RESUMEN
We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Proteasas 3C de Coronavirus , Inhibidores de Proteasa de Coronavirus , Descubrimiento de Drogas , SARS-CoV-2 , Humanos , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/química , Simulación del Acoplamiento Molecular , Inhibidores de Proteasa de Coronavirus/síntesis química , Inhibidores de Proteasa de Coronavirus/química , Inhibidores de Proteasa de Coronavirus/farmacología , Relación Estructura-Actividad , Cristalografía por Rayos XRESUMEN
A phosphine-catalyzed domino assembly of six units of 2-bromomethyl acrylates afforded polyalkenyl adducts containing two cyclohexenyl rings. This reaction occurs under mild conditions providing the final product by formation of seven carbon-carbon bonds and four stereocenters. Experimental and computational studies support an initial dimerization of the substrate, which in turn trimerizes involving two totally regio- and stereocontrolled Diels-Alder cycloadditions. The yield of the hexamerization of the 2-bromomethyl acrylates depends on the size of the ester function. The protocol has also proved to be practicable on a gram scale.
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Many studies have demonstrated how the pyrrolidine nucleus is more efficient than the corresponding piperidine or morpholine as organocatalysts in the condensation of aldehydes with electrophiles via enamine. Focussing on morpholine-enamines, their low reactivity is ascribed to the presence of oxygen on the ring and to the pronounced pyramidalisation of nitrogen, decreasing the nucleophilicity of the enamine. Thus, the selection of efficient morpholine organocatalysts appears to be a difficult challenge. Herein, we reported on the synthesis of new organocatalysts belonging to the class of ß-morpholine amino acids that were tested in a model reaction, i.e., the 1,4-addition reaction of aldehydes to nitroolefins. Starting from commercially available amino acids and epichlorohydrin, we designed an efficient synthesis for the aforementioned catalysts, controlling the configuration and the substitution pattern. Computational studies indeed disclosed the transition state of the reaction, explaining why, despite all the limitations of the morpholine ring for enamine catalysis, our best catalyst works efficiently, affording condensation products with excellent yields, diastereoselection and good-to-exquisite enantioselectivity.
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Though the bulk of the COVID-19 pandemic is behind, the search for effective and safe anti-SARS-CoV-2 drugs continues to be relevant. A highly pursued approach for antiviral drug development involves targeting the viral spike (S) protein of SARS-CoV-2 to prevent its attachment to the cellular receptor ACE2. Here, we exploited the core structure of polymyxin B, a naturally occurring antibiotic, to design and synthesize unprecedented peptidomimetics (PMs), intended to target contemporarily two defined, non-overlapping regions of the S receptor-binding domain (RBD). Monomers 1, 2, and 8, and heterodimers 7 and 10 bound to the S-RBD with micromolar affinity in cell-free surface plasmon resonance assays (KD ranging from 2.31 µM to 2.78 µM for dimers and 8.56 µM to 10.12 µM for monomers). Although the PMs were not able to fully protect cell cultures from infection with authentic live SARS-CoV-2, dimer 10 exerted a minimal but detectable inhibition of SARS-CoV-2 entry in U87.ACE2+ and A549.ACE2.TMPRSS2+ cells. These results validated a previous modeling study and provided the first proof-of-feasibility of using medium-sized heterodimeric PMs for targeting the S-RBD. Thus, heterodimers 7 and 10 may serve as a lead for the development of optimized compounds, which are structurally related to polymyxin, with improved S-RBD affinity and anti-SARS-CoV-2 potential.
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COVID-19 , Peptidomiméticos , Humanos , SARS-CoV-2 , Peptidomiméticos/farmacología , Sitios de Unión , Enzima Convertidora de Angiotensina 2/química , Polimixinas , Pandemias , Unión ProteicaRESUMEN
Accelerated molecular dynamics (aMD) protocols were assessed on predicting the secondary structure of eight peptides, of which two are helical, three are ß-hairpins, and three are disordered. Protocols consisted of combinations of three force fields (ff99SB, ff14SB, ff19SB) and two explicit solvation models (TIP3P and OPC), and were evaluated in two independent aMD simulations, one starting from an extended conformation, the other starting from a misfolded conformation. The results of these analyses indicate that all three combinations performed well on helical peptides. As for ß-hairpins, ff19SB performed well with both solvation methods, with a slight preference for the TIP3P solvation model, even though performance was dependent on both peptide sequence and initial conformation. The ff19SB/OPC combination had the best performance on intrinsically disordered peptides. In general, ff14SB/TIP3P suffered the strongest helical bias.
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Benchmarking , Simulación de Dinámica Molecular , Solventes/química , Péptidos/química , Estructura Secundaria de Proteína , Agua/químicaRESUMEN
Structures composed of alternating α and ß amino acids can give rise to peculiar secondary structural motifs, which could self-assemble into complex structures of controlled geometries. This work describes the self-assembly properties of an α,ß-peptide, containing three units of syn H2-(2-F-Phe)-h-PheGly-OH, able to self-organize on surfaces into a fascinating supramolecular rope. This material was characterized by AFM, electronic conduction and fluorescence measurements. Molecular dynamics simulations showed that this hexapeptide can self-assemble into an antiparallel ß-sheet layer, stabilized by intermolecular H-bonds, which, in turn, can self-assemble into many side-by-side layers, due to π-π interactions. As a matter of fact, we demonstrated that in this system, the presence of aromatic residues at the intramolecular interface promoted by the alternation of α,ß-amino-acids in the primary sequence, endorses the formation of a super-secondary structure where the aromatic groups are close to each other, conferring to the system good electron conduction properties. This work demonstrates the capability and future potential of designing and fabricating distinctive nanostructures and efficient bioelectronic interfaces based on an α,ß-peptide, by controlling structure and interaction processes beyond those obtained with α- or ß-peptides alone.
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The design and synthesis of a series of peptide derivatives based on a short ACE2 α-helix 1 epitope and subsequent [i - i+4] stapling of the secondary structure resulted in the identification of a 9-mer peptide capable to compete with recombinant ACE2 towards Spike RBD in the micromolar range. Specifically, SARS-CoV-2 Spike inhibitor screening based on colorimetric ELISA assay and structural studies by circular dichroism showed the ring-closing metathesis cyclization being capable to stabilize the helical structure of the 9-mer 34HEAEDLFYQ42 epitope better than the triazole stapling via click chemistry. MD simulations showed the stapled peptide being able not only to bind the Spike RBD, sterically interfering with ACE2, but also showing higher affinity to the target as compared to parent epitope.
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COVID-19 , Glicoproteína de la Espiga del Coronavirus , Humanos , Enzima Convertidora de Angiotensina 2 , SARS-CoV-2 , Epítopos , Péptidos/farmacología , Unión ProteicaRESUMEN
Asparagine endopeptidase (AEP), also called legumain, is a pH-dependent endolysosomal cysteine protease that cleaves its substrates after asparagine residues. Recent studies showed that it possesses δ-secretase activity and that it is implicated in numerous neurological diseases such as Alzheimer's disease (AD). Following evidence of aryl-morpholines as useful asparagine endopeptidase inhibitors, a series of morpholinoanilines with diverse substituents at ortho position were synthesized in view of improving the potency and scope of this molecular scaffold, allowing to identify ethyl 2-isonipecotate-4-morpholinoaniline possessing inhibition potency in the nanomolar range. CNS MPO (CNS MultiParameter Optimization) calculations revealed that most of the compounds developed in this work show physicochemical parameters in the desirable range for CNS drug candidates.
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Enfermedad de Alzheimer , Cisteína Endopeptidasas , Enfermedad de Alzheimer/tratamiento farmacológico , Fármacos del Sistema Nervioso Central/uso terapéutico , Cisteína Endopeptidasas/química , Humanos , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/uso terapéuticoRESUMEN
Malaria accounts for millions of cases and thousands of deaths every year. In the absence of an effective vaccine, drugs are still the most important tool in the fight against the disease. Plasmodium parasites developed resistance to all classes of known antimalarial drugs. Thus, the search for antimalarial drugs with novel mechanisms of action is compelling. The human GTPase Rac1 plays a role in parasite invasion of the host cell in many intracellular pathogens. Also, in Plasmodium falciparum, the involvement of Rac1 during both the invasion process and parasite intracellular development was suggested. The aim of this work is to test a panel of Rac1 inhibitors as potential antimalarial drugs. Fourteen commercially available or newly synthesized inhibitors of Rac1 were tested for antimalarial activity. Among these, EHop-016 was the most effective against P. falciparum in vitro, with nanomolar 50% inhibitory concentrations (IC50s) (138.8 ± 16.0 nM on the chloroquine-sensitive D10 strain and 321.5 ± 28.5 nM on the chloroquine-resistant W2 strain) and a selectivity index of 37.8. EHop-016 did not inhibit parasite invasion of red blood cells but affected parasite growth inside them. Among the tested Rac1 inhibitors, EHop-016 showed promising activity that raises attention to this class of molecules as potential antimalarials and deserves further investigation.
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Antimaláricos , GTP Fosfohidrolasas , Malaria Falciparum , Proteína de Unión al GTP rac1 , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Cloroquina/farmacología , Cloroquina/uso terapéutico , GTP Fosfohidrolasas/antagonistas & inhibidores , Humanos , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum , Proteína de Unión al GTP rac1/antagonistas & inhibidoresRESUMEN
In our previous study, we had identified a 9-mer peptide (FSHß (89-97)) derived from seat belt loop of human FSHß and demonstrated its ability to function as FSHR antagonist in vivo. Structure analysis revealed that the four central residues 91STDC94 within this peptide may not be critical for receptor binding. In the present study, 91STDC94 residues were substituted with alanine to generate ΔFSHß 89-97(91STDC94/AAAA) peptide. Analogous to the parent peptide, ΔFSHß 89-97(91STDC94/AAAA) peptide inhibited binding of iodinated FSH to rat FSHR and reduced FSH-induced cAMP production. The peptide could impede granulosa cell proliferation leading to reduction in FSH-mediated ovarian weight gain in immature female rats. In these rats, peptide administration further downregulated androgen receptor and estrogen receptor-alpha expression and upregulated estrogen receptor-beta expression. The results indicate that substitution of 91STDC94 with alanine did not significantly alter FSHR antagonist activity of FSHß (89-97) peptide implying that these residues are not critical for FSH-FSHR interaction and can be replaced with non-peptidic moieties for development of more potent peptidomimetics.
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Diseño de Fármacos , Hormona Folículo Estimulante/farmacología , Péptidos/farmacología , Peptidomiméticos , Receptores de HFE/antagonistas & inhibidores , Animales , Sitios de Unión/efectos de los fármacos , Femenino , Hormona Folículo Estimulante/química , Humanos , Modelos Moleculares , Ovario/efectos de los fármacos , Péptidos/químicaRESUMEN
Finasteride, a 5-alpha reductase (5α-R) inhibitor, is a widely used drug for treating androgen-dependent conditions. However, its use is associated with sexual, psychological, and physical complaints, suggesting that other mechanisms, in addition to 5α-R inhibition, may be involved. Here, a multidisciplinary approach has been used to identify potential finasteride off-target proteins. SPILLO-PBSS software suggests an additional inhibitory activity of finasteride on phenylethanolamine N-methyltransferase (PNMT), the limiting enzyme in formation of the stress hormone epinephrine. The interaction of finasteride with PNMT was supported by docking and molecular dynamics analysis and by in vitro assay, confirming the inhibitory nature of the binding. Finally, this inhibition was also confirmed in an in vivo rat model. Literature data indicate that PNMT activity perturbation may be correlated with sexual and psychological side effects. Therefore, results here obtained suggest that the binding of finasteride to PNMT might have a role in producing the side effects exerted by finasteride treatment.
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Inhibidores de 5-alfa-Reductasa/química , Finasterida/química , Feniletanolamina N-Metiltransferasa/metabolismo , Inhibidores de 5-alfa-Reductasa/metabolismo , Inhibidores de 5-alfa-Reductasa/farmacología , Animales , Sitios de Unión , Unión Competitiva , Catecolaminas/análisis , Catecolaminas/metabolismo , Cromatografía Líquida de Alta Presión , Bases de Datos de Proteínas , Epinefrina/metabolismo , Finasterida/metabolismo , Finasterida/farmacología , Humanos , Masculino , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Feniletanolamina N-Metiltransferasa/química , Unión Proteica , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem , TermodinámicaRESUMEN
The chemical structure of oligonucleotide analogues dictates the conformation of oligonucleotide analogue oligomers, their ability to hybridize complementary DNA and RNA, their stability to degradation and their pharmacokinetic properties. In a study aimed at investigating new analogues featuring a neutral backbone, we explored the ability of oligomers containing a morpholino-peptide backbone to bind oligonucleotides. Circular Dichroism studies revealed the ability of our oligomers to interact with DNA, molecular modelling studies revealed the interaction responsible for complex stabilization.
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ADN/metabolismo , Morfolinos/síntesis química , Morfolinos/metabolismo , Péptidos/síntesis química , Péptidos/metabolismo , Dicroismo Circular , ADN/química , Modelos Moleculares , Conformación Molecular , Morfolinos/química , Péptidos/químicaRESUMEN
The lack of accurate and easily applicable methods for the diagnosis of liver fibrosis, a disease characterized by an accumulation of the extracellular matrix released by activated hepatic stellate cells (HSCs), has been a major limitation for the clinical management of liver diseases. The identification of biomarkers specific to liver microstructure alterations, combined with a non-invasive optical imaging modality, could guide clinicians towards a therapeutic strategy. In this study, structural information of the insulin-like growth factor 2 receptor (IGF2R), an overexpressed protein on activated HSCs, was used for in silico screening of novel IGF2R-specific peptide ligands. Molecular dynamics simulations, followed by computational alanine scanning of the IGF2R/IGF2 complex, led to the identification of a putative peptide sequence containing the most relevant amino acids for the receptor-ligand interaction (IGF2 E12-C21). The Residue Scan tool, implemented in the MOE software, was then used to optimize the binding affinity of this sequence by amino acid mutations. The designed peptides and their associated scrambled sequences were fluorescently labelled and their binding affinity to LX-2 cells (model for activated human HSCs) was tested using flow cytometry and confocal microscopy. In vitro binding was verified for all sequences (KD ≤ 13.2 µM). With respect to the putative binding sequence, most mutations led to an increased affinity. All sequences have shown superior binding compared to their associated scrambled sequences. Using HPLC, all peptides were tested in vitro for their proteolytic resistance and showed a stability of ≥60% intact after 24 h at 37 °C in 50% v/v FBS. In view of their prospective diagnostic application, a comparison of binding affinity was performed in perpetuated and quiescent-like LX-2 cells. Furthermore, the IGF2R expression for different cell phenotypes was analysed by a quantitative mass spectrometric approach. Our peptides showed increased binding to the perpetuated cell state, indicating their good selectivity for the diagnostically relevant phenotype. In summary, the increased binding affinity of our peptides towards perpetuated LX-2 cells, as well as the satisfactory proteolytic stability, proves that the in silico designed sequences offer a new potential strategy for the targeting of hepatic fibrosis.
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Transdiferenciación Celular , Simulación por Computador , Células Estrelladas Hepáticas/patología , Cirrosis Hepática/patología , Péptidos/metabolismo , Receptor IGF Tipo 2/metabolismo , Línea Celular , Humanos , Factor II del Crecimiento Similar a la Insulina/química , Factor II del Crecimiento Similar a la Insulina/metabolismo , Ligandos , Simulación de Dinámica Molecular , Péptidos/química , Conformación Proteica , Estabilidad Proteica , Receptor IGF Tipo 2/químicaRESUMEN
The pandemic caused by SARS-CoV-2 is currently representing a major health and economic threat to humanity. So far, no specific treatment to this viral infection has been developed and the emergency still requires an efficient intervention. In this work, we used virtual screening to facilitate drug repurposing against SARS-CoV-2, targeting viral main proteinase and spike protein with 3000 existing drugs. We used a protocol based on a docking step followed by a short molecular dynamic simulation and rescoring by the Nwat-MMGBSA approach. Our results provide suggestions for prioritizing in vitro and/or in vivo tests of already available compounds.
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Antivirales , Reposicionamiento de Medicamentos/métodos , Péptido Hidrolasas , Glicoproteína de la Espiga del Coronavirus , Antivirales/química , Antivirales/metabolismo , Betacoronavirus/química , Betacoronavirus/enzimología , Betacoronavirus/metabolismo , COVID-19 , Infecciones por Coronavirus/virología , Humanos , Simulación de Dinámica Molecular , Pandemias , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Neumonía Viral/virología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
A new non-natural ß-amino acid, named 3-Ar-ß-Morph, was designed and synthesized via a regio- and diastereoselective Pd-catalyzed C(sp3)H-arylation of the corresponding 2S,6S-(6-methoxymorpholin-2-yl)carboxylic acid, readily available from glucose. According to the computational prevision and confirmed by IR and NMR data, the insertion of 3-Ar-ß-Morph in a model foldamer represents a way to stabilize a PPII-like helix through the presence of two γ-turns, secondary structure motifs induced by the morpholine ring, and the trans-tertiary amide bond.
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Despite a high degree of structural similarity, it is known that MMP2 and MMP9 have distinct roles in the angiogenic switch and in cell migration, as they activate diverse signaling pathways. Indeed, inhibition of MMP2 and MMP9 can show beneficial or detrimental effects depending on the stage of tumor progression. Thus, the selective inhibition of gelatinases is of relevance for a successful drug lead, which has to be achieved despite the high structural similarity of the two gelatinases. Herein, the synthesis and evaluation of d-proline-derived hydroxamic acids containing amino appendages at C-4 as gelatinase inhibitors are reported. Inhibition assays enabled the identification of a > 200-fold selective MMP9 inhibitor when Lys was considered as a C-4 substituent, thus addressing gelatinase selectivity beyond the S1' subsite, which is a major driver for selectivity. Molecular docking studies revealed the basic moiety of Lys as detrimental for inhibition of MMP2 as compared to MMP9.
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Descubrimiento de Drogas , Gelatinasas/antagonistas & inhibidores , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Peptidomiméticos/farmacología , Relación Dosis-Respuesta a Droga , Gelatinasas/metabolismo , Humanos , Inhibidores de la Metaloproteinasa de la Matriz/síntesis química , Inhibidores de la Metaloproteinasa de la Matriz/química , Simulación del Acoplamiento Molecular , Estructura Molecular , Peptidomiméticos/síntesis química , Peptidomiméticos/química , Relación Estructura-ActividadRESUMEN
FSH-FSHR interaction is critical for folliculogenesis, spermatogenesis and progression of several cancers. Therefore, FSHR is an attractive target for fertility regulation and cancer therapeutics. Based on homology and structural analysis of hFSH-FSHR(ECD) complex, a minimal continuous stretch within FSHß seat-belt loop (FSHß (89-97)) was identified to be crucial for FSHR interaction. The ability of FSHß (89-97) peptide to neutralize FSHR activity was evaluated by a panel of in vitro and in vivo experiments. The synthetic peptide significantly inhibited binding of [125I]-FSH to rat Fshr as well as FSH-induced cAMP production. In immature rats, FSHß (89-97) peptide administration reduced FSH-mediated increase in ovarian weight. The peptide inhibited transition of follicles from pre-antral to antral stage and hindered the cell cycle progression of granulosa cells beyond G0/G1 phase. In adult rats, administration of the peptide inhibited estradiol synthesis and significantly perturbed folliculogenesis.
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Hormona Folículo Estimulante de Subunidad beta/metabolismo , Células de la Granulosa/efectos de los fármacos , Oligopéptidos/farmacología , Folículo Ovárico/efectos de los fármacos , Ovario/metabolismo , Receptores de HFE/antagonistas & inhibidores , Animales , Cristalografía por Rayos X/métodos , Estradiol/biosíntesis , Femenino , Células de la Granulosa/metabolismo , Oligopéptidos/química , Folículo Ovárico/metabolismo , Ovario/efectos de los fármacos , Elementos Estructurales de las Proteínas , Ratas , Ratas Sprague-Dawley , Receptores de HFE/metabolismo , Relación Estructura-ActividadRESUMEN
With the potential of improving virtual screening outcome, MM-PB/GBSA has become a disputed method that requires extensive testing and tuning to provide the optimal results. One of the tuning factors is the internal or solute dielectric constant. We have applied three test sets with receptors of different categories and libraries from different sources to investigate the underlying issue related to this constant. We discovered that increasing internal dielectric value does not improve the virtual screening enrichment qualitatively. More interestingly, nonpolar and polar calculated energies act differently in libraries with different molecular weight distributions. From this work, the performance of MM-PBSA rescoring in virtual screening is more library- than receptor-dependent.