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1.
Nucleic Acids Res ; 52(11): 6441-6458, 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38499483

RESUMO

Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14's exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.


Assuntos
Metiltransferases , RNA Viral , SARS-CoV-2 , Proteínas não Estruturais Virais , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/química , Metiltransferases/metabolismo , Metiltransferases/genética , Metiltransferases/química , Metilação , RNA Viral/metabolismo , RNA Viral/química , RNA Viral/genética , Exorribonucleases/metabolismo , Exorribonucleases/genética , Humanos , Ligação Proteica , Capuzes de RNA/metabolismo , Capuzes de RNA/genética , Regulação Alostérica , COVID-19/virologia , COVID-19/genética , Multimerização Proteica , Replicação Viral/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/química , Proteínas Virais Reguladoras e Acessórias
2.
Mol Cancer ; 22(1): 150, 2023 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-37679783

RESUMO

Recent advances in immuno-oncology have opened up new and impressive treatment options for cancer. Notwithstanding, overcoming the limitations of the current FDA-approved therapies with monoclonal antibodies (mAbs) that block the PD-1/PD-L1 pathway continues to lead to the testing of multiple approaches and optimizations. Recently, a series of macrocyclic peptides have been developed that exhibit binding strengths to PD-L1 ranging from sub-micromolar to micromolar. In this study, we present the most potent non-antibody-based PD-1/PD-L1 interaction inhibitor reported to date. The structural and biological characterization of this macrocyclic PD-L1 targeting peptide provides the rationale for inhibition of both PD-1/PD-L1 and CD80/PD-L1 complexes. The IC50 and EC50 values obtained in PD-L1 binding assays indicate that the pAC65 peptide has potency equivalent to the current FDA-approved mAbs and may have similar activity to the BMS986189 peptide, which entered the clinical trial and has favorable safety and pharmacokinetic data. The data presented here delineate the generation of similar peptides with improved biological activities and applications not only in the field of cancer immunotherapy but also in other disorders related to the immune system.


Assuntos
Antígeno B7-H1 , Receptor de Morte Celular Programada 1 , Humanos , Anticorpos Monoclonais/farmacologia , Inibidores de Checkpoint Imunológico , Peptídeos/farmacologia
3.
Biochem J ; 477(7): 1345-1362, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32207815

RESUMO

We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein (NAP) involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15 bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported the identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like NAP that likely influences phage excision-integration reactions or bacterial gene expression.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Sítios de Ligação , Simulação por Computador , DNA/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fator Proteico para Inversão de Estimulação/química , Fator Proteico para Inversão de Estimulação/genética , Expressão Gênica , Proteínas Mutantes/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Multimerização Proteica/genética , Proteínas Virais/química
4.
Int J Mol Sci ; 22(21)2021 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-34769226

RESUMO

Targeting the programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) interaction has become an established strategy for cancer immunotherapy. Although hundreds of small-molecule, peptide, and peptidomimetic inhibitors have been proposed in recent years, only a limited number of drug candidates show good PD-1/PD-L1 blocking activity in cell-based assays. In this article, we compare representative molecules from different classes in terms of their PD-1/PD-L1 dissociation capacity measured by HTRF and in vitro bioactivity determined by the immune checkpoint blockade (ICB) co-culture assay. We point to recent discoveries that underscore important differences in the mechanisms of action of these molecules and also indicate one principal feature that needs to be considered, which is the eventual human PD-L1 specificity.


Assuntos
Antígeno B7-H1/antagonistas & inibidores , Inibidores de Checkpoint Imunológico , Peptidomiméticos , Animais , Antígeno B7-H1/metabolismo , Células CHO , Cricetulus , Avaliação de Medicamentos , Humanos , Inibidores de Checkpoint Imunológico/química , Inibidores de Checkpoint Imunológico/farmacologia , Células Jurkat , Peptidomiméticos/química , Peptidomiméticos/farmacologia
5.
Molecules ; 25(13)2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32630327

RESUMO

Screening for small-molecule fragments that can lead to potent inhibitors of protein-protein interactions (PPIs) is often a laborious step as the fragments cannot dissociate the targeted PPI due to their low µM-mM affinities. Here, we describe an NMR competition assay called w-AIDA-NMR (weak-antagonist induced dissociation assay-NMR), which is sensitive to weak µM-mM ligand-protein interactions and which can be used in initial fragment screening campaigns. By introducing point mutations in the complex's protein that is not targeted by the inhibitor, we lower the effective affinity of the complex, allowing for short fragments to dissociate the complex. We illustrate the method with the compounds that block the Mdm2/X-p53 and PD-1/PD-L1 oncogenic interactions. Targeting the PD-/PD-L1 PPI has profoundly advanced the treatment of different types of cancers.


Assuntos
Antígeno B7-H1/antagonistas & inibidores , Espectroscopia de Ressonância Magnética/métodos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Humanos , Receptor de Morte Celular Programada 1/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo
6.
Angew Chem Int Ed Engl ; 59(13): 5235-5241, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-31944488

RESUMO

Stapled peptides are chemical entities in-between biologics and small molecules, which have proven to be the solution to high affinity protein-protein interaction antagonism, while keeping control over pharmacological performance such as stability and membrane penetration. We demonstrate that the multicomponent reaction-based stapling is an effective strategy for the development of α-helical peptides with highly potent dual antagonistic action of MDM2 and MDMX binding p53. Such a potent inhibitory activity of p53-MDM2/X interactions was assessed by fluorescence polarization, microscale thermophoresis, and 2D NMR, while several cocrystal structures with MDM2 were obtained. This MCR stapling protocol proved efficient and versatile in terms of diversity generation at the staple, as evidenced by the incorporation of both exo- and endo-cyclic hydrophobic moieties at the side chain cross-linkers. The interaction of the Ugi-staple fragments with the target protein was demonstrated by crystallography.


Assuntos
Peptídeos/química , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/química , Proteína Supressora de Tumor p53/química , Aldeídos/química , Aminas/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Cianetos/química , Polarização de Fluorescência , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação Proteica
7.
PLoS Pathog ; 13(10): e1006686, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29040325

RESUMO

Restriction factors are important components of intrinsic cellular defense mechanisms against viral pathogens. TRIM5α is a restriction factor that intercepts the incoming capsid cores of retroviruses such as HIV and provides an effective species-specific barrier to retroviral infection. The TRIM5α SPRY domain directly binds the capsid with only very weak, millimolar-level affinity, and productive capsid recognition therefore requires both TRIM5α dimerization and assembly of the dimers into a multivalent hexagonal lattice to promote avid binding. Here, we explore the important unresolved question of whether the SPRY domains are flexibly linked to the TRIM lattice or more precisely positioned to maximize avidity. Biochemical and biophysical experiments indicate that the linker segment connecting the SPRY domain to the coiled-coil domain adopts an α-helical fold, and that this helical portion mediates interactions between the two domains. Targeted mutations were generated to disrupt the putative packing interface without affecting dimerization or higher-order assembly, and we identified mutant proteins that were nevertheless deficient in capsid binding in vitro and restriction activity in cells. Our studies therefore support a model wherein substantial avidity gains during assembly-mediated capsid recognition by TRIM5α come in part from tailored spacing of tethered recognition domains.


Assuntos
Capsídeo/imunologia , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Retroviridae/imunologia , Animais , Fatores de Restrição Antivirais , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases
8.
Biotechnol Bioeng ; 116(1): 76-86, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30252938

RESUMO

Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z-domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small-angle X-ray scattering data of the antibody-protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three-dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismo , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Ligação Proteica , Conformação Proteica , Espalhamento a Baixo Ângulo
9.
Molecules ; 24(15)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374878

RESUMO

CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins - important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 - a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide-57.


Assuntos
Antígeno B7-H1/antagonistas & inibidores , Imunoterapia , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Antígenos B7/antagonistas & inibidores , Antígenos B7/química , Antígeno B7-H1/química , Humanos , Espectroscopia de Ressonância Magnética , Neoplasias/imunologia , Ligação Proteica/efeitos dos fármacos
10.
J Sep Sci ; 41(22): 4122-4132, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30240534

RESUMO

Protein-A chromatography is the most widely used chromatography step in downstream processing of antibodies. A deeper understanding of the influence of the surface topology on a molecular/nanoscale level on adsorption is essential for further improvement. It is not clear if the binding is homogenous throughout the entire bead network. We followed the protein absorption process and observed the formation of a protein layer on fibers of chromatography resin in a time-resolved manner in nanoscale. To characterize the changes in the antibody-protein-A ligand complex, small angle X-ray scattering was employed using a miniaturized X-ray-transparent chromatography column packed with a MabSelect SuRe resin. Antibody-free MabSelect SuRe resin fiber had an average radius of 12 nm and the protein layer thickness resulting from antibody adsorption was 5.5 and 10.4 nm for fiber and junctions, respectively under applied native conditions. We hypothesize that an average of 1.2 antibodies were adsorbed per protein-A ligand tetramer bound to the outermost units. In contrast to previous studies, it was therefore possible for the first time to directly correlate the nanostructure changes inside the column, which is otherwise a black box, with the adsorption and elution process.


Assuntos
Anticorpos Monoclonais/química , Nanoestruturas/análise , Proteína Estafilocócica A/química , Adsorção , Cromatografia de Afinidade , Tamanho da Partícula , Espalhamento a Baixo Ângulo , Propriedades de Superfície , Difração de Raios X
11.
Int J Biol Macromol ; 262(Pt 2): 130136, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38354926

RESUMO

Alphaviruses pose a significant threat to public health. Capsid protein encoded in the alphaviral genomes constitutes an interesting therapy target, as it also serves as a protease (CP). Remarkably, it undergoes autoproteolysis, leading to the generation of the C-terminal tryptophan that localizes to the active pocket, deactivating the enzyme. Lack of activity hampers the viral replication cycle, as the virus is not capable of producing the infectious progeny. We investigated the structure and function of the CP encoded in the genome of O'nyong'nyong virus (ONNV), which has instigated outbreaks in Africa. Our research provides a high-resolution crystal structure of the ONNV CP in its active state and evaluates the enzyme's activity. Furthermore, we demonstrated a dose-dependent reduction in ONNV CP proteolytic activity when exposed to indole, suggesting that tryptophan analogs may be a promising basis for developing small molecule inhibitors. It's noteworthy that the capsid protease plays an essential role in virus assembly, binding viral glycoproteins through its glycoprotein-binding hydrophobic pocket. We showed that non-aromatic cyclic compounds like dioxane disrupt this vital interaction. Our findings provide deeper insights into ONNV's biology, and we believe they will prove instrumental in guiding the development of antiviral strategies against arthritogenic alphaviruses.


Assuntos
Alphavirus , Proteínas do Capsídeo , Humanos , Proteínas do Capsídeo/química , Capsídeo/química , Capsídeo/metabolismo , Vírus O'nyong-nyong/metabolismo , Peptídeo Hidrolases/metabolismo , Ideação Suicida , Triptofano/metabolismo , Alphavirus/metabolismo , Endopeptidases/metabolismo
12.
Int J Biol Macromol ; 280(Pt 4): 135510, 2024 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-39304044

RESUMO

Peroxisomal protein import has been identified as a valid target in trypanosomiases, an important health threat in Central and South America. The importomer is built of multiple peroxins (Pex) and structural characterization of these proteins facilitates rational inhibitor development. We report crystal structures of the Trypanosoma brucei and T. cruzi tetratricopeptide repeat domain (TPR) of the cytoplasmic peroxisomal targeting signal 1 (PTS1) receptor Pex5. The structure of the TPR domain of TbPex5 represents an apo-form of the receptor which, together with the previously determined structure of the complex of TbPex5 TPR and PTS1 demonstrate significant receptor dynamics associated with signal peptide recognition. The structure of the complex of TPR domain of TcPex5 with PTS1 provided in this study details the molecular interactions that guide signal peptide recognition at the atomic level in the pathogenic species currently perceived as the most relevant among Trypanosoma. Small - angle X - ray scattering (SAXS) data obtained in solution supports the crystallographic findings on the compaction of the TPR domains of TbPex5 and TcPex5 upon interaction with the cargo.

13.
ACS Med Chem Lett ; 15(6): 828-836, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38894909

RESUMO

Therapeutic antibodies directed against either programmed cell death-1 protein (PD-1) or its ligand PD-L1 have demonstrated efficacy in the treatment of various cancers. In contrast with antibodies, small molecules have the potential for increased tissue penetration; better pharmacology; and therefore, improved antitumor activity. A series of nonsymmetric C2 inhibitors were synthesized and evaluated for PD-1/PD-L1 interaction inhibition. These compounds induced PD-L1 dimerization and effectively blocked PD-L1/PD-1 interaction in a homogeneous time-resolved fluorescence (HTRF) assay with most inhibitors exhibiting IC50 values in the single-digit nM range and below. Their high inhibitory potency was also demonstrated in a cell-based coculture PD-1 signaling assay where 2 exhibited an EC50 inhibitory activity of 21.8 nM, which approached that of the PD-L1 antibody durvalumab (EC50 = 0.3-1.8 nM). Structural insight into how these inhibitors interact with PD-L1 was gained by using NMR and X-ray cocrystal structure studies. These data support further preclinical evaluation of these compounds as antibody alternatives.

14.
RSC Med Chem ; 15(4): 1210-1215, 2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38665826

RESUMO

The progress in cancer survival and treatment has witnessed a remarkable transformation through the innovative approach of targeting the inhibitory immune checkpoint protein PD-1/PD-L1 complex by mAbs, e.g. pembrolizumab (Keytruda). While generating 17.2 billion U.S. dollars in revenue in 2021, the true significance of these developments lies in their ability to enhance cancer patient outcomes. Despite the proven efficacy of mAbs in inhibiting the PD-1/PD-L1 signaling pathways, they face significant challenges, including limited response rates, high production costs, missing oral bioavailability, and extended half-lives that can lead to immune-related adverse effects. A promising alternative approach involves the use of small molecules acting as PD-1/PD-L1 antagonists to stimulate PD-L1 dimerization. However, the precise mechanisms of action of these molecules remain partially understood, posing challenges to their development. In this context, our research focuses on the creation of a novel scaffold based on the Ugi tetrazole four-component reaction (UT-4CR) to develop low-molecular-weight inhibitors of PD-L1. Employing structure-based methods, we synthesized a library of small compounds using biphenyl vinyl isocyanide, leading to the discovery of a structure-activity relationship among 1,5-disubstituted tetrazole-based inhibitors. Supported by a cocrystal structure with PD-L1, these inhibitors underwent biophysical testing, including HTRF and protein NMR experiments, resulting in the identification of potent candidates with sub-micromolar PD-L1 affinities. This finding opens opportunities to the further development of a new class of PD-L1 antagonists, holding promise for improved cancer immunotherapy strategies.

15.
J Chromatogr A ; 1730: 465102, 2024 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-38941799

RESUMO

Staphylococcal protein-A affinity chromatography has been optimized for antibody purification, achieving a current capacity of up to 90 mg/ml in packed bed. The morphology of the particles, the number of antibodies bound per ligand and the spatial arrangement of the ligands were assessed by in-situ Small-angle X-ray scattering (SAXS) and scanning electron microscopy (SEM) combined with measurement of adsorption isotherms. We employed SAXS measurements to probe the nanoscale structure of the chromatographic resin. From scanning electron microcopy, the morphology and area of the beads were obtained. The adsorption isotherm revealed a bi-Langmuirian behavior where the association constant varied with the critical bulk concentration, indicating multilayer adsorption. Determining the antibody-ligand stoichiometry was crucial for understanding the adsorption mechanism, which was estimated to be 4 at lower concentrations and 4.5 at higher concentrations, suggestive of reversible protein-protein interactions. The same results were reached from the in-situ small angle X-ray scattering measurements. A stoichiometry of 6 cannot be achieved since the two protein A monomers are anchored to the stationary phase and thus sterically hindered. Normalization through ellipsoids facilitated SAXS analysis, enabling the determination of distances between ligands and antibody-ligand complexes. Density fluctuations were examined by subtracting the elliptical fit, providing insights into ligand density distribution. The dense ligand packing of TOYOPEARL® AF-rProtein A HC was confirmed, making further increases in ligand density impractical. Additionally, SAXS analysis revealed structural rearrangements of the antibody-ligand complex with increasing antibody surface load, suggesting reversible association of antibodies.


Assuntos
Cromatografia de Afinidade , Espalhamento a Baixo Ângulo , Proteína Estafilocócica A , Difração de Raios X , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismo , Ligantes , Cromatografia de Afinidade/métodos , Adsorção , Anticorpos/química , Microscopia Eletrônica de Varredura
16.
Int J Biol Macromol ; 267(Pt 1): 131392, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38582483

RESUMO

The main protease (Mpro) of SARS-CoV-2 is critical in the virus's replication cycle, facilitating the maturation of polyproteins into functional units. Due to its conservation across taxa, Mpro is a promising target for broad-spectrum antiviral drugs. Targeting Mpro with small molecule inhibitors, such as nirmatrelvir combined with ritonavir (Paxlovid™), which the FDA has approved for post-exposure treatment and prophylaxis, can effectively interrupt the replication process of the virus. A key aspect of Mpro's function is its ability to form a functional dimer. However, the mechanics of dimerization and its influence on proteolytic activity remain less understood. In this study, we utilized biochemical, structural, and molecular modelling approaches to explore Mpro dimerization. We evaluated critical residues, specifically Arg4 and Arg298, that are essential for dimerization. Our results show that changes in the oligomerization state of Mpro directly affect its enzymatic activity and dimerization propensity. We discovered a synergistic relationship influencing dimer formation, involving both intra- and intermolecular interactions. These findings highlight the potential for developing allosteric inhibitors targeting Mpro, offering promising new directions for therapeutic strategies.


Assuntos
Antivirais , Proteases 3C de Coronavírus , Multimerização Proteica , SARS-CoV-2 , SARS-CoV-2/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Humanos , Antivirais/farmacologia , Antivirais/química , Tratamento Farmacológico da COVID-19 , Modelos Moleculares , COVID-19/virologia , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química
17.
Eur J Med Chem ; 276: 116683, 2024 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-39032403

RESUMO

A series of novel 2-arylmethoxy-4-(2-fluoromethyl-biphenyl-3-ylmethoxy) benzylamine derivatives was designed, synthesized, and evaluated for their antitumor effects as PD-1/PD-L1 inhibitors both in vitro and in vivo. Firstly, the ability of these compounds to block the PD-1/PD-L1 immune checkpoint was assessed using the homogeneous time-resolved fluorescence (HTRF) assay. Two of the compounds can strongly block the PD-1/PD-L1 interaction, with IC50 values of less than 10 nM, notably, compound HD10 exhibited significant clinical potential by inhibiting the PD-1/PD-L1 interaction with an IC50 value of 3.1 nM. Further microscale thermophoresis (MST) analysis demonstrated that HD10 had strong interaction with PD-L1 protein. Co-crystal structure (2.7 Å) analysis of HD10 in complex with the PD-L1 protein revealed a strong affinity between the compound and the target PD-L1 dimer. This provides a solid theoretical basis for further in vitro and in vivo studies. Next, a typical cell-based experiment demonstrated that HD10 could remarkably prevent the interaction of hPD-1 293 T cells from human recombinant PD-L1 protein, effectively restoring T cell function, and promoting IFN-γ secretion in a dose-dependent manner. Moreover, HD10 was effective in suppressing tumor growth (TGI = 57.31 %) in a PD-1/PD-L1 humanized mouse model without obvious toxicity. Flow cytometry, qPCR, and immunohistochemistry data suggested that HD10 inhibits tumor growth by activating the immune system in vivo. Based on these results, it seems likely that HD10 is a promising clinical candidate that should be further investigated.


Assuntos
Antineoplásicos , Antígeno B7-H1 , Benzilaminas , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Receptor de Morte Celular Programada 1 , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Animais , Camundongos , Relação Estrutura-Atividade , Benzilaminas/farmacologia , Benzilaminas/química , Benzilaminas/síntese química , Estrutura Molecular , Relação Dose-Resposta a Droga , Proliferação de Células/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/síntese química , Inibidores de Checkpoint Imunológico/química , Linhagem Celular Tumoral , Feminino , Modelos Moleculares
18.
ACS Med Chem Lett ; 15(1): 36-44, 2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-38229762

RESUMO

Although heavily studied, the subject of anti-PD-L1 small-molecule inhibitors is still elusive. Here we present a systematic overview of the principles behind successful anti-PD-L1 small-molecule inhibitor design on the example of the m-terphenyl scaffold, with a particular focus on the neglected influence of the solubilizer tag on the overall affinity toward PD-L1. The inhibitor developed according to the proposed guidelines was characterized through its potency in blocking PD-1/PD-L1 complex formation in homogeneous time-resolved fluorescence and cell-based assays. The affinity is also explained based on the crystal structure of the inhibitor itself and its costructure with PD-L1 as well as a molecular modeling study. Our results structuralize the knowledge related to the strong pharmacophore feature of the m-terphenyl scaffold preferential geometry and the more complex role of the solubilizer tag in PD-L1 homodimer stabilization.

19.
Antiviral Res ; 219: 105731, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37838220

RESUMO

Despite the advances in contemporary medicine and availability of numerous innovative therapies, effective treatment and prevention of SARS-CoV-2 infections pose a challenge. In the search for new anti-SARS-CoV-2 drug candidates, natural products are frequently explored. Here, fifteen cyanopeptolins (CPs) were isolated from the Baltic cyanobacterium Nostoc edaphicum and tested against SARS-CoV-2. Of these depsipeptides, the Arg-containing structural variants showed the strongest inhibition of the Delta SARS-CoV-2 infection in A549ACE2/TMPRSS2 cells. The functional assays indicated a direct interaction of the Arg-containing CP978 with the virions. CP978 also induced a significant decline in virus replication in the primary human airway epithelial cells (HAE). Of the four tested SARS-CoV-2 variants, Wuhan, Alpha, Omicron and Delta, only Wuhan was not affected by CP978. Finally, the analyses with application of confocal microscopy and with the SARS-CoV-2 pseudoviruses showed that CP978-mediated inhibition of viral infection results from the direct binding of the cyanopeptolin with the coronaviral S protein. Considering the potency of viral inhibition and the mode of action of CP978, the significance of the peptide as antiviral drug candidate should be further explored.


Assuntos
COVID-19 , Nostoc , Humanos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus
20.
Front Immunol ; 14: 1124894, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37138886

RESUMO

Spondyloarthropathies (SpA) are a family of rheumatic disorders that could be divided into axial (axSpA) and peripheral (perSpA) sub-forms depending on the disease clinical presentation. The chronic inflammation is believed to be driven by innate immune cells such as monocytes, rather than self-reactive cells of adaptive immune system. The aim of the study was to investigate the micro-RNA (miRNA) profiles in monocyte subpopulations (classical, intermediate and non-classical subpopulations) acquired from SpA patients or healthy individuals in search for prospective disease specific and/or disease subtype differentiating miRNA markers. Several SpA-specific and axSpA/perSpA differentiating miRNAs have been identified that appear to be characteristic for specific monocyte subpopulation. For classical monocytes, upregulation of miR-567 and miR-943 was found to be SpA-specific, whereas downregulation of miR-1262 could serve as axSpA-differentiating, and the expression pattern of miR-23a, miR-34c, mi-591 and miR-630 as perSpA-differentiating markers. For intermediate monocytes, expression levels of miR-103, miR-125b, miR-140, miR-374, miR-376c and miR-1249 could be used to distinguish SpA patients from healthy donors, whereas the expression pattern of miR-155 was identified as characteristic for perSpA. For non-classical monocytes, differential expression of miR-195 was recognized as general SpA indicator, while upregulation of miR-454 and miR-487b could serve as axSpA-differentiating, and miR-1291 as perSpA-differentiating markers. Our data indicate for the first time that in different SpA subtypes, monocyte subpopulations bear disease-specific miRNA signatures that could be relevant for SpA diagnosis/differentiation process and may help to understand SpA etiopathology in the context of already known functions of monocyte subpopulations.


Assuntos
MicroRNAs , Espondiloartropatias , Humanos , Monócitos , Estudos Prospectivos , MicroRNAs/genética , MicroRNAs/metabolismo , Diferenciação Celular , Espondiloartropatias/diagnóstico , Espondiloartropatias/genética , Espondiloartropatias/metabolismo
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