RESUMEN
Despite the approval of vaccines, monoclonal antibodies and restrictions during the pandemic, the demand for new efficacious and safe antivirals is compelling to boost the therapeutic arsenal against the COVID-19. The viral 3-chymotrypsin-like protease (3CLpro) is an essential enzyme for replication with high homology in the active site across CoVs and variants showing an almost unique specificity for Leu-Gln as P2-P1 residues, allowing the development of broad-spectrum inhibitors. The design, synthesis, biological activity, and cocrystal structural information of newly conceived peptidomimetic covalent reversible inhibitors are herein described. The inhibitors display an aldehyde warhead, a Gln mimetic at P1 and modified P2-P3 residues. Particularly, functionalized proline residues were inserted at P2 to stabilize the ß-turn like bioactive conformation, modulating the affinity. The most potent compounds displayed low/sub-nM potency against the 3CLpro of SARS-CoV-2 and MERS-CoV and inhibited viral replication of three human CoVs, i.e. SARS-CoV-2, MERS-CoV, and HCoV 229 in different cell lines. Particularly, derivative 12 exhibited nM-low µM antiviral activity depending on the virus, and the highest selectivity index. Some compounds were co-crystallized with SARS-CoV-2 3CLpro validating our design. Altogether, these results foster future work toward broad-spectrum 3CLpro inhibitors to challenge CoVs related pandemics.
Asunto(s)
COVID-19 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Peptidomiméticos , Humanos , SARS-CoV-2 , Inhibidores de Proteasas/química , Peptidomiméticos/farmacología , Peptidomiméticos/química , Rayos X , Péptido Hidrolasas , Antivirales/químicaRESUMEN
Cyanoacrylates define a class of inhibitors which are capable to form a transient covalent bond with a cysteine flanking the binding site, thereby increasing the residence time and prolonging the inhibitory effect on the target protein under nonequilibrium conditions. Herein, we describe the synthetic access to cyanoacrylate-based HDAC4 inhibitors and the procedures for the characterization of the transient nature of the covalent bond between cyanoacrylates and thiols or cysteines in HDAC4.
Asunto(s)
Cianoacrilatos , Cisteína , Cisteína/metabolismo , Sitios de Unión , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/químicaRESUMEN
KRAS is the most frequently mutated oncogene and plays a predominant role in driving initiation and progression of multiple cancers. Attempts to degrade the oncogene KRASG12C with PROTAC strategy have been considered as an alternative strategy to combate cancers. However, the irreversible PROTACs may compromise the substoichiometric activity to decrease the potency. Herein, we report the development of YF135, the first reversible-covalent PROTAC capable of recruiting VHL mediated proteasomal degradation of KRASG12C. YF135 induces the rapid and sustained degradation of endogenous KRASG12C and attenuates pERK signaling in H358 and H23 cells in a reversible manner.
Asunto(s)
Neoplasias , Oligopéptidos/farmacología , Proteínas Proto-Oncogénicas p21(ras) , Carcinogénesis , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Oncogenes , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
As one of the principal etiological agents of hand, foot, and mouth disease (HFMD), enterovirus 71 (EV71) is associated with severe neurological complications or fatal diseases, while without effective medications thus far. Here we applied dually activated Michael acceptor to develop a series of reversible covalent compounds for EV71 3C protease (3Cpro), a promising antiviral drug target that plays an essential role during viral replication by cleaving the precursor polyprotein, inhibiting host protein synthesis, and evading innate immunity. Among them, cyanoacrylate and Boc-protected cyanoarylamide derivatives (SLQ-4 and SLQ-5) showed effective antiviral activity against EV71. The two inhibitors exhibited broad antiviral effects, acting on RD, 293T, and Vero cell lines, as well as on EV71 A, B, C, CVA16, and CVB3 viral strains. We further determined the binding pockets between the two inhibitors and 3Cpro based on docking studies. These results, together with our previous studies, provide evidence to elucidate the mechanism of action of these two reversible covalent inhibitors and contribute to the development of clinically effective medicines to treat EV71 infections.
Asunto(s)
Proteasas Virales 3C/antagonistas & inhibidores , Antivirales/farmacología , Enterovirus Humano A/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Proteasas Virales 3C/química , Acrilamidas/química , Acrilamidas/farmacología , Animales , Antivirales/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cianoacrilatos/química , Cianoacrilatos/farmacología , Enterovirus/clasificación , Enterovirus/efectos de los fármacos , Infecciones por Enterovirus/virología , Humanos , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/química , Replicación Viral/efectos de los fármacosRESUMEN
The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro ) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro ), we have designed and synthesized a series of SC2MPro inhibitors that contain ß-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active-site cysteine C145. All inhibitors display high potency with Ki values at or below 100â nM. The most potent compound, MPI3, has as a Ki value of 8.3â nM. Crystallographic analyses of SC2MPro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5â µM and A549/ACE2 cells at 0.16-0.31â µM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with ultra-high antiviral potency.
Asunto(s)
Antivirales/farmacología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , SARS-CoV-2/efectos de los fármacos , Células A549 , Alanina/análogos & derivados , Alanina/metabolismo , Alanina/farmacología , Animales , Antivirales/síntesis química , Antivirales/metabolismo , Dominio Catalítico , Chlorocebus aethiops , Proteasas 3C de Coronavirus/química , Proteasas 3C de Coronavirus/metabolismo , Cisteína/química , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/metabolismo , Humanos , Pruebas de Sensibilidad Microbiana , Unión Proteica , Pirrolidinonas/síntesis química , Pirrolidinonas/metabolismo , Pirrolidinonas/farmacología , SARS-CoV-2/enzimología , Células VeroRESUMEN
Based on the similarity between the active sites of the deubiquitylating and deneddylating enzyme ChlaDub1 (Cdu1) and the evolutionarily related protease adenain, a target-hopping screening approach on a focused set of adenain inhibitors was investigated. The cyanopyrimidine-based inhibitors identified represent the first active-site-directed small-molecule inhibitors of Cdu1. High-resolution crystal structures of Cdu1 in complex with two covalently bound cyanopyrimidines, as well as with its substrate ubiquitin, were obtained. These structural data were complemented by enzymatic assays and covalent docking studies to provide insight into the substrate recognition of Cdu1, active-site pocket flexibility and potential hotspots for ligand interaction. Combined, these data provide a strong basis for future structure-guided medicinal chemistry optimization of this cyanopyrimidine scaffold into more potent and selective Cdu1 inhibitors.
Asunto(s)
Chlamydia trachomatis/enzimología , Enzimas Desubicuitinizantes/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Proteínas Fúngicas/antagonistas & inhibidores , Pirimidinas/química , Secuencia de Aminoácidos , Dominio Catalítico , Chlamydia trachomatis/química , Cisteína Endopeptidasas/química , Enzimas Desubicuitinizantes/química , Proteínas Fúngicas/química , Humanos , Simulación del Acoplamiento Molecular , Oligopéptidos/química , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Especificidad por SustratoRESUMEN
A reversible Michael addition reaction between thiol nucleophiles and cyanoenones has been previously postulated to be the mechanism-of-action of a new family of reversible covalent drugs. However, the hypothetical Michael adducts in this mechanism have only been detected by spectroscopic methods in solution. Herein, the crystallographic observation of reversible Michael addition with a potent cyanoenone drug candidate by means of the crystalline-sponge method is reported. After inclusion of the cyanoenone substrate, the sponge crystal was treated with a thiol solution. Subsequent crystallographic analysis confirmed the single-crystal-to-single-crystal transformation of the substrate into the impermanent Michael adduct.
Asunto(s)
Preparaciones Farmacéuticas/química , Compuestos de Sulfhidrilo/química , Cristalización , Cristalografía por Rayos X , Modelos Moleculares , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
In light of the major contribution of the reactive warhead to the binding energy trend in reversible covalent transition-state analog inhibitors of serine and cysteine hydrolases, would it be possible to rationally design and quickly filter such warheads, especially for large-scale screening? The previously defined W1 and W2 covalent descriptors quantitatively account for the energetic effect of the covalent bonds reorganization, accompanying enzyme-inhibitor covalent binding. The quantum mechanically calculated W1 and W2 reflect the warhead binding energy by modeling of the enzyme-inhibitor reaction core. Here, we demonstrate the use of these descriptors for warhead filtering, and examine its scope and limitations. The W1 and W2 descriptors provide a tool for rational design of various warheads as universal building blocks of real inhibitors without the requirement of 3D structural information about the target enzyme or QSAR studies. These warheads could then be used as hit structural templates in the subsequent optimization of inhibitors recognition sites.
Asunto(s)
Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Hidrolasas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Sitios de Unión , Hidrolasas/metabolismo , Estructura Molecular , Inhibidores de Proteasas/aislamiento & purificación , Relación Estructura-Actividad CuantitativaRESUMEN
7,8-Diaminopelargonic acid synthase (BioA) of Mycobacterium tuberculosis is a recently validated target for therapeutic intervention in the treatment of tuberculosis (TB). Using biophysical fragment screening and structural characterization of compounds, we have identified a potent aryl hydrazine inhibitor of BioA that reversibly modifies the pyridoxal-5'-phosphate (PLP) cofactor, forming a stable quinonoid. Analogous hydrazides also form covalent adducts that can be observed crystallographically but are incapable of inactivating the enzyme. In the X-ray crystal structures, small molecules induce unexpected conformational remodeling in the substrate binding site. We compared these conformational changes to those induced upon binding of the substrate (7-keto-8-aminopelargonic acid), and characterized the inhibition kinetics and the X-ray crystal structures of BioA with the hydrazine compound and analogues to unveil the mechanism of this reversible covalent modification.