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We have previously shown computationally that Mycolactone (MLN), a toxin produced by Mycobacterium ulcerans, strongly binds to Munc18b and other proteins, presumably blocking degranulation and exocytosis of blood platelets and mast cells. We investigated the effect of MLN on endocytosis using similar approaches, and it bound strongly to the N-terminal of the clathrin protein and a novel SARS-CoV-2 fusion protein. Experimentally, we found 100% inhibition up to 60 nM and 84% average inhibition at 30 nM in SARS-CoV-2 live viral assays. MLN was also 10× more potent than remdesivir and molnupiravir. MLN's toxicity against human alveolar cell line A549, immortalized human fetal renal cell line HEK293, and human hepatoma cell line Huh7.1 were 17.12%, 40.30%, and 36.25%, respectively. The cytotoxicity IC50 breakpoint ratio versus anti-SARS-CoV-2 activity was more than 65-fold. The IC50 values against the alpha, delta, and Omicron variants were all below 0.020 µM, and 134.6 nM of MLN had 100% inhibition in an entry and spread assays. MLN is eclectic in its actions through its binding to Sec61, AT2R, and the novel fusion protein, making it a good drug candidate for treating and preventing COVID-19 and other similarly transmitted enveloped viruses and pathogens.
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COVID-19 , Humanos , SARS-CoV-2 , Antivirais/farmacologia , Células HEK293RESUMO
The ubiquitin (Ub) kinase-ligase pair PINK1-PRKN mediates the degradation of damaged mitochondria by macroautophagy/autophagy (mitophagy). PINK1 surveils mitochondria and upon stress accumulates on the mitochondrial surface where it phosphorylates serine 65 of Ub to activate PRKN and to drive mitochondrial turnover. While loss of either PINK1 or PRKN is genetically linked to Parkinson disease (PD) and activating the pathway seems to have great therapeutic potential, there is no formal proof that stimulation of mitophagy is always beneficial. Here we used biochemical and cell biological methods to study single nucleotide variants in the activation loop of PINK1 to modulate the enzymatic function of this kinase. Structural modeling and in vitro kinase assays were used to investigate the molecular mechanism of the PINK1 variants. In contrast to the PD-linked PINK1G411S mutation that diminishes Ub kinase activity, we found that the PINK1G411A variant significantly boosted Ub phosphorylation beyond levels of PINK1 wild type. This resulted in augmented PRKN activation, mitophagy rates and increased viability after mitochondrial stress in midbrain-derived, gene-edited neurons. Mechanistically, the G411A variant stabilizes the kinase fold of PINK1 and transforms Ub to adopt the preferred, C-terminally retracted conformation for improved substrate turnover. In summary, we identify a critical role of residue 411 for substrate receptivity that may now be exploited for drug discovery to increase the enzymatic function of PINK1. The genetic substitution of Gly411 to Ala increases mitophagy and may be useful to confirm neuroprotection in vivo and might serve as a critical positive control during therapeutic development.Abbreviations: ATP: adenosine triphosphate; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; Ub-CR: ubiquitin with C-terminally retracted tail; CTD: C-terminal domain (of PINK1); ELISA: enzyme-linked immunosorbent assay; HCI: high-content imaging; IB: immunoblot; IF: immunofluorescence; NPC: neuronal precursor cells; MDS: molecular dynamics simulation; PD: Parkinson disease; p-S65-Ub: ubiquitin phosphorylated at Ser65; RMSF: root mean scare fluctuation; TOMM: translocase of outer mitochondrial membrane; TVLN: ubiquitin with T66V and L67N mutation, mimics Ub-CR; Ub: ubiquitin; WT: wild-type.
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Doença de Parkinson , Proteínas Quinases , Humanos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Doença de Parkinson/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Autofagia , Ubiquitina/metabolismoRESUMO
COVID-19 is a devastating respiratory and inflammatory illness caused by a new coronavirus that is rapidly spreading throughout the human population. Over the past 12 months, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, has already infected over 160 million (>20% located in United States) and killed more than 3.3 million people around the world (>20% deaths in USA). As we face one of the most challenging times in our recent history, there is an urgent need to identify drug candidates that can attack SARS-CoV-2 on multiple fronts. We have therefore initiated a computational dynamics drug pipeline using molecular modeling, structure simulation, docking and machine learning models to predict the inhibitory activity of several million compounds against two essential SARS-CoV-2 viral proteins and their host protein interactors-S/Ace2, Tmprss2, Cathepsins L and K, and Mpro-to prevent binding, membrane fusion and replication of the virus, respectively. All together, we generated an ensemble of structural conformations that increase high-quality docking outcomes to screen over >6 million compounds including all FDA-approved drugs, drugs under clinical trial (>3000) and an additional >30 million selected chemotypes from fragment libraries. Our results yielded an initial set of 350 high-value compounds from both new and FDA-approved compounds that can now be tested experimentally in appropriate biological model systems. We anticipate that our results will initiate screening campaigns and accelerate the discovery of COVID-19 treatments.
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Antivirais/uso terapêutico , Tratamento Farmacológico da COVID-19 , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Descoberta de Drogas , Reposicionamento de Medicamentos , Humanos , Aprendizado de Máquina , Simulação de Acoplamento Molecular , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
The titular member of the MAX network of proteins, MYC-associated factor X (MAX), serves an important regulatory function in transcription of E-box genes associated with cell proliferation, differentiation, and apoptosis. Wild type MAX dimerizes with both MYC and MAD, both of which are members of the MAX network, and can promote or repress cell functions as needed. However, pathogenic variants in MAX are known to upset this balance, leading to uncontrolled oncogenic activity and disease phenotypes such as paragangliomas and pheochromocytomas. We report a 58-year-old male and his 32-year-old daughter, both of which have a history of pheochromocytoma and the unique nonsense MAX variant c.271C>T (p.Q91X). These individuals were diagnosed with pheochromocytomas in their early twenties that were later removed through corrective surgery. The father now presents with recurrent symptoms of hypertension, hyperhidrosis, and headaches, which accompany new pheochromocytomas of his remaining adrenal gland. Pathogenicity of this MAX variant is proven through molecular modeling. The case of this father-daughter pair supports both heritability of pheochromocytoma and the paternal parent-of-origin effect for MAX pathogenic variants.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Predisposição Genética para Doença , Impressão Genômica , Feocromocitoma/genética , Adulto , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Códon sem Sentido , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , LinhagemRESUMO
Diseases are often caused by mutant proteins. Many drugs have limited effectiveness and/or toxic side effects because of a failure to selectively target the disease-causing mutant variant, rather than the functional wild type protein. Otherwise, the drugs may even target different proteins with similar structural features. Designing drugs that successfully target mutant proteins selectively represents a major challenge. Decades of cancer research have led to an abundance of potential therapeutic targets, often touted to be "master regulators". For many of these proteins, there are no FDA-approved drugs available; for others, off-target effects result in dose-limiting toxicity. Cancer-related proteins are an excellent medium to carry the story of mutant-specific targeting, as the disease is both initiated and sustained by mutant proteins; furthermore, current chemotherapies generally fail at adequate selective distinction. This review discusses some of the challenges associated with selective targeting from a structural biology perspective, as well as some of the developments in algorithm approach and computational workflow that can be applied to address those issues. One of the most widely researched proteins in cancer biology is p53, a tumor suppressor. Here, p53 is discussed as a specific example of a challenging target, with contemporary drugs and methodologies used as examples of burgeoning successes. The oncogene KRAS, which has been described as "undruggable", is another extensively investigated protein in cancer biology. This review also examines KRAS to exemplify progress made towards selective targeting of diseasecausing mutant proteins. Finally, possible future directions relevant to the topic are discussed.
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Antineoplásicos/farmacologia , Desenho de Fármacos/métodos , Proteínas Mutantes/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Antineoplásicos/uso terapêutico , Humanos , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular/métodos , Proteínas Mutantes/genética , Neoplasias/genética , Medicina de Precisão/métodos , Estrutura Terciária de Proteína/genética , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Fluxo de TrabalhoRESUMO
Chloroethylagelastatin A (CEAA) is an analogue of agelastatin A (AA), a natural alkaloid derived from a marine sponge. It is under development for therapeutic use against brain tumors as it has excellent central nervous system (CNS) penetration and pre-clinical therapeutic activity against brain tumors. Recently, AA was shown to inhibit protein synthesis by binding to the ribosomal A-site. In this study, we developed a novel virtual screening platform to perform a comprehensive screening of various AA analogues showing that AA analogues with proven therapeutic activity including CEAA have significant ribosomal binding capacity whereas therapeutically inactive analogues show poor ribosomal binding and revealing structural fingerprint features essential for drug-ribosome interactions. In particular, CEAA was found to have greater ribosomal binding capacity than AA. Biological tests showed that CEAA binds the ribosome and contributes to protein synthesis inhibition. Our findings suggest that CEAA may possess ribosomal inhibitor activity and that our virtual screening platform may be a useful tool in discovery and development of novel ribosomal inhibitors.
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Alcaloides , Antineoplásicos , Neoplasias Encefálicas , Poríferos/classificação , Ribossomos , Alcaloides/química , Alcaloides/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Proteínas de Neoplasias/biossíntese , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/química , Ribossomos/metabolismoRESUMO
Persistent protein obstacles on genomic DNA, such as DNA-protein crosslinks (DPCs) and tight nucleoprotein complexes, can block replication forks. DPCs can be removed by the proteolytic activities of the metalloprotease SPRTN or the proteasome in a replication-coupled manner; however, additional proteolytic mechanisms may exist to cope with the diversity of protein obstacles. Here, we show that FAM111A, a PCNA-interacting protein, plays an important role in mitigating the effect of protein obstacles on replication forks. This function of FAM111A requires an intact trypsin-like protease domain, the PCNA interaction, and the DNA-binding domain that is necessary for protease activity in vivo. FAM111A, but not SPRTN, protects replication forks from stalling at poly(ADP-ribose) polymerase 1 (PARP1)-DNA complexes trapped by PARP inhibitors, thereby promoting cell survival after drug treatment. Altogether, our findings reveal a role of FAM111A in overcoming protein obstacles to replication forks, shedding light on cellular responses to anti-cancer therapies.
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Replicação do DNA , Receptores Virais/metabolismo , Tripsina/química , Camptotecina/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA , DNA Topoisomerases Tipo I/metabolismo , DNA de Cadeia Simples/metabolismo , Humanos , Mutação/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Receptores Virais/química , Receptores Virais/genéticaRESUMO
Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme overexpressed by many different tumor types. QSOX1 catalyzes the formation of disulfide bonds in proteins. Because short hairpin knockdowns (KD) of QSOX1 have been shown to suppress tumor growth and invasion in vitro and in vivo, we hypothesized that chemical compounds inhibiting QSOX1 enzymatic activity would also suppress tumor growth, invasion, and metastasis. High throughput screening using a QSOX1-based enzymatic assay revealed multiple potential QSOX1 inhibitors. One of the inhibitors, known as "SBI-183," suppresses tumor cell growth in a Matrigel-based spheroid assay and inhibits invasion in a modified Boyden chamber, but does not affect viability of nonmalignant cells. Oral administration of SBI-183 inhibits tumor growth in 2 independent human xenograft mouse models of renal cell carcinoma. We conclude that SBI-183 warrants further exploration as a useful tool for understanding QSOX1 biology and as a potential novel anticancer agent in tumors that overexpress QSOX1.
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Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Renais/tratamento farmacológico , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/uso terapêutico , Animais , Feminino , Humanos , Camundongos , Camundongos SCIDRESUMO
Chondrogenic tumors involving the temporal bone are rare and typically arise spontaneously with unilateral presentation. Somatic IDH mutations are common in these tumors, but germline inheritance has not been documented to our knowledge. We describe familial chondrosarcoma, grade 1, of the mastoid with unilateral presentation in the mother and bilateral presentation in each of her two children. Each individual presented with headaches, facial paresis, and conductive hearing loss between the ages of 9-12. Exome sequencing of all three affected family members identified a shared germline heterozygous c.299G>A (p.Arg100Gln) missense variant in IDH1. The p.Arg100Gln variant has only rarely been observed as a somatic mutation in glial tumors, and previous in vitro experiments have shown that p.Arg100Gln produces small amounts of the oncometabolite D-2-hydroxyglutarate (D2HG). Biochemical testing in all three affected family members on urine and plasma was unable to detect increases in D2HG in these sample types. Due to insufficient tumor for methylation studies, we performed genome-wide methylation analysis of an IDH1 p.Arg100Gln mutant brain tumor from an unrelated individual to functionally evaluate this variant. These studies demonstrated a global hypermethylation phenotype consistent with other known isocitrate dehydrogenase (IDH) mutant brain tumors, suggesting that this variant has neomorphic activity despite low-level production of D2HG. The bones of the facial skeleton are formed by membranous ossification and we hypothesize that abnormal embryonic cartilage that rests within the suture lines may be involved in this tumor entity. Testing of additional individuals with similar presentations is needed to confirm this finding and clarify the associated phenotypes.
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Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Nanomedicine, however, offers new opportunities to facilitate drug delivery in PDAC. Our previous work has shown that poly(ethylene glycol)-functionalized nanodiamond (ND) mediated drug delivery offered a considerable improvement over free drug in PDAC. Inspired by this result and guided by molecular simulations, we opted for simultaneous loading of irinotecan and curcumin in ultra-small PEGylated NDs (ND-IRT + CUR). We observed that ND-IRT + CUR was more efficacious in killing AsPC-1 and PANC-1 cells than NDs with single drugs. Using NDs functionalized with a near-infrared (NIR) dye, we demonstrated the preferential localization of the NDs in tumors and metastatic lesions. We further demonstrate that ND-IRT + CUR is capable of producing pronounced anti-tumor effects in two different clinically relevant, immune-competent genetic models of PDAC. Cytokine profiling indicated that NDs with or without drugs downregulated the expression of IL-10, a key modulator of the tumor microenvironment. Thus, using a combination of in silico, in vitro, and in vivo approaches, we show for the first time the remarkable anti-tumor efficacy of PEGylated NDs carrying a dual payload of irinotecan plus curcumin. These results highlight the potential use of such nano-carriers in the treatment of patients with pancreatic cancer.
Assuntos
Curcumina , Portadores de Fármacos , Nanodiamantes , Neoplasias Pancreáticas , Animais , Linhagem Celular Tumoral , Curcumina/química , Curcumina/farmacocinética , Curcumina/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Humanos , Camundongos , Camundongos Mutantes , Nanodiamantes/química , Nanodiamantes/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Polietilenoglicóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias PancreáticasRESUMO
Pancreatic ductal adenocarcinoma (PDAC) has one of the highest mortality rates among cancers. Chemotherapy is the standard first-line treatment, but only modest survival benefits are observed. With the advent of targeted therapies, epidermal growth factor receptor (EGFR) has been acknowledged as a prospective target in PDAC since it is overexpressed in up to 60% of cases. Similarly, the tyrosine-protein kinase Met (cMET) is also overexpressed in PDAC (27-60%) and is a prognostic marker for poor survival. Interestingly, EGFR and cMET share some common signaling pathways including PI3K/Akt and MAPK pathways. Small molecule inhibitors or bispecific antibodies that can target both EGFR and cMET are therefore emerging as novel options for cancer therapy. We previously developed a dual EGFR and cMET inhibitor (N19) that was able to inhibit tumor growth in nonsmall cell lung cancer models resistant to EGFR tyrosine kinase inhibitors (TKI). Here, we report the development of a novel liposomal formulation of N19 (LN19) and showed significant growth inhibition and increased sensitivity toward gemcitabine in the pancreatic adenocarcinoma orthotopic xenograft model. Taken together, our results suggest that LN19 can be valued as an effective combination therapy with conventional chemotherapy such as gemcitabine for PDAC patients.
Assuntos
Adenocarcinoma/patologia , Desenho de Fármacos , Lipossomos/química , Neoplasias Pancreáticas/patologia , Polietilenoglicóis/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/farmacologia , Composição de Medicamentos , Receptores ErbB/metabolismo , Feminino , Humanos , Camundongos , Metástase Neoplásica , Proteólise/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Gencitabina , Neoplasias PancreáticasRESUMO
BACKGROUND: Transient receptor potential cation channel subfamily V member 4 (TRPV4) is an ion channel permeable to Ca2+ that is sensitive to physical, hormonal, and chemical stimuli. This protein is expressed in many cell types, including osteoclasts, chondrocytes, and sensory neurons. As such, pathogenic variants of this gene are associated with skeletal dysplasias and neuromuscular disorders. Pathogenesis of these phenotypes is not yet completely understood, but it is known that genotype-phenotype correlations for TRPV4 pathogenic variants often are not present. METHODS: Newly characterized, suspected pathogenic variant in TRPV4 was analyzed using protein informatics and personalized protein-level molecular studies, genomic exome analysis, and clinical study. RESULTS: This statement is demonstrated in the family of our proband, a 47-year-old female having the novel c.2401A>G (p.K801E) variant of TRPV4. We discuss the common symptoms between the proband, her father, and her daughter, and compare her phenotype to known TRPV4-associated skeletal dysplasias. CONCLUSIONS: Protein informatics and molecular modeling are used to confirm the pathogenicity of the unique TRPV4 variant found in this family. Multiple data were combined in a comprehensive manner to give complete overall perspective on the patient disease and prognosis.
Assuntos
Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Osteocondrodisplasias/genética , Fenótipo , Canais de Cátion TRPV/genética , Feminino , Humanos , Mutação com Perda de Função , Pessoa de Meia-Idade , Osteocondrodisplasias/patologia , Linhagem , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismoRESUMO
The CHEK2 gene and its encoded protein Chk2 have a well-known role in cancers, especially those related to breast cancer mediated through the BRCA1 gene. Additionally Chk2 has a crucial role in DNA repair, apoptosis and the cell cycle, which is why classification of variants of uncertain significance (VUS) is an area highly sought for a better elucidation of the "genomic effect" that results. Because it can often take years before enough clinical data is accumulated, and the costly and expensive functional analysis for individual variants presents a significant hurdle, it is important to identify other tools to help aid in clarifying the impact of specific variants on a protein's function and eventually the patient's health outcome. Here we describe a newly identified CHEK2 variant and analyze with an integrated approach combining genomics (whole exome analysis), clinical study, radiographic imaging, and protein informatics to identify and predict the functional impact of the VUS on the protein's behavior and predicted impact on the related pathways. The observed and analyzed defects in the protein were consistent with the expected clinical effect. Here, we support the use of personalized protein modeling and informatics and further our goal of developing a large-scale protein deposition archive for all protein-level VUS.
Assuntos
Quinase do Ponto de Checagem 2/genética , Biologia Computacional/métodos , Genômica , Imageamento Tridimensional , Adulto , Carcinogênese/genética , Carcinogênese/patologia , Quinase do Ponto de Checagem 2/química , Feminino , Humanos , Masculino , Modelos Moleculares , Neoplasias/genética , Linhagem , Fatores de Risco , Eletricidade EstáticaRESUMO
RNA-binding protein aggregation is a pathological hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To gain better insight into the molecular interactions underlying this process, we investigated FUS, which is mutated and aggregated in both ALS and FTLD. We generated a Drosophila model of FUS toxicity and identified a previously unrecognized synergistic effect between the N-terminal prion-like domain and the C-terminal arginine-rich domain to mediate toxicity. Although the prion-like domain is generally considered to mediate aggregation of FUS, we find that arginine residues in the C-terminal low-complexity domain are also required for maturation of FUS in cellular stress granules. These data highlight an important role for arginine-rich domains in the pathology of RNA-binding proteins.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/toxicidade , Drosophila melanogaster/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/toxicidade , Sequência de Aminoácidos , Animais , Arginina/metabolismo , Linhagem Celular Tumoral , Proteínas de Drosophila/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Humanos , Atividade Motora , Neurônios Motores/patologia , Degeneração Neural/patologia , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
Sensory ataxic neuropathy with dysarthria and ophthalmoparesis (SANDO) is a rare phenotype resulting from pathogenic variants of mitochondrial DNA polymerase gamma (POLG). We modeled a novel POLG variant, T599P, that causes the SANDO phenotype and another variant at the same residue, p.T599E, to observe their effect on protein function and confirm the pathogenicity of T599P. Through neoteric molecular modeling techniques, we show that changes at the T599 residue position introduce extra rigidity into the surrounding helix-loop-helix, which places steric pressure on nearby nucleotides. We also provide a clinical description of the T599P variant, which was found in a 42-year-old female proband. The proband presented a 1-year history of progressive gait instability, dysarthria and foot numbness. Her neurologic examination revealed ataxic dysarthria, restricted eye movements, head and palatal tremors, reduced lower limb reflexes, distal multimodal sensory loss and a wide, unsteady ataxic gait. Electromyography studies indicated a sensory neuropathy. Whole-exome sequencing was pursued after tests for infectious, inflammatory and paraneoplastic causes were negative.
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Here we present an innovative computational-based drug discovery strategy, coupled with machine-based learning and functional assessment, for the rational design of novel small molecule inhibitors of the lipogenic enzyme stearoyl-CoA desaturase 1 (SCD1). Our methods resulted in the discovery of several unique molecules, of which our lead compound SSI-4 demonstrates potent anti-tumor activity, with an excellent pharmacokinetic and toxicology profile. We improve upon key characteristics, including chemoinformatics and absorption/distribution/metabolism/excretion (ADME) toxicity, while driving the IC50 to 0.6 nM in some instances. This approach to drug design can be executed in smaller research settings, applied to a wealth of other targets, and paves a path forward for bringing small-batch based drug programs into the Clinic.
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BACKGROUND: The CYP11A1 gene encodes the cytochrome P450 side-chain cleavage enzyme, which is essential for steroid formation. Recessive variants in this gene can lead to impairment of sexual differentiation caused by a complete or partial loss of steroid hormone production. The phenotypic spectrum in affected 46XY males may vary from surgically repairable defects including cryptorchidism and hypospadias to complete feminization of external gonads, accompanied by symptoms of adrenal dysfunction. METHODS: Whole-exome sequencing (WES) of a 12-year-old male proband and his parents was performed after a protracted diagnostic odyssey failed to uncover the cause of his primary adrenal insufficiency. Of note, the proband had early symptomatology and corrective surgery for hypospadias, raising suspicion for a disorder of steroidogenesis. RESULTS: WES identified compound heterozygous variants in CYP11A1 including a novel canonical splice site variant (c.425+1G>A) and a previously reported p.E314K variant, which were consistent with a diagnosis of congenital adrenal insufficiency with partial 46XY sex reversal. CONCLUSION: Congenital adrenal insufficiency with 46XY sex reversal is a rare disorder that is characterized by dysregulation of steroid hormone synthesis, leading to adrenal and gonadal dysfunction. In this report, we describe a patient with adrenal insufficiency, hypospadias, and skin hyperpigmentation who was found to have a novel c.425+1G>A variant in trans with the p.E314K variant in CYP11A1. We performed structural analyses to examine the effect of the p.E314K variant on protein function and show that it falls in the core of the protein may disrupt cholesterol binding in the active site.
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Insuficiência Adrenal/congênito , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Insuficiência Adrenal/diagnóstico , Insuficiência Adrenal/genética , Criança , Enzima de Clivagem da Cadeia Lateral do Colesterol/química , Análise Mutacional de DNA , Heterozigoto , Humanos , Masculino , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Estrutura Terciária de Proteína , Sítios de Splice de RNA/genética , Sequenciamento do ExomaRESUMO
An astute modification of the plectin-1-targeting peptide KTLLPTP by introducing a C-terminal cysteine preceded by a tyrosine residue imparted a reducing property to the peptide. This novel property is then exploited to fabricate gold nanoparticles (GNP) via an in situ reduction of gold(iii) chloride in a one-pot, green synthesis. The modified peptide KTLLPTPYC also acts as a template to generate highly monodispersed, spherical GNPs with a narrow size distribution and improved stability. Plectin-1 is known to be aberrantly expressed in the surface of pancreatic ductal adenocarcinoma (PDAC) cells while showing cytoplasmic expression in normal cells. The synthesized GNPs are thus in situ surface modified with the peptides via the cysteine residue leaving the N-terminal KTLLPTP sequence free for targeting plectin-1. The visual molecular dynamics based simulations support the experimental observations like particle size, gemcitabine conjugation and architecture of the peptide-grafted nanoassembly. Additionally, GNPs conjugated to gemcitabine demonstrate significantly higher cytotoxicity in vitro in two established PDAC cell lines (AsPC-1 and PANC-1) and an admirable in vivo antitumor efficacy in a PANC-1 orthotopic xenograft model through selective uptake in PDAC tumor tissues. Altogether, this strategy represents a unique method for the fabrication of a GNP based targeted drug delivery platform using a multifaceted peptide that acts as reducing agent, template for GNP synthesis and targeting agent to display remarkable selectivity towards PDAC.
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Desoxicitidina/análogos & derivados , Portadores de Fármacos/síntese química , Ouro , Nanopartículas Metálicas , Neoplasias Pancreáticas/tratamento farmacológico , Plectina/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Humanos , Peptídeos , GencitabinaRESUMO
PRSS3/mesotrypsin is an atypical isoform of trypsin, the upregulation of which has been implicated in promoting tumor progression. To date there are no mesotrypsin-selective pharmacological inhibitors which could serve as tools for deciphering the pathological role of this enzyme, and could potentially form the basis for novel therapeutic strategies targeting mesotrypsin. A virtual screen of the Natural Product Database (NPD) and Food and Drug Administration (FDA) approved Drug Database was conducted by high-throughput molecular docking utilizing crystal structures of mesotrypsin. Twelve high-scoring compounds were selected for testing based on lowest free energy docking scores, interaction with key mesotrypsin active site residues, and commercial availability. Diminazene (CID22956468), along with two similar compounds presenting the bis-benzamidine substructure, was validated as a competitive inhibitor of mesotrypsin and other human trypsin isoforms. Diminazene is the most potent small molecule inhibitor of mesotrypsin reported to date with an inhibitory constant (Ki) of 3.6±0.3 µM. Diminazene was subsequently co-crystalized with mesotrypsin and the crystal structure was solved and refined to 1.25 Å resolution. This high resolution crystal structure can now offer a foundation for structure-guided efforts to develop novel and potentially more selective mesotrypsin inhibitors based on similar molecular substructures.
Assuntos
Descoberta de Drogas , Simulação de Acoplamento Molecular , Inibidores da Tripsina/farmacologia , Tripsina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Bases de Dados de Produtos Farmacêuticos , Diminazena/química , Diminazena/farmacologia , Relação Dose-Resposta a Droga , Descoberta de Drogas/métodos , Escherichia coli , Humanos , Ligação de Hidrogênio , Estrutura Molecular , Eletricidade Estática , Tripsina/química , Tripsina/genética , Inibidores da Tripsina/química , Estados Unidos , United States Food and Drug AdministrationRESUMO
Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC50(app) = 63 nM), nontoxic at concentrations up to 100 µM, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets.