RESUMO
The DNA damage binding protein 1 (DDB1) is an essential component of protein complexes involved in DNA damage repair and the ubiquitin-proteasome system (UPS) for protein degradation. As an adaptor protein specific to Cullin-RING E3 ligases, DDB1 binds different receptors that poise protein substrates for ubiquitination and subsequent degradation by the 26S proteasome. Examples of DDB1-binding protein receptors are Cereblon (CRBN) and the WD-repeat containing DDB1- and CUL4-associated factors (DCAFs). Cognate substrates of CRBN and DCAFs are involved in cancer-related cellular processes or are mimicked by viruses to reprogram E3 ligases for the ubiquitination of antiviral host factors. Thus, disrupting interactions of DDB1 with receptor proteins might be an effective strategy for anticancer and antiviral drug discovery. Here, we developed fluorescence polarization (FP)-based peptide displacement assays that utilize full-length DDB1 and fluorescein isothiocyanate (FITC)-labeled peptide probes derived from the specific binding motifs of DDB1 interactors. A general FP-based assay condition applicable to diverse peptide probes was determined and optimized. Mutagenesis and biophysical analyses were then employed to identify the most suitable peptide probe. The FITC-DCAF15 L49A peptide binds DDB1 with a dissociation constant of 68 nM and can be displaced competitively by unlabeled peptides at sub-µM to low nM concentrations. These peptide displacement assays can be used to screen small molecule libraries to identify novel modulators that could specifically antagonize DDB1 interactions toward development of antiviral and cancer therapeutics.
Assuntos
Proteínas de Ligação a DNA , Peptídeos , Humanos , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Polarização de Fluorescência/métodos , Peptídeos/química , Peptídeos/farmacologia , Ligação Proteica , Ubiquitina-Proteína Ligases/metabolismoRESUMO
We herewith applied a priori a generic hit identification method (POEM) for difficult targets of known three-dimensional structure, relying on the simple knowledge of physicochemical and topological properties of a user-selected cavity. Searching for local similarity to a set of fragment-bound protein microenvironments of known structure, a point cloud registration algorithm is first applied to align known subpockets to the target cavity. The resulting alignment then permits us to directly pose the corresponding seed fragments in a target cavity space not typically amenable to classical docking approaches. Last, linking potentially connectable atoms by a deep generative linker enables full ligand enumeration. When applied to the WD40 repeat (WDR) central cavity of leucine-rich repeat kinase 2 (LRRK2), an unprecedented binding site, POEM was able to quickly propose 94 potential hits, five of which were subsequently confirmed to bind in vitro to LRRK2-WDR.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Simulação de Acoplamento Molecular , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Sítios de Ligação , Domínios Proteicos , Humanos , Ligantes , Ligação Proteica , Repetições WD40 , AlgoritmosRESUMO
The gene-encoding Indole-3-glycerol phosphate synthase, a key enzyme involved in the cyclization of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate, from Pyrococcus furiosus was cloned and expressed in Escherichia coli. The gene product was produced in the soluble and active form. The recombinant protein, purified to apparent homogeneity, displayed highest activity at 100 °C and pH of 5.5. The recombinant enzyme followed Michaelis-Menten kinetics exhibiting apparent Vmax and Km values of 20 ± 0.5 µmol min-1 mg-1 and 140 ± 10 µM, respectively. The activation energy, determined from the linear Arrhenius plot, was 17 ± 0.5 kJ mol-1. A unique property of PfInGPS is its stability against denaturants and temperature. There was no significant change in activity even in the presence of 8 M urea or 5 M guanidine hydrochloride. Furthermore, recombinant PfInGPS was highly thermostable with a half-life of 200 min at 100 °C. To the best of our knowledge, this is the most stable indole-3-glycerol phosphate synthase characterized to date.
Assuntos
Proteínas Arqueais/metabolismo , Indol-3-Glicerolfosfato Sintase/metabolismo , Desnaturação Proteica , Pyrococcus furiosus/enzimologia , Proteínas Arqueais/química , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Indol-3-Glicerolfosfato Sintase/químicaRESUMO
Analysis of the genome sequence of Pyrobaculum calidifontis revealed the presence of an open reading frame Pcal_1127 annotated as ribose-5-phosphate pyrophosphokinase. To examine the properties of Pcal_1127 the coding gene was cloned, expressed in Escherichia coli, and the purified gene product was characterized. Pcal_1127 exhibited higher activity when ATP was replaced by dATP as pyrophosphate donor. Phosphate and EDTA activated the enzyme activity and equivalent amount of activity was detected with ATP and dATP in their presence. Recombinant Pcal_1127 could utilize all the four nucleotides as pyrophosphate donors with a marked preference for ATP. Optimum temperature and pH for the enzyme activity were 55 °C and 10.5, respectively. A unique feature of Pcal_1127 was its stability against temperature as well as denaturants. Pcal_1127 exhibited more than 95 % residual activity after heating for 4 h at 90 °C and a half-life of 15 min in the boiling water. The enzyme activity was not affected by the presence of 8 M urea or 4 M guanidinium chloride. Pcal_1127 was a highly efficient enzyme with a catalytic efficiency of 5183 mM-1 s-1. These features make Pcal_1127, a novel and unique ribose-5-phosphate pyrophosphokinase.
Assuntos
Proteínas de Bactérias/genética , Temperatura Alta , Pyrobaculum/enzimologia , Ribose-Fosfato Pirofosfoquinase/genética , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Pyrobaculum/genética , Ribose-Fosfato Pirofosfoquinase/química , Ribose-Fosfato Pirofosfoquinase/metabolismoRESUMO
The 5'-mRNA-cap formation is a conserved process in protection of mRNA in eukaryotic cells, resulting in mRNA stability and efficient translation. In humans, two methyltransferases, RNA cap guanine-N7 methyltransferase (hRNMT) and cap-specific nucleoside-2'-O-methyltransferase 1 (hCMTr1) methylate the mRNA resulting in cap0 (N7mGpppN-RNA) and cap1 (N7mGpppN2'-Om-RNA) formation, respectively. Coronaviruses mimic this process by capping their RNA to evade human immune systems. The coronaviral nonstructural proteins, nsp14 and nsp10-nsp16, catalyze the same reactions as hRNMT and hCMTr1, respectively. These two viral enzymes are important targets for development of inhibitor-based antiviral therapeutics. However, assessing the selectivity of such inhibitors against human corresponding proteins is crucial. Human RNMTs have been implicated in proliferation of cancer cells and are also potential targets for development of anticancer therapeutics. Here, we report the development and optimization of a radiometric assay for hRNMT, full kinetic characterization of its activity, and optimization of the assay for high-throughput screening with a Z-factor of 0.79. This enables selectivity determination for a large number of hits from various screening of coronaviral methyltransferases, and also screening hRNMT for discovery of inhibitors and chemical probes that potentially could be used to further investigate the roles RNMTs play in cancers.
Assuntos
Infecções por Coronavirus , Coronavirus , Humanos , Coronavirus/genética , Guanina/metabolismo , Metiltransferases/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Viral/genética , Proteínas não Estruturais Virais/genéticaRESUMO
Arginine and lysine, frequently appearing as a pair on histones, have been proven to carry diverse modifications and execute various epigenetic regulatory functions. However, the most context-specific and transient effectors of these marks, while significant, have evaded study as detection methods have thus far not reached a standard to capture these ephemeral events. Herein, a pair of complementary photo-arginine/δ-photo-lysine (R-dz/K-dz) probes is developed and involve these into histone peptide, nucleosome, and chromatin substrates to capture and explore the interactomes of Arg and Lys hPTMs. By means of these developed tools, this study identifies that H3R2me2a can recruit MutS protein homolog 6 (MSH6), otherwise repelDouble PHD fingers 2 (DPF2), Retinoblastoma binding protein 4/7 (RBBP4/7). And it is disclosed that H3R2me2a inhibits the chromatin remodeling activity of the cBAF complex by blocking the interaction between DPF2 (one component of cBAF) and the nucleosome. In addition, the novel pairs of H4K5 PTMs and respective readers are highlighted, namely H4K5me-Lethal(3)malignant brain tumor-like protein 2 (L3MBTL2), H4K5me2-L3MBTL2, and H4K5acK8ac-YEATS domain-containing protein 4 (YEATS4). These powerful tools pave the way for future investigation of related epigenetic mechanisms including but not limited to hPTMs.
Assuntos
Lisina , Nucleossomos , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Histonas/metabolismo , Cromatina , Arginina/metabolismoRESUMO
Despite their widespread impact on human health there are no approved drugs for combating alphavirus infections. The heterocyclic ß-aminomethyl vinyl sulfone RA-0002034 (1a) is a potent irreversible covalent inhibitor of the alphavirus nsP2 cysteine protease with broad spectrum antiviral activity. Analogs of 1a that varied each of three regions of the molecule were synthesized to establish structure-activity relationships for inhibition of Chikungunya (CHIKV) nsP2 protease and viral replication. The covalent warhead was highly sensitive to modifications of the sulfone or vinyl substituents. However, numerous alterations to the core 5-membered heterocycle and its aryl substituent were well tolerated and several analogs were identified that enhanced CHIKV nsP2 binding. For example, the 4-cyanopyrazole analog 8d exhibited a kinact /Ki ratio >10,000 M-1s-1. 3-Arylisoxazole was identified an isosteric replacement for the 5-membered heterocycle, which circumvented the intramolecular cyclization that complicated the synthesis of pyrazole-based inhibitors like 1a. The accumulated structure-activity data was used to build a ligand-based model of the enzyme active site, which can be used to guide the design of covalent nsP2 protease inhibitors as potential therapeutics against alphaviruses.
RESUMO
The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to significant global morbidity and mortality. A crucial viral protein, the non-structural protein 14 (nsp14), catalyzes the methylation of viral RNA and plays a critical role in viral genome replication and transcription. Due to the low mutation rate in the nsp region among various SARS-CoV-2 variants, nsp14 has emerged as a promising therapeutic target. However, discovering potential inhibitors remains a challenge. In this work, we introduce a computational pipeline for the rapid and efficient identification of potential nsp14 inhibitors by leveraging virtual screening and the NCI open compound collection, which contains 250,000 freely available molecules for researchers worldwide. The introduced pipeline provides a cost-effective and efficient approach for early-stage drug discovery by allowing researchers to evaluate promising molecules without incurring synthesis expenses. Our pipeline successfully identified seven promising candidates after experimentally validating only 40 compounds. Notably, we discovered NSC620333, a compound that exhibits a strong binding affinity to nsp14 with a dissociation constant of 427 ± 84 nM. In addition, we gained new insights into the structure and function of this protein through molecular dynamics simulations. We identified new conformational states of the protein and determined that residues Phe367, Tyr368, and Gln354 within the binding pocket serve as stabilizing residues for novel ligand interactions. We also found that metal coordination complexes are crucial for the overall function of the binding pocket. Lastly, we present the solved crystal structure of the nsp14-MTase complexed with SS148 (PDB:8BWU), a potent inhibitor of methyltransferase activity at the nanomolar level (IC50 value of 70 ± 6 nM). Our computational pipeline accurately predicted the binding pose of SS148, demonstrating its effectiveness and potential in accelerating drug discovery efforts against SARS-CoV-2 and other emerging viruses.
RESUMO
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has been responsible for numerous large-scale outbreaks in the last twenty years. Currently, there are no FDA-approved therapeutics for any alphavirus infection. CHIKV non-structural protein 2 (nsP2), which contains a cysteine protease domain, is essential for viral replication, making it an attractive target for a drug discovery campaign. Here, we optimized a CHIKV nsP2 protease (nsP2pro) biochemical assay for the screening of a 6,120-compound cysteine-directed covalent fragment library. Using a 50% inhibition threshold, we identified 153 hits (2.5% hit rate). In dose-response follow up, RA-0002034, a covalent fragment that contains a vinyl sulfone warhead, inhibited CHIKV nsP2pro with an IC 50 of 58 ± 17 nM, and further analysis with time-dependent inhibition studies yielded a k inact /K I of 6.4 x 10 3 M -1 s -1 . LC-MS/MS analysis determined that RA-0002034 covalently modified the catalytic cysteine in a site-specific manner. Additionally, RA-0002034 showed no significant off-target reactivity against a panel of cysteine proteases. In addition to the potent biochemical inhibition of CHIKV nsP2pro activity and exceptional selectivity, RA-0002034 was tested in cellular models of alphavirus infection and effectively inhibited viral replication of both CHIKV and related alphaviruses. This study highlights the discovery and characterization of the chemical probe RA-0002034 as a promising hit compound from covalent fragment-based screening for development toward a CHIKV or pan-alphavirus therapeutic. Significance Statement: Chikungunya virus is one of the most prominent and widespread alphaviruses and has caused explosive outbreaks of arthritic disease. Currently, there are no FDA-approved drugs to treat disease caused by chikungunya virus or any other alphavirus-caused infection. Here, we report the discovery of a covalent small molecule inhibitor of chikungunya virus nsP2 protease activity and viral replication of four diverse alphaviruses. This finding highlights the utility of covalent fragment screening for inhibitor discovery and represents a starting point towards the development of alphavirus therapeutics targeting nsP2 protease.
RESUMO
We have developed a novel chemical handle (PFI-E3H1) and a chemical probe (PFI-7) as ligands for the Gid4 subunit of the human E3 ligase CTLH degradation complex. Through an efficient initial hit-ID campaign, structure-based drug design (SBDD) and leveraging the sizeable Pfizer compound library, we identified a 500 nM ligand for this E3 ligase through file screening alone. Further exploration identified a vector that is tolerant to addition of a linker for future chimeric molecule design. The chemotype was subsequently optimized to sub-100 nM Gid4 binding affinity for a chemical probe. These novel tools, alongside the suitable negative control also identified, should enable the interrogation of this complex human E3 ligase macromolecular assembly.
RESUMO
Seven coronaviruses have infected humans (HCoVs) to-date. SARS-CoV-2 caused the current COVID-19 pandemic with the well-known high mortality and severe socioeconomic consequences. MERS-CoV and SARS-CoV caused epidemic of MERS and SARS, respectively, with severe respiratory symptoms and significant fatality. However, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43 cause respiratory illnesses with less severe symptoms in most cases. All coronaviruses use RNA capping to evade the immune systems of humans. Two viral methyltransferases, nsp14 and nsp16, play key roles in RNA capping and are considered valuable targets for development of anti-coronavirus therapeutics. But little is known about the kinetics of nsp10-nsp16 methyltransferase activities of most HCoVs, and reliable assays for screening are not available. Here, we report the expression, purification, and kinetic characterization of nsp10-nsp16 complexes from six HCoVs in parallel with previously characterized SARS-CoV-2. Probing the active sites of all seven by SS148 and WZ16, the two recently reported dual nsp14 / nsp10-nsp16 inhibitors, revealed pan-inhibition. Overall, our study show feasibility of developing broad-spectrum dual nsp14 / nsp10-nsp16-inhibitor therapeutics.
Assuntos
COVID-19 , Humanos , Metiltransferases/química , Pandemias , RNA , SARS-CoV-2/genéticaRESUMO
Cbl-b is a RING-type E3 ubiquitin ligase that is expressed in several immune cell lineages, where it negatively regulates the activity of immune cells. Cbl-b has specifically been identified as an attractive target for cancer immunotherapy due to its role in promoting an immunosuppressive tumor environment. A Cbl-b inhibitor, Nx-1607, is currently in phase I clinical trials for advanced solid tumor malignancies. Using a suite of biophysical and cellular assays, we confirm potent binding of C7683 (an analogue of Nx-1607) to the full-length Cbl-b and its N-terminal fragment containing the TKBD-LHR-RING domains. To further elucidate its mechanism of inhibition, we determined the co-crystal structure of Cbl-b with C7683, revealing the compound's interaction with both the TKBD and LHR, but not the RING domain. Here, we provide structural insights into a novel mechanism of Cbl-b inhibition by a small-molecule inhibitor that locks the protein in an inactive conformation by acting as an intramolecular glue.
Assuntos
Neoplasias , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/química , Ligação Proteica , Conformação Molecular , FosforilaçãoRESUMO
SARS-CoV-2 non-structural protein 13 (nsp13) is a highly conserved helicase and RNA 5'-triphosphatase. It uses the energy derived from the hydrolysis of nucleoside triphosphates for directional movement along the nucleic acids and promotes the unwinding of double-stranded nucleic acids. Nsp13 is essential for replication and propagation of all human and non-human coronaviruses. Combined with its defined nucleotide binding site and druggability, nsp13 is one of the most promising candidates for the development of pan-coronavirus therapeutics. Here, we report the development and optimization of bioluminescence assays for kinetic characterization of nsp13 ATPase activity in the presence and absence of single-stranded DNA. Screening of a library of 5000 small molecules in the presence of single-stranded DNA resulted in the discovery of six nsp13 small-molecule inhibitors with IC50 values ranging from 6 ± 0.5 to 50 ± 6 µM. In addition to providing validated methods for high-throughput screening of nsp13 in drug discovery campaigns, the reproducible screening hits we present here could potentially be chemistry starting points toward the development of more potent and selective nsp13 inhibitors, enabling the discovery of antiviral therapeutics.
Assuntos
Metiltransferases/metabolismo , RNA Helicases/metabolismo , SARS-CoV-2/química , Proteínas não Estruturais Virais/metabolismo , Adenosina Trifosfatases , COVID-19/virologia , DNA de Cadeia Simples , Humanos , Metiltransferases/antagonistas & inibidores , Ácidos Nucleicos/metabolismo , RNA Helicases/antagonistas & inibidores , SARS-CoV-2/fisiologia , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
Anemia is a common complication of certain chronic diseases and can be treated by stimulating hematopoietic cells to increase red blood cell count, and this action is achieved by recombinant human erythropoietin. In this review article, we have discussed about hypertension, which develops as a result of erythropoietin therapy. We have explored the pathogenesis of erythropoietin-induced hypertension and discussed some ways to prevent and treat this condition. Also, an attempt has been made to find out the role of blood viscosity in erythropoietin-induced hypertension. We conducted a comprehensive review of literature by collecting data from online databases like PubMed and Google Scholar. We mainly studied clinical trials that unraveled the mechanism of hypertension caused by erythropoietin. Hypertension is mainly caused due to enhanced vascular responsiveness to constrictors and impaired action of vasodilators. Role of blood viscosity in the pathogenesis of hypertension is doubtful due to the lack of consistency in the studies. Incidence of hypertension can be reduced by achieving slow correction of anemia and by switching to subcutaneous route of administration. Conventional anti-hypertensives have been found to be beneficial in the treatment. In some severe and persistent cases, temporary discontinuation of erythropoietin may be needed.
RESUMO
Toxic shock syndrome (TSS) is an uncommon complication of infection caused by streptococci and staphylococci. It is associated with a high mortality rate. When evaluating patients with shock symptoms from skin or soft tissue sources, a high index of suspicion for TSS must be maintained. Prompt diagnosis and integrative management with surgical intervention, antibiotics, hemodynamic stabilization, and adjuvants like intravenous immunoglobulins improve survival.
RESUMO
Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, necessitate the development of therapeutics that could be easily and effectively administered worldwide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA capping through its 2'-O-methylation activity. Like other methyltransferases, the SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for the methyltransferase activity of the nsp10-nsp16 complex in a 384-well format, kinetic characterization, and optimization of the assay for HTS (Z' factor = 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting the SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.
Assuntos
Adenosina/análogos & derivados , Ensaios de Triagem em Larga Escala , Capuzes de RNA/antagonistas & inibidores , RNA Viral/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Adenosina/química , Adenosina/farmacologia , COVID-19/virologia , Clonagem Molecular , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Metilação , Metiltransferases , Modelos Moleculares , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , Trítio , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process, including the nonstructural protein 16 (nsp16), which is an S-adenosyl-l-methionine (SAM)-dependent 2'-O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in a 384-well format with a Z' factor of 0.6, suitable for high-throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, the product of the reaction, S-adenosyl-l-homocysteine (SAH), and a common methyltransferase inhibitor, sinefungin, using isothermal titration calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high-throughput method for screening the nsp10-nsp16 complex for RNA competitive inhibitors toward developing COVID-19 therapeutics.
Assuntos
Antivirais/farmacologia , Ensaios de Triagem em Larga Escala , RNA Viral/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Adenosina/análogos & derivados , Adenosina/farmacologia , Ligação Competitiva , COVID-19/virologia , Inibidores Enzimáticos/farmacologia , Polarização de Fluorescência , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Metiltransferases , Ligação Proteica , Capuzes de RNA/antagonistas & inibidores , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Transdução de Sinais , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação Viral , Tratamento Farmacológico da COVID-19RESUMO
In this study, we have focused on the structure-based design of the inhibitors of one of the two SARS-CoV-2 methyltransferases (MTases), nsp14. This MTase catalyzes the transfer of the methyl group from S-adenosyl-l-methionine (SAM) to cap the guanosine triphosphate moiety of the newly synthesized viral RNA, yielding the methylated capped RNA and S-adenosyl-l-homocysteine (SAH). As the crystal structure of SARS-CoV-2 nsp14 is unknown, we have taken advantage of its high homology to SARS-CoV nsp14 and prepared its homology model, which has allowed us to identify novel SAH derivatives modified at the adenine nucleobase as inhibitors of this important viral target. We have synthesized and tested the designed compounds in vitro and shown that these derivatives exert unprecedented inhibitory activity against this crucial enzyme. The docking studies nicely explain the contribution of an aromatic part attached by a linker to the position 7 of the 7-deaza analogues of SAH.
Assuntos
COVID-19 , Metiltransferases , Exorribonucleases , Humanos , Ligantes , Metiltransferases/genética , SARS-CoV-2 , Proteínas não Estruturais ViraisRESUMO
The COVID-19 pandemic has clearly brought the healthcare systems worldwide to a breaking point, along with devastating socioeconomic consequences. The SARS-CoV-2 virus, which causes the disease, uses RNA capping to evade the human immune system. Nonstructural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small-molecule inhibitors of nsp14 methyltransferase (MTase) activity, we developed and employed a radiometric MTase assay to screen a library of 161 in-house synthesized S-adenosylmethionine (SAM) competitive MTase inhibitors and SAM analogs. Among six identified screening hits, SS148 inhibited nsp14 MTase activity with an IC50 value of 70 ± 6 nM and was selective against 20 human protein lysine MTases, indicating significant differences in SAM binding sites. Interestingly, DS0464 with an IC50 value of 1.1 ± 0.2 µM showed a bisubstrate competitive inhibitor mechanism of action. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein MTases. The structure-activity relationship provided by these compounds should guide the optimization of selective bisubstrate nsp14 inhibitors and may provide a path toward a novel class of antivirals against COVID-19, and possibly other coronaviruses.
Assuntos
COVID-19/genética , Exorribonucleases/genética , Ligação Proteica/genética , SARS-CoV-2/genética , Proteínas não Estruturais Virais/genética , Antivirais/farmacologia , Sítios de Ligação/genética , COVID-19/virologia , Humanos , Metilação , Pandemias , RNA Viral/genética , SARS-CoV-2/patogenicidade , Replicação Viral/genéticaRESUMO
The COVID-19 pandemic has clearly brought the healthcare systems world-wide to a breaking point along with devastating socioeconomic consequences. The SARS-CoV-2 virus which causes the disease uses RNA capping to evade the human immune system. Non-structural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small molecule inhibitors of nsp14 methyltransferase (MT) activity, we developed and employed a radiometric MT assay to screen a library of 161 in house synthesized S-adenosylmethionine (SAM) competitive methyltransferase inhibitors and SAM analogs. Among seven identified screening hits, SS148 inhibited nsp14 MT activity with an IC 50 value of 70 ± 6 nM and was selective against 20 human protein lysine methyltransferases indicating significant differences in SAM binding sites. Interestingly, DS0464 with IC 50 value of 1.1 ± 0.2 µM showed a bi-substrate competitive inhibitor mechanism of action. Modeling the binding of this compound to nsp14 suggests that the terminal phenyl group extends into the RNA binding site. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein methyltransferases. The structure-activity relationship provided by these compounds should guide the optimization of selective bi-substrate nsp14 inhibitors and may provide a path towards a novel class of antivirals against COVID-19, and possibly other coronaviruses.