RESUMO
Phylogenetic analysis has demonstrated that some positive-sense RNA viruses can be classified into the picornavirus-like supercluster, which includes picornaviruses, caliciviruses, and coronaviruses. These viruses possess 3C or 3C-like proteases (3Cpro or 3CLpro, respectively), which contain a typical chymotrypsin-like fold and a catalytic triad (or dyad) with a Cys residue as a nucleophile. The conserved key sites of 3Cpro or 3CLpro may serve as attractive targets for the design of broad-spectrum antivirals for multiple viruses in the supercluster. We previously reported the structure-based design and synthesis of potent protease inhibitors of Norwalk virus (NV), a member of the Caliciviridae family. We report herein the broad-spectrum antiviral activities of three compounds possessing a common dipeptidyl residue with different warheads, i.e., an aldehyde (GC373), a bisulfite adduct (GC376), and an α-ketoamide (GC375), against viruses that belong to the supercluster. All compounds were highly effective against the majority of tested viruses, with half-maximal inhibitory concentrations in the high nanomolar or low micromolar range in enzyme- and/or cell-based assays and with high therapeutic indices. We also report the high-resolution X-ray cocrystal structures of NV 3CLpro-, poliovirus 3Cpro-, and transmissible gastroenteritis virus 3CLpro- GC376 inhibitor complexes, which show the compound covalently bound to a nucleophilic Cys residue in the catalytic site of the corresponding protease. We conclude that these compounds have the potential to be developed as antiviral therapeutics aimed at a single virus or multiple viruses in the picornavirus-like supercluster by targeting 3Cpro or 3CLpro.
Assuntos
Antivirais/farmacologia , Coronavirus/efeitos dos fármacos , Norovirus/efeitos dos fármacos , Picornaviridae/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Animais , Antivirais/química , Linhagem Celular , Coronavirus/enzimologia , Cristalografia por Raios X , Cisteína Endopeptidases/química , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Modelos Moleculares , Norovirus/enzimologia , Picornaviridae/enzimologia , Inibidores de Proteases/química , Conformação Proteica , Proteínas Virais/químicaRESUMO
Noroviruses are the most common cause of acute viral gastroenteritis, accounting for >21 million cases annually in the US alone. Norovirus infections constitute an important health problem for which there are no specific antiviral therapeutics or vaccines. In this study, a series of bisulfite adducts derived from representative transition state inhibitors (dipeptidyl aldehydes and α-ketoamides) was synthesized and shown to exhibit anti-norovirus activity in a cell-based replicon system. The ED(50) of the most effective inhibitor was 60 nM. This study demonstrates for the first time the utilization of bisulfite adducts of transition state inhibitors in the inhibition of norovirus 3C-like protease in vitro and in a cell-based replicon system. The approach described herein can be extended to the synthesis of the bisulfite adducts of other classes of transition state inhibitors of serine and cysteine proteases, such as α-ketoheterocycles and α-ketoesters.
Assuntos
Antivirais/química , Norovirus/enzimologia , Peptídeo Hidrolases/química , Inibidores de Proteases/química , Sulfitos/química , Proteínas Virais/antagonistas & inibidores , Animais , Antivirais/síntese química , Antivirais/metabolismo , Células CHO , Cricetinae , Cricetulus , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/síntese química , Inibidores de Proteases/metabolismo , Ligação Proteica , Sulfitos/síntese química , Sulfitos/metabolismo , Proteínas Virais/metabolismoRESUMO
The design, synthesis, and in vitro evaluation of the first macrocyclic inhibitor of 3C and 3C-like proteases of picornavirus, norovirus, and coronavirus are reported. The in vitro inhibitory activity (50% effective concentration) of the macrocyclic inhibitor toward enterovirus 3C protease (CVB3 Nancy strain), and coronavirus (SARS-CoV) and norovirus 3C-like proteases, was determined to be 1.8, 15.5 and 5.1 µM, respectively.
Assuntos
Coronavirus/enzimologia , Compostos Macrocíclicos/farmacologia , Norovirus/enzimologia , Peptídeo Hidrolases/metabolismo , Picornaviridae/enzimologia , Inibidores de Proteases/farmacologia , Relação Dose-Resposta a Droga , Desenho de Fármacos , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Modelos Moleculares , Conformação Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Relação Estrutura-AtividadeRESUMO
The design, synthesis, and evaluation of a series of dipeptidyl α-hydroxyphosphonates is reported. The synthesized compounds displayed high anti-norovirus activity in a cell-based replicon system, as well as high enzyme selectivity.
Assuntos
Antivirais/química , Antivirais/farmacologia , Desenho de Fármacos , Norovirus/efeitos dos fármacos , Organofosfonatos/química , Organofosfonatos/farmacologia , Infecções por Caliciviridae/tratamento farmacológico , Humanos , Simulação de Acoplamento Molecular , Norovirus/enzimologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Relação Estrutura-AtividadeRESUMO
There is currently an unmet need for the development of small-molecule therapeutics for norovirus infection. The piperazine scaffold, a privileged structure embodied in many pharmacological agents, was used to synthesize an array of structurally-diverse derivatives which were screened for anti-norovius activity in a cell-based replicon system. The studies described herein demonstrate for the first time that functionalized piperazine derivatives possess anti-norovirus activity. Furthermore, these studies have led to the identification of two promising compounds (6a and 9l) that can be used as a launching pad for the optimization of potency, cytotoxicity, and drug-like characteristics.
Assuntos
Antivirais/farmacologia , Infecções por Caliciviridae/tratamento farmacológico , Norovirus/metabolismo , Piperazinas/farmacologia , Motivos de Aminoácidos , Linhagem Celular , Química Farmacêutica/métodos , Desenho de Fármacos , Humanos , Modelos Químicos , Norovirus/efeitos dos fármacos , Piperazinas/química , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
A series of structurally-diverse α-ketoamides and α-ketoheterocycles was synthesized and subsequently investigated for inhibitory activity against norovirus 3CL protease in vitro, as well as anti-norovirus activity in a cell-based replicon system. The synthesized compounds were found to inhibit norovirus 3CL protease in vitro and to also exhibit potent anti-norovirus activity in a cell-based replicon system.
Assuntos
Amidas/química , Cisteína Endopeptidases/química , Compostos Heterocíclicos/química , Norovirus/enzimologia , Peptídeos/química , Inibidores de Proteases/química , Amidas/farmacologia , Cisteína Endopeptidases/farmacologia , Compostos Heterocíclicos/farmacologia , Modelos Moleculares , Estrutura Molecular , Norovirus/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Relação Estrutura-AtividadeRESUMO
The development of small molecule therapeutics to combat norovirus infection is of considerable interest from a public health perspective because of the highly contagious nature of noroviruses. A series of amino acid-derived acyclic sulfamide-based norovirus inhibitors has been synthesized and evaluated using a cell-based replicon system. Several compounds were found to display potent anti-norovirus activity, low toxicity, and good aqueous solubility. These compounds are suitable for further optimization of pharmacological and ADMET properties.
Assuntos
Aminoácidos/química , Aminoácidos/farmacologia , Antivirais/química , Antivirais/farmacologia , Norovirus/efeitos dos fármacos , Sulfonamidas/química , Sulfonamidas/farmacologia , Aminoácidos/síntese química , Animais , Antivirais/síntese química , Infecções por Caliciviridae/tratamento farmacológico , Linhagem Celular , Desenho de Fármacos , Humanos , Sulfonamidas/síntese químicaRESUMO
The first series of peptidyl aldehyde inhibitors that incorporate in their structure a glutamine surrogate has been designed and synthesized based on the known substrate specificity of Norwalk virus 3C protease. The inhibitory activity of the compounds with the protease and with a norovirus cell-based replicon system was investigated. Members of this class of compounds exhibited noteworthy activity both in vitro and in a cell-based replicon system.
Assuntos
Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/farmacologia , Desenho de Fármacos , Vírus Norwalk/enzimologia , Técnicas de Química Sintética , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/química , Relação Dose-Resposta a Droga , Modelos Moleculares , Conformação Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A series of broad-spectrum antifungal agents based on the 1,2-benzisothiazol-3(2H)-one scaffold is reported. Preliminary structure-activity relationship studies have established the importance of the presence of the heterocyclic ring, a methyl group, and a phenyl ring for optimal manifestation of antifungal activity.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Fungos/efeitos dos fármacos , Tiazóis/química , Tiazóis/farmacologia , Antifúngicos/síntese química , Compostos Heterocíclicos/química , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade , Tiazóis/síntese químicaRESUMO
A scaffold hopping strategy was employed to identify new chemotypes that inhibit noroviruses. The replacement of the cyclosulfamide scaffold by an array of heterocyclic scaffolds lead to the identification of additional series of compounds that possessed anti-norovirus activity in a cell-based replicon system.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Norovirus/efeitos dos fármacos , Amidas/síntese química , Amidas/química , Amidas/farmacologia , Antivirais/química , Linhagem Celular , Compostos Heterocíclicos/química , Humanos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
A new class of compounds that exhibit anti-norovirus activity in a cell-based system and embody in their structure a cyclosulfamide scaffold has been identified. The structure of the initial hit (compound 2a, ED(50) 4 µM, TD(50) 50 µM) has been prospected by exploiting multiple points of diversity and generating appropriate structure-activity relationships.
Assuntos
Amidas/química , Amidas/farmacologia , Vírus Norwalk/efeitos dos fármacos , Ácidos Sulfônicos/química , Ácidos Sulfônicos/farmacologia , Linhagem Celular Tumoral , Humanos , Estrutura Molecular , Vírus Norwalk/química , Relação Estrutura-AtividadeRESUMO
Norovirus infection constitutes the primary cause of acute viral gastroenteritis. There are currently no vaccines or norovirus-specific antiviral therapeutics available for the management of norovirus infection. Norovirus 3C-like protease is essential for viral replication, consequently, inhibition of this enzyme is a fruitful avenue of investigation that may lead to the emergence of antinorovirus therapeutics. We describe herein the optimization of dipeptidyl inhibitors of norovirus 3C-like protease using iterative SAR, X-ray crystallographic, and enzyme and cell-based studies. We also demonstrate herein in vivo efficacy of an inhibitor using the murine model of norovirus infection.
Assuntos
Norovirus/enzimologia , Peptídeo Hidrolases/química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Proteínas Virais/química , Animais , Antivirais/química , Antivirais/farmacologia , Linhagem Celular/efeitos dos fármacos , Técnicas de Química Sintética , Proteases 3C de Coronavírus , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Inibidores da Dipeptidil Peptidase IV/química , Inibidores da Dipeptidil Peptidase IV/farmacologia , Relação Dose-Resposta a Droga , Desenho de Fármacos , Feminino , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Camundongos Endogâmicos BALB C , Modelos Moleculares , Norovirus/efeitos dos fármacos , Norovirus/patogenicidade , Peptídeo Hidrolases/metabolismo , Conformação Proteica , Relação Estrutura-Atividade , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismoRESUMO
Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against a feline coronavirus in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC(50) in a nanomolar range) and, furthermore, combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in a cell culture system.