Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 13 de 13
Filter
1.
J Biol Chem ; 299(11): 105276, 2023 11.
Article in English | MEDLINE | ID: mdl-37739035

ABSTRACT

Imbalanced immune responses are a prominent hallmark of cancer and autoimmunity. Myeloid cells can be overly suppressive, inhibiting protective immune responses or inactive not controlling autoreactive immune cells. Understanding the mechanisms that induce suppressive myeloid cells, such as myeloid-derived suppressor cells (MDSCs) and tolerogenic dendritic cells (TolDCs), can facilitate the development of immune-restoring therapeutic approaches. MDSCs are a major barrier for effective cancer immunotherapy by suppressing antitumor immune responses in cancer patients. TolDCs are administered to patients to promote immune tolerance with the intent to control autoimmune disease. Here, we investigated the development and suppressive/tolerogenic activity of human MDSCs and TolDCs to gain insight into signaling pathways that drive immunosuppression in these different myeloid subsets. Moreover, monocyte-derived MDSCs (M-MDSCs) generated in vitro were compared to M-MDSCs isolated from head-and-neck squamous cell carcinoma patients. PI3K-AKT signaling was identified as being crucial for the induction of human M-MDSCs. PI3K inhibition prevented the downregulation of HLA-DR and the upregulation of reactive oxygen species and MerTK. In addition, we show that the suppressive activity of dexamethasone-induced TolDCs is induced by ß-catenin-dependent Wnt signaling. The identification of PI3K-AKT and Wnt signal transduction pathways as respective inducers of the immunomodulatory capacity of M-MDSCs and TolDCs provides opportunities to overcome suppressive myeloid cells in cancer patients and optimize therapeutic application of TolDCs. Lastly, the observed similarities between generated- and patient-derived M-MDSCs support the use of in vitro-generated M-MDSCs as powerful model to investigate the functionality of human MDSCs.


Subject(s)
Dendritic Cells , Myeloid-Derived Suppressor Cells , Phosphatidylinositol 3-Kinases , Signal Transduction , Wnt Signaling Pathway , Humans , Dendritic Cells/immunology , Immunomodulation/immunology , Immunotherapy , Myeloid-Derived Suppressor Cells/immunology , Neoplasms/immunology , Neoplasms/therapy , Phosphatidylinositol 3-Kinases/immunology , Proto-Oncogene Proteins c-akt/immunology , Signal Transduction/immunology , Wnt Signaling Pathway/immunology , Tumor Cells, Cultured
2.
Bioconjug Chem ; 32(2): 301-310, 2021 02 17.
Article in English | MEDLINE | ID: mdl-33476135

ABSTRACT

Functionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theranostics, and antibody-drug conjugates (ADC). In addition, therapeutic and theranostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labeling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogeneous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab' fragments bearing two distinct site-specific labeling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab' molecule (DTFab'), which can be easily isolated. To demonstrate feasibility, we functionalized the DTFab' with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theranostics, and next-generation ADCs.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Hybridomas/chemistry , Immunoglobulin Fab Fragments/chemistry , Antibodies, Monoclonal/chemistry , Immunoconjugates/chemistry , Stochastic Processes
3.
Molecules ; 26(1)2021 Jan 02.
Article in English | MEDLINE | ID: mdl-33401754

ABSTRACT

Using the classical Ugi four-component reaction to fuse an amine, ketone, carboxylic acid, and isocyanide, here we prepared a short library of N-alkylated α,α-dialkylglycine derivatives. Due to the polyfunctionality of the dipeptidic scaffold, this highly steric hindered system shows an interesting acidolytic cleavage of the C-terminal amide. In this regard, we studied the structure-acid lability relationship of the C-terminal amide bond (cyclohexylamide) of N-alkylated α,α-dialkylglycine amides 1a-n in acidic media and, afterward, it was established that the most important structural features related to its cleavage. Then, it was demonstrated that electron-donating effects in the aromatic amines, flexible acyl chains (Gly) at the N-terminal and the introduction of cyclic compounds into dipeptide scaffolds, increased the rate of acidolysis. All these effects are related to the ease with which the oxazolonium ion intermediate forms and they promote the proximity of the central carbonyl group to the C-terminal amide, resulting in C-terminal amide cleavage. Consequently, these findings could be applied for the design of new protecting groups, handles for solid-phase synthesis, and linkers for conjugation, due to its easily modulable and the fact that it allows to fine tune its acid-lability.


Subject(s)
Dipeptides/chemistry , Glycine/analogs & derivatives , Glycine/chemistry , Alkylation , Amides/chemistry , Amines/chemistry , Chemistry Techniques, Synthetic , Cyanides/chemistry , Hydrolysis , Molecular Structure , Oxazines/chemistry , Solubility
4.
Bioconjug Chem ; 29(4): 1199-1208, 2018 04 18.
Article in English | MEDLINE | ID: mdl-29433317

ABSTRACT

The design and generation of complex multifunctional macromolecular structures by bioconjugation is a hot topic due to increasing interest in conjugates with therapeutic applications. In this regard, the development of efficient, selective, and safe conjugation methods is a major objective. In this report, we describe the use of the bis(bromomethyl)benzene scaffold as a linker for bioconjugation with special emphasis on antibody conjugation. We first performed the monothioalkylation of 1,3,5-tris(bromomethyl)benzene, which rendered the reactive dibromotrimethylbenzyl derivatives to be used in thiol bis-alkylation. Next, we introduced into the linker either a bis(Cys)-containing peptide or anti-CD4 and -CD13 monoclonal antibodies, previously subjected to partial reduction of disulfide bonds. Mass spectrometry, UV-vis spectra, and SDS-PAGE experiments revealed that this bis-alkylating agent for bioconjugation preserved both antibody integrity and antibody-antigen binding affinity, as assessed by flow cytometry. Taken together, our results show that the mesitylene scaffold is a suitable linker for thiol-based bioconjugation reactions. This linker could be applicable in the near future for the preparation of antibody drug conjugates.


Subject(s)
Antibodies, Monoclonal/chemistry , Benzene Derivatives/chemistry , Immunoconjugates/chemistry , Peptides/chemistry , Sulfhydryl Compounds/chemistry , Alkylation , Benzene Derivatives/chemical synthesis , Models, Molecular , Oxidation-Reduction , Peptides/chemical synthesis , Solid-Phase Synthesis Techniques , Sulfhydryl Compounds/chemical synthesis
5.
Molecules ; 20(4): 5409-22, 2015 Mar 26.
Article in English | MEDLINE | ID: mdl-25822080

ABSTRACT

Here the aromatic formylation mediated by TiCl4 and dichloromethyl methyl ether previously described by our group has been explored for a wide range of aromatic rings, including phenols, methoxy- and methylbenzenes, as an excellent way to produce aromatic aldehydes. Here we determine that the regioselectivity of this process is highly promoted by the coordination between the atoms present in the aromatic moiety and those in the metal core.


Subject(s)
Bis(Chloromethyl) Ether/chemistry , Polycyclic Aromatic Hydrocarbons/chemistry , Titanium/chemistry , Models, Chemical , Models, Molecular , Molecular Structure
6.
Molecules ; 18(5): 5155-62, 2013 May 06.
Article in English | MEDLINE | ID: mdl-23648593

ABSTRACT

Cys-disulfide bonds contribute to the stabilization of peptide and protein structures. The synthesis of these molecules requires a proper protection of Cys residues, which is crucial to prevent side-reactions and also to achieve the correct Cys connectivity. Here we undertook a mechanistic study of a set of well-known acid-labile Cys protecting groups, as well other new promising groups, in order to better understand the nature of their acid-lability. The stability of the carbocation generated during the acid treatment was found to have a direct impact on the removal of the protective groups from the corresponding protected Cys-containing peptides. Hence a combination of steric and conjugative effects determines the stability of the carbocations generated. Here we propose diphenylmethyl (Dpm) as a promising protecting group on the basis of its intermediate relative carbocation stability. All the optimized geometries and energies presented in this study were determined using a B3LYP/6-31G(d,p) calculation. The results discussed herein may be of broader applicability for the development of new protecting groups.


Subject(s)
Cysteine/chemistry , Peptides/chemistry , Peptides/chemical synthesis
7.
Chempluschem ; 88(5): e202300028, 2023 05.
Article in English | MEDLINE | ID: mdl-37134299

ABSTRACT

Cysteine-based perfluoroaromatic (hexafluorobenzene (HFB) and decafluorobiphenyl (DFBP)) were synthesized and established as a chemoselective and available core to construct molecular systems ranging from small molecules to biomolecules with interesting properties. The DFBP was found more effective than HFB for the monoalkylation of decorated thiol molecules. As proof of concept of the potential application of perfluorinated derivatives as non-cleavable linkers, some antibody-perfluorinated conjugates were prepared via thiol through two different strategies, i) using thiol from reduced cystamine coupling to carboxylic acids from mAb by amide bond, and ii) using thiols from reduction of mAb disulfide bond. Conjugates cell binding analysis demonstrated that the bioconjugation does not affect the macromolecular entity. Besides, some molecular properties of synthesized compounds are evaluated through spectroscopic characterization (FTIR and 19 F NMR chemical shifts) and theoretical calculations. The comparison of calculated and experimental 19 F NMR shifts and IR wavenumbers give excellent correlations, asserting as powerful tools in structurally identifying HFB and DFBP derivatives. Moreover, molecular docking was also developed to predict cysteine-based perfluorated derivatives' affinity against topoisomerase Il and cyclooxygenase 2 (COX-2). The results suggested that mainly cysteine-based DFBP derivatives could be potential topoisomerase II α and COX-2 binders, becoming potential anticancer agents and candidates for anti-inflammatory treatment.


Subject(s)
Antineoplastic Agents , Cysteine , Cysteine/chemistry , Molecular Docking Simulation , Cyclooxygenase 2/metabolism , Antineoplastic Agents/chemistry , Sulfhydryl Compounds/chemistry
8.
J Immunother Cancer ; 10(4)2022 04.
Article in English | MEDLINE | ID: mdl-35428705

ABSTRACT

BACKGROUND: Type 1 conventional dendritic cells (cDC1s) are characterized by their ability to induce potent CD8+ T cell responses. In efforts to generate novel vaccination strategies, notably against cancer, human cDC1s emerge as an ideal target to deliver antigens. cDC1s uniquely express XCR1, a seven transmembrane G protein-coupled receptor. Due to its restricted expression and endocytic nature, XCR1 represents an attractive receptor to mediate antigen-delivery to human cDC1s. METHODS: To explore tumor antigen delivery to human cDC1s, we used an engineered version of XCR1-binding lymphotactin (XCL1), XCL1(CC3). Site-specific sortase-mediated transpeptidation was performed to conjugate XCL1(CC3) to an analog of the HLA-A*02:01 epitope of the cancer testis antigen New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1). While poor epitope solubility prevented isolation of stable XCL1-antigen conjugates, incorporation of a single polyethylene glycol (PEG) chain upstream of the epitope-containing peptide enabled generation of soluble XCL1(CC3)-antigen fusion constructs. Binding and chemotactic characteristics of the XCL1-antigen conjugate, as well as its ability to induce antigen-specific CD8+ T cell activation by cDC1s, was assessed. RESULTS: PEGylated XCL1(CC3)-antigen conjugates retained binding to XCR1, and induced cDC1 chemoattraction in vitro. The model epitope was efficiently cross-presented by human cDC1s to activate NY-ESO-1-specific CD8+ T cells. Importantly, vaccine activity was increased by targeting XCR1 at the surface of cDC1s. CONCLUSION: Our results present a novel strategy for the generation of targeted vaccines fused to insoluble antigens. Moreover, our data emphasize the potential of targeting XCR1 at the surface of primary human cDC1s to induce potent CD8+ T cell responses.


Subject(s)
Antigens, Neoplasm , Cancer Vaccines , Dendritic Cells , Esophageal Neoplasms , Esophageal Squamous Cell Carcinoma , Lymphokines , Membrane Proteins , Sialoglycoproteins , Antigens, Neoplasm/administration & dosage , Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/administration & dosage , Cancer Vaccines/immunology , Cross-Priming , Dendritic Cells/immunology , Epitopes/immunology , Esophageal Neoplasms/immunology , Esophageal Neoplasms/therapy , Esophageal Squamous Cell Carcinoma/immunology , Esophageal Squamous Cell Carcinoma/therapy , Humans , Lymphokines/administration & dosage , Lymphokines/immunology , Male , Membrane Proteins/administration & dosage , Membrane Proteins/immunology , Sialoglycoproteins/administration & dosage , Sialoglycoproteins/immunology
9.
ACS Omega ; 5(13): 7424-7431, 2020 Apr 07.
Article in English | MEDLINE | ID: mdl-32280884

ABSTRACT

Bioconjugation is a key approach for the development of novel molecular entities with clinical applications. The biocompatibility and specificity of biomolecules such as peptides, proteins, and antibodies make these macromolecules ideal carriers for selective targeted therapies. In this context, there is a need to develop new molecular units that cover the requirements of the next generation of targeted pharmaceuticals. Here, we present the design and development of a versatile and stable linker based on a N-alkylated α,α-dialkyl dipeptide for bioconjugation, with a particular focus on antibody-drug conjugates (ADCs). Starting with the well-known Ugi multicomponent reaction, the convenient chemical modification of the prepared adducts allowed us the obtention of versatile bifunctional linkers for bioconjugation. A conjugation strategy was tested to demonstrate the efficiency of the linker. In addition, a novel cytotoxic anti-HER2 ADC was prepared using the Ugi-linker approach.

10.
ChemistryOpen ; 6(2): 206-210, 2017 04.
Article in English | MEDLINE | ID: mdl-28413752

ABSTRACT

The use of the tetrahydropyranyl (Thp) group for the protection of serine and threonine side-chain hydroxyl groups in solid-phase peptide synthesis has not been widely investigated. Ser/Thr side-chain hydroxyl protection with this acid-labile and non-aromatic moiety is presented here. Although Thp reacts with free carboxylic acids, it can be concluded that to introduce Thp ethers at the hydroxyl groups of N-protected Ser and Thr, protection of the C-terminal carboxyl group is unnecessary due to the lability of Thp esters. Thp-protected Ser/Thr-containing tripeptides are synthesized and the removal of Thp studied in low concentrations of trifluoroacetic acid in the presence of cation scavengers. Given its general stability to most non-acidic reagents, improved solubility of its conjugates and ease with which it can be removed, Thp emerges as an effective protecting group for the hydroxyl groups of Ser and Thr in solid-phase peptide synthesis.

11.
ChemistryOpen ; 6(2): 168-177, 2017 04.
Article in English | MEDLINE | ID: mdl-28413747

ABSTRACT

Tetrahydropyranyl (Thp) is recognized as a useful protecting group for alcohols in organic synthesis. It has several advantages, including low cost, ease of introduction, general stability to most non-acidic reagents, it confers good solubility, and the ease with which it can be removed if the functional group it protects requires manipulation. However, little attention has been paid to Thp in peptide chemistry. Provided here is a concise analysis of the Thp protection of various amino acid functionalities (OH, SH, NH and COOH) and its application to peptide synthesis. Thp is a useful moiety for the side-chain protection of serine, threonine and cysteine and is suitable for the Fmoc/tBu solid-phase peptide synthesis strategy. The immobilized version of 3,4-dihydro-2H-pyran, the so-called Ellman resin, is also discussed as a useful solid support for anchoring the side chains of serine, threonine and tryptophan residues.

12.
Org Lett ; 17(7): 1680-3, 2015 Apr 03.
Article in English | MEDLINE | ID: mdl-25764355

ABSTRACT

Tetrahydropyranyl (Thp), which exploits the concept of being an S,O-acetal nonaromatic protecting group for cysteine, has been shown to be superior to Trt, Dpm, Acm, and StBu in solid-phase peptide synthesis using the Fmoc/tBu strategy. Thus, Cys racemization and C-terminal 3-(1-piperidinyl)alanine formation were minimized when the Cys was protected with Thp. This nonaromatic protecting group also improved the solubility of Cys-containing protected peptides.


Subject(s)
Alanine/analogs & derivatives , Amino Acids/chemistry , Cysteine/chemistry , Fluorenes/chemistry , Peptides/chemistry , Pyrans/chemistry , Alanine/chemistry , Molecular Structure , Solid-Phase Synthesis Techniques
13.
Org Lett ; 14(21): 5472-5, 2012 Nov 02.
Article in English | MEDLINE | ID: mdl-23075170

ABSTRACT

To address the existing gap in the current set of acid-labile Cys-protecting groups for the Fmoc/tBu strategy, diverse Fmoc-Cys(PG)-OH derivatives were prepared and incorporated into a model tripeptide to study their stability against TFA. S-Dpm proved to be compatible with the commonly used S-Trt group and was applied for the regioselecive construction of disulfide bonds.


Subject(s)
Cysteine/chemistry , Fluorenes/chemistry , Amino Acids/chemistry , Combinatorial Chemistry Techniques , Cysteine/analogs & derivatives , Models, Molecular , Molecular Structure , Peptides/chemistry
SELECTION OF CITATIONS
SEARCH DETAIL