxylic acid (ß2), and (1R,2R)-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (Mpro). The resultant peptides, BM3 and BM7, bearing one ß2 and two ß1, exhibited potent inhibitory activities with IC50 values of 40 and 20â nM, respectively. BM3 and BM7 also showed remarkable serum stability with half-lives of 48 and >168â h, respectively. Notably, BM3A and BM7A, wherein the cßAAs were substituted with alanine, lost their inhibitory activities against Mpro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cßAA to the activity and stability of peptides. Overall, our results highlight the potential of cßAA in generating potent and highly stable macrocyclic peptides with drug-like properties.