RESUMO
A hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia, or c9ALS/FTD. The RNA transcribed from the expansion, r(G4C2)exp, causes various pathologies, including intron retention, aberrant translation that produces toxic dipeptide repeat proteins (DPRs), and sequestration of RNA-binding proteins (RBPs) in RNA foci. Here, we describe a small molecule that potently and selectively interacts with r(G4C2)exp and mitigates disease pathologies in spinal neurons differentiated from c9ALS patient-derived induced pluripotent stem cells (iPSCs) and in two c9ALS/FTD mouse models. These studies reveal a mode of action whereby a small molecule diminishes intron retention caused by the r(G4C2)exp and allows the liberated intron to be eliminated by the nuclear RNA exosome, a multi-subunit degradation complex. Our findings highlight the complexity of mechanisms available to RNA-binding small molecules to alleviate disease pathologies and establishes a pipeline for the design of brain penetrant small molecules targeting RNA with novel modes of action in vivo.
Assuntos
Exossomos , Demência Frontotemporal , Animais , Camundongos , Demência Frontotemporal/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , RNA/genética , Exossomos/metabolismo , Barreira Hematoencefálica/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Encéfalo/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA NuclearRESUMO
Starting from an already known MMP-13 inhibitor, 1, we pursued an SAR-approach focusing on optimizing interactions close to the Zn2+ binding site of the enzyme. We found the oxetane containing compound 32 (MMP-13 IC50 = 42 nM), which exhibited complete inhibition of collagenolysis in in vitro studies and an excellent selectivity profile among the MMP family. Interestingly, docking studies propose that the oxetane ring in 32 is oriented towards the Zn2+ ion for chelating the metal ion. Chelating properties of MMP13-inhibitors are often connected with non-selectivity within the enzyme family. Compound 32 demonstrates a rare example where the selectivity can be explained via combinatorial effects of interactions within the S1' loop and a chelating effect of the oxetane moiety. Furthermore, in vivo pharmacokinetic studies were performed demonstrating a concentration of 1.97 µM of 32 within the synovial fluid of the rat knee joint, which makes the compound a promising lead compound for further optimization and development for osteoarthritis.
Assuntos
Éteres Cíclicos , Inibidores de Metaloproteinases de Matriz , Ratos , Animais , Metaloproteinase 13 da Matriz/química , Metaloproteinase 13 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/química , Quelantes/farmacologia , Quelantes/química , Zinco/químicaRESUMO
Over the last two decades, activity-based protein profiling (ABPP) has been established as a tremendously useful proteomic tool for measuring the activity of proteins in their cellular context, annotating the function of uncharacterized proteins, and investigating the target profile of small-molecule inhibitors. Unlike hydrolases and other enzyme classes, which exhibit a characteristic nucleophilic residue, oxidoreductases have received much less attention in ABPP. In this minireview, the state of the art of ABPP of oxidoreductases is described and the scope and limitations of the existing approaches are discussed. It is noted that several ABPP probes have been described for various oxidases, but none so far for a reductase, which gives rise to opportunities for future research.
Assuntos
Descoberta de Drogas , Oxirredutases/antagonistas & inibidores , Proteoma/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , HumanosRESUMO
Combretastatin A-4 (CA-4) is a highly cytotoxic natural product and several derivatives have been prepared which underwent clinical trial. These investigations revealed that the cis-stilbene moiety of the natural product is prone to undergo cis/trans isomerization under physiological conditions, reducing the overall activity of the drug candidates. Herein, we report the preparation of cis-restrained carbocyclic analogs of CA-4. The compounds, which differ by the size and hybridization of the carbocyclic ring have been evaluated for their cytotoxic properties and their ability to inhibit tubulin polymerization. Biological data, supported by molecular docking studies, identified cyclobutenyl and cyclobutyl derivatives of the natural product as highly promising drug candidates.
Assuntos
Antineoplásicos/farmacologia , Estilbenos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Estilbenos/síntese química , Estilbenos/metabolismo , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacologiaRESUMO
A structure-activity/structure-property relationship study based on the physicochemical as well as in vitro pharmacokinetic properties of a first generation matrix metalloproteinase (MMP)-13 inhibitor (2) was undertaken. After systematic variation of inhibitor 2, compound 31 was identified which exhibited microsomal half-life higher than 20â¯min, kinetic solubility higher than 20⯵M, and a permeability coefficient greater than 20â¯×â¯10-6â¯cm/s. Compound 31 also showed excellent in vivo PK properties after IV dosing (Cmaxâ¯=â¯56.8⯵M, T1/2 (plasma)â¯=â¯3.0â¯h, Clâ¯=â¯0.23â¯mL/min/kg) and thus is a suitable candidate for in vivo efficacy studies in an OA animal model.
Assuntos
Metaloproteinase 13 da Matriz/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/farmacologia , Animais , Inibidores das Enzimas do Citocromo P-450/farmacologia , Meia-Vida , Humanos , Concentração Inibidora 50 , Cinética , Inibidores de Metaloproteinases de Matriz/farmacocinética , Camundongos , Microssomos Hepáticos/metabolismo , Modelos Animais , Ratos , Solubilidade , Relação Estrutura-AtividadeRESUMO
Multiple myeloma promotes systemic skeletal bone disease that greatly contributes to patient morbidity. Resorption of type I collagen-rich bone matrix by activated osteoclasts results in the release of sequestered growth factors that can drive progression of the disease. Matrix metalloproteinase-13 (MMP13) is a collagenase expressed predominantly in the skeleton by mesenchymal stromal cells (MSC) and MSC-derived osteoblasts. Histochemical analysis of human multiple myeloma specimens also demonstrated that MMP13 largely localizes to the stromal compartment compared with CD138+ myeloma cells. In this study, we further identified that multiple myeloma induces MMP13 expression in bone stromal cells. Because of its ability to degrade type I collagen, we examined whether bone stromal-derived MMP13 contributed to myeloma progression. Multiple myeloma cells were inoculated into wild-type or MMP13-null mice. In independent in vivo studies, MMP13-null mice demonstrated significantly higher overall survival rates and lower levels of bone destruction compared with wild-type controls. Unexpectedly, no differences in type I collagen processing between the groups were observed. Ex vivo stromal coculture assays showed reduced formation and activity in MMP13-null osteoclasts. Analysis of soluble factors from wild-type and MMP13-null MSCs revealed decreased bioavailability of various osteoclastogenic factors including CXCL7. CXCL7 was identified as a novel MMP13 substrate and regulator of osteoclastogenesis. Underscoring the importance of host MMP13 catalytic activity in multiple myeloma progression, we demonstrate the in vivo efficacy of a novel and highly selective MMP13 inhibitor that provides a translational opportunity for the treatment of this incurable disease. SIGNIFICANCE: Genetic and pharmacologic approaches show that bone stromal-derived MMP13 catalytic activity is critical for osteoclastogenesis, bone destruction, and disease progression. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/9/2415/F1.large.jpg.
Assuntos
Metaloproteinase 13 da Matriz/metabolismo , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/mortalidade , Osteólise/genética , Transdução de Sinais/genética , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Quimiocinas CXC/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Masculino , Metaloproteinase 13 da Matriz/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , Taxa de SobrevidaRESUMO
The most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) is an expanded G4C2 RNA repeat [r(G4C2)exp] in chromosome 9 open reading frame 72 (C9orf72), which elicits pathology through several mechanisms. Here, we developed and characterized a small molecule for targeted degradation of r(G4C2)exp. The compound was able to selectively bind r(G4C2)exp's structure and to assemble an endogenous nuclease onto the target, provoking removal of the transcript by native RNA quality control mechanisms. In c9ALS patientderived spinal neurons, the compound selectively degraded the mutant C9orf72 allele with limited off-targets and reduced quantities of toxic dipeptide repeat proteins (DPRs) translated from r(G4C2)exp. In vivo work in a rodent model showed that abundance of both the mutant allele harboring the repeat expansion and DPRs were selectively reduced by this compound. These results demonstrate that targeted small-molecule degradation of r(G4C2)exp is a strategy for mitigating c9ALS/FTD-associated pathologies and studying disease-associated pathways in preclinical models.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Expansão das Repetições de DNA , Demência Frontotemporal/genética , Humanos , RibonucleasesRESUMO
The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an expanded G4C2 repeat [(G4C2)exp] in C9ORF72. ALS/FTD-associated toxicity has been traced to the RNA transcribed from the repeat expansion [r(G4C2)exp], which sequesters RNA-binding proteins (RBPs) and undergoes repeat-associated non-ATG (RAN) translation to generate toxic dipeptide repeats. Using in vitro and cell-based assays, we identified a small molecule (4) that selectively bound r(G4C2)exp, prevented sequestration of an RBP, and inhibited RAN translation. Indeed, biophysical characterization showed that 4 selectively bound the hairpin form of r(G4C2)exp, and nuclear magnetic resonance spectroscopy studies and molecular dynamics simulations defined this molecular recognition event. Cellular imaging revealed that 4 localized to r(G4C2)exp cytoplasmic foci, the putative sites of RAN translation. Collectively, these studies highlight that the hairpin structure of r(G4C2)exp is a therapeutically relevant target and small molecules that bind it can ameliorate c9ALS/FTD-associated toxicity.
Assuntos
Proteína C9orf72/genética , Expansão das Repetições de DNA/genética , Bibliotecas de Moléculas Pequenas/química , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Sítios de Ligação , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Humanos , Cinética , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , TermodinâmicaRESUMO
Structure-based virtual screening (SBVS) is a common method for the fast identification of hit structures at the beginning of a medicinal chemistry program in drug discovery. The SBVS, described in this manuscript, is focused on finding small molecule hits that can be further utilized as a starting point for the development of inhibitors of matrix metalloproteinase 13 (MMP-13) via structure-based molecular design. We intended to identify a set of structurally diverse hits, which occupy all subsites (S1'-S3', S2, and S3) centering the zinc containing binding site of MMP-13, by the virtual screening of a chemical library comprising more than ten million commercially available compounds. In total, 23 compounds were found as potential MMP-13 inhibitors using Glide docking followed by the analysis of the structural interaction fingerprints (SIFt) of the docked structures.
Assuntos
Inibidores de Metaloproteinases de Matriz/farmacologia , Simulação de Acoplamento Molecular/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação , Biologia Computacional , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Metaloproteinase 13 da Matriz/química , Metaloproteinase 13 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/química , Modelos Moleculares , Conformação Proteica , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
We describe the use of comparative structural analysis and structure-guided molecular design to develop potent and selective inhibitors (10d and (S)-17b) of matrix metalloproteinase 13 (MMP-13). We applied a three-step process, starting with a comparative analysis of the X-ray crystallographic structure of compound 5 in complex with MMP-13 with published structures of known MMP-13·inhibitor complexes followed by molecular design and synthesis of potent but nonselective zinc-chelating MMP inhibitors (e.g., 10a and 10b). After demonstrating that the pharmacophores of the chelating inhibitors (S)-10a, (R)-10a, and 10b were binding within the MMP-13 active site, the Zn2+ chelating unit was replaced with nonchelating polar residues that bridged over the Zn2+ binding site and reached into a solvent accessible area. After two rounds of structural optimization, these design approaches led to small molecule MMP-13 inhibitors 10d and (S)-17b, which bind within the substrate-binding site of MMP-13 and surround the catalytically active Zn2+ ion without chelating to the metal. These compounds exhibit at least 500-fold selectivity versus other MMPs.
Assuntos
Metaloproteinase 13 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/farmacologia , Quelantes/química , Quelantes/farmacologia , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Metaloproteinase 13 da Matriz/química , Modelos Moleculares , Zinco/químicaRESUMO
The design of selective matrix metalloproteinase (MMP) inhibitors that also possess favorable solubility properties has proved to be especially challenging. A prior approach using collagen-model templates combined with transition state analogs produced a first generation of triple-helical peptide inhibitors (THPIs) that were effective in vitro against discrete members of the MMP family. These THPI constructs were also highly water-soluble. The present study sought improvements in the first generation THPIs by enhancing thermal stability and selectivity. A THPI selective for MMP-2 and MMP-9 was redesigned to incorporate non-native amino acids (Flp and mep), resulting in an increase of 18 °C in thermal stability. This THPI was effective in vivo in a mouse model of multiple sclerosis, reducing clinical severity and weight loss. Two other THPIs were developed to be more selective within the collagenolytic members of the MMP family. One of these THPIs was serendipitously more effective against MMP-8 than MT1-MMP and was utilized successfully in a mouse model of sepsis. The THPI targeting MMP-8 minimized lung damage, increased production of the anti-inflammatory cytokine IL-10, and vastly improved mouse survival.