RESUMO
Fabry disease is an X-linked lysosomal storage disorder caused by loss of alpha-galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of glycosphingolipids in multiple cells and tissues. FLT190, an investigational gene therapy, is currently being evaluated in a Phase 1/2 clinical trial in patients with Fabry disease (NCT04040049). FLT190 consists of a potent, synthetic capsid (AAVS3) containing an expression cassette with a codon-optimized human GLA cDNA under the control of a liver-specific promoter FRE1 (AAV2/S3-FRE1-GLAco). For mouse studies FLT190 genome was pseudotyped with AAV8 for efficient transduction. Preclinical studies in a murine model of Fabry disease (Gla-deficient mice), and non-human primates (NHPs) showed dose-dependent increases in plasma α-Gal A with steady-state observed 2 weeks following a single intravenous dose. In Fabry mice, AAV8-FLT190 treatment resulted in clearance of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) in plasma, urine, kidney, and heart; electron microscopy analyses confirmed reductions in storage inclusion bodies in kidney and heart. In NHPs, α-Gal A expression was consistent with the levels of hGLA mRNA in liver, and no FLT190-related toxicities or adverse events were observed. Taken together, these studies demonstrate preclinical proof-of-concept of liver-directed gene therapy with FLT190 for the treatment of Fabry disease.
Assuntos
Doença de Fabry , Terapia Genética , Animais , Humanos , Camundongos , Células Cultivadas , Doença de Fabry/genética , Doença de Fabry/terapia , Fibroblastos , Vetores Genéticos , Fígado/metabolismo , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismoRESUMO
Synergism among several intertwined catalytic cycles allows for selective, room temperature oxidation of primary amines to the corresponding nitriles in 85-98% isolated yield. This metal-free, scalable, operationally simple method employs a catalytic quantity of 4-acetamido-TEMPO (ACT; TEMPO=2,2,6,6-tetramethylpiperidine N-oxide) radical and the inexpensive, environmentally benign triple salt oxone as the terminal oxidant under mild conditions. Simple filtration of the reaction mixture through silica gel affords pure nitrile products.
RESUMO
Although fluorine often plays an influential role in molecular recognition, little is known about the effect of aliphatic fluorine on the CH-π interaction in solution. A series of molecular balances were synthesized that contain fluorinated and nonfluorinated alkyl groups. Our findings indicate that fluorine's polarizing ability does enhance CH-π binding and depends on molecular orientation. Surprisingly, when the terminal end of the alkyl group is completely fluorinated, the balance tips toward fluorophobicity and assumes an unusual constrained conformation.
RESUMO
The rapK gene required for biosynthesis of the DHCHC starter acid that initiates rapamycin biosynthesis was deleted from strain BIOT-3410, a derivative of Streptomyces rapamycinicus which had been subjected to classical strain and process development and capable of robust rapamycin production at titres up to 250mg/L. The resulting strain BIOT-4010 could no longer produce rapamycin, but when supplied exogenously with DHCHC produced rapamycin at titres equivalent to its parent strain. This strain enabled mutasynthetic access to new rapalogs that could not readily be isolated from lower titre strains when fed DHCHC analogs. Mutasynthesis of some rapalogs resulted predominantly in compounds lacking late post polyketide synthase biosynthetic modifications. To enhance the relative production of fully elaborated rapalogs, genes encoding late-acting biosynthetic pathway enzymes which failed to act efficiently on the novel compounds were expressed ectopically to give strain BIOT-4110. Strains BIOT-4010 and BIOT-4110 represent valuable tools for natural product lead optimization using biosynthetic medicinal chemistry and for the production of rapalogs for pre-clinical and early stage clinical trials.
Assuntos
Melhoramento Genético/métodos , Mutagênese Sítio-Dirigida/métodos , Recombinação Genética/genética , Sirolimo/metabolismo , Streptomyces/fisiologia , Sirolimo/isolamento & purificação , Especificidade da Espécie , Streptomyces/classificaçãoRESUMO
We are developing tablet dosage forms for implantation directly into the subconjunctival space of the eye. The matrix metalloproteinase inhibitor, ilomastat, has previously been shown to be efficacious at suppressing scarring following glaucoma filtration surgery (GFS). We report on the physical characterisation of ilomastat which is being developed for ocular implantation. Since ilomastat is being considered for implantation it is necessary to examine its polymorphs and their influence on aspects of the in vitro drug release profile. X-ray powder diffraction identified two polymorphs of ilomastat from different commercial batches of the compound. Tablets were prepared from the two different polymorphs. Isothermal perfusion calorimetry was used to show that amorphous content is not increased during tablet formulation. The melting points of the two polymorphs are 188 and 208°C as determined by differential scanning calorimetry. Utilising single crystal X-ray diffraction, the structural conformations and packing arrangements of the different polymorphs were determined. The orthorhombic crystal crystallised as a monohydrate while the second monoclinic crystal form is non-solvated. Ilomastat tablets prepared from the two different solid forms exhibited similar drug release profiles in vitro under conditions mimicking the aqueous composition, volume and flow of the subconjunctival space after GFS. This suggests that a reproducible dose at each time point during release after implantation should be achievable in vivo with ilomastat tablets prepared from the two polymorphs identified.
Assuntos
Indóis/administração & dosagem , Indóis/química , Implantes Absorvíveis , Administração Oftálmica , Varredura Diferencial de Calorimetria/métodos , Química Farmacêutica/métodos , Cristalização/métodos , Preparações de Ação Retardada/química , Ácidos Hidroxâmicos , Pós/química , Comprimidos/administração & dosagem , Comprimidos/química , Difração de Raios X/métodosRESUMO
Gene therapy is an exciting therapeutic concept that offers the promise of a cure for an array of inherited and acquired disorders. The liver has always been a key target for gene therapy as it controls essential biological processes including digestion, metabolism, detoxification, immunity, and blood coagulation. Metabolic disorders of hepatic origin number several hundreds, and for many, liver transplantation remains the only cure. Liver-targeted gene therapy is an attractive treatment modality for many of these conditions. After years of failure, substantial progress in this field in the past decade has resulted in promising clinical efficacy and safety in patients with monogenetic disorders with Valoctocogene roxaparvovec (Roctavian), the first gene therapy for treatment for hemophilia A, to be approved in Europe. Another, Etranacogene dezaparvovec (AMT-061) for hemophilia B is also in the final stages of approval. A number of other liver targeted gene therapy products are at an advanced stage of development, thus heralding a new era of potentially curative molecular medicine. This review explores the recent clinical advances in liver targeted gene therapy as well as the challenges that need to be overcome for the widespread adoption of this new treatment paradigm.
Assuntos
Hemofilia A , Hemofilia B , Terapia Genética/métodos , Vetores Genéticos , Hemofilia A/genética , Hemofilia A/terapia , Hemofilia B/genética , Hemofilia B/terapia , Humanos , FígadoRESUMO
The glycosylation of natural product scaffolds with highly modified deoxysugars is often essential for their biological activity, being responsible for specific contacts to molecular targets and significantly affecting their pharmacokinetic properties. In order to provide tools for the targeted alteration of natural product glycosylation patterns, significant strides have been made to understand the biosynthesis of activated deoxysugars and their transfer. We report here efforts towards the production of plasmid-borne biosynthetic gene cassettes capable of producing TDP-activated forms of D-mycaminose, D-angolosamine and D-desosamine. We additionally describe the transfer of these deoxysugars to macrolide aglycones using the glycosyl transferases EryCIII, TylMII and AngMII, which display usefully broad substrate tolerance.
Assuntos
Glucosamina/análogos & derivados , Macrolídeos/química , Macrolídeos/metabolismo , Clonagem Molecular , Engenharia Genética , Glucosamina/química , Glucosamina/metabolismo , Estrutura Molecular , Família Multigênica/genética , Análise de Sequência , Streptomyces/química , Streptomyces/genética , Streptomyces/metabolismoRESUMO
This study presents the in vitro hydrodynamic assessment of the TRISKELE, a new system suitable for transcatheter aortic valve implantation (TAVI), aiming to mitigate the procedural challenges experienced with current technologies. The TRISKELE valve comprises three polymeric leaflet and an adaptive sealing cuff, supported by a novel fully retrievable self-expanding nitinol wire frame. Valve prototypes were manufactured in three sizes of 23, 26, and 29 mm by automated dip-coating of a biostable polymer, and tested in a hydrodynamic bench setup in mock aortic roots of 21, 23, 25, and 27 mm annulus, and compared to two reference valves suitable for equivalent implantation ranges: Edwards SAPIEN XT and Medtronic CoreValve. The TRISKELE valves demonstrated a global hydrodynamic performance comparable or superior to the controls with significant reduction in paravalvular leakage. The TRISKELE valve exhibits enhanced anchoring and improved sealing. The valve is currently under preclinical investigation.
Assuntos
Valva Aórtica/cirurgia , Próteses Valvulares Cardíacas , Hemodinâmica , Stents Metálicos Autoexpansíveis , Substituição da Valva Aórtica Transcateter/instrumentação , Ligas , Valva Aórtica/fisiopatologia , Humanos , Hidrodinâmica , Teste de Materiais , Desenho de Prótese , Aço InoxidávelRESUMO
We report the directed biosynthesis of borrelidin analogues and their selective anti-proliferative activity against human cancer cell lines.
Assuntos
Inibidores da Angiogênese/biossíntese , Neoplasias/tratamento farmacológico , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Ensaios de Seleção de Medicamentos Antitumorais , Álcoois Graxos/química , Álcoois Graxos/metabolismo , Álcoois Graxos/farmacologia , Humanos , Conformação Molecular , EstereoisomerismoRESUMO
AIMS: The aim of this study was to introduce and demonstrate the feasibility in an acute preclinical model of a new transcatheter heart valve concept with a self-expanding wire frame, polymeric leaflets and a sealing component. METHODS AND RESULTS: The TRISKELE valve was developed based on a previously validated polymeric leaflet design, an adaptive sealing cuff and a novel nitinol wire frame which reduces stress on the leaflets and radial pressure on the surrounding tissue. A valve prototype of 26 mm nominal diameter was manufactured by automated dip coating of a biostable polymer. The prototype was implanted via brachiocephalic approach in orthotopic position in an acute ovine model through a highly controllable multistage deployment process. The atraumatic retrievability of the valve after full expansion was verified in situ before final release in the optimal position. Observation indicated secure valve anchoring, adequate leaflet motion, and no interference of coronary flow or mitral valve function. CONCLUSIONS: The TRISKELE valve system has the potential to mitigate complications related to imprecise valve positioning, and may offer a safer and more economical TAVI solution to a broad range of patients. The valve is currently under preclinical investigation for its long-term function and durability.
Assuntos
Próteses Valvulares Cardíacas , Substituição da Valva Aórtica Transcateter/instrumentação , Animais , Estudos de Viabilidade , Modelos Animais , Desenho de Prótese , OvinosRESUMO
BACKGROUND: Acute demyelinating optic neuritis, a common feature of multiple sclerosis, can damage vision through neurodegeneration in the optic nerve and in its fibres in the retina. Inhibition of voltage-gated sodium channels is neuroprotective in preclinical models. In this study we aimed to establish whether sodium-channel inhibition with phenytoin is neuroprotective in patient with acute optic neuritis. METHODS: We did a randomised, placebo-controlled, double-blind phase 2 trial at two UK academic hospitals in London and Sheffield. Patients with acute optic neuritis aged 18-60 years, presenting within 2 weeks of onset, with visual acuity of 6/9 or worse, were randomly assigned (1:1) by minimisation via a web-based service to oral phenytoin (maintenance dose 4 mg/kg per day if randomised before or on July 16, 2013, and 6 mg/kg per day if randomised on or after July 17, 2013) or placebo for 3 months, stratified by time from onset, centre, previous multiple sclerosis diagnosis, use of disease-modifying treatment, and use of corticosteroids for acute optic neuritis. Participants and treating and assessing physicians were masked to group assignment. The primary outcome was retinal nerve fibre layer (RNFL) thickness in the affected eye at 6 months, adjusted for fellow-eye RNFL thickness at baseline, analysed in a modified intention-to-treat population of all randomised participants who were followed up at 6 months. Safety was analysed in the entire population, including those who were lost to follow-up. The trial is registered with ClinicalTrials.gov, number NCT 01451593. FINDINGS: We recruited 86 participants between Feb 3, 2012, and May 22, 2014 (42 assigned to phenytoin and 44 to placebo). 29 were assigned to phenytoin 4 mg/kg and 13 to phenytoin 6 mg/kg. Five participants were lost to follow-up, so the primary analysis included 81 participants (39 assigned to phenytoin and 42 to placebo). Mean 6-month RNFL thickness in the affected eye at 6 months was 81.46 µm (SD 16.27) in the phenytoin group (a mean decrease of 16.69 µm [SD 13.73] from baseline) versus 74.29 µm (15.14) in the placebo group (a mean decrease of 23.79 µm [13.97] since baseline; adjusted 6-month difference of 7.15 µm [95% CI 1.08-13.22]; p=0.021), corresponding to a 30% reduction in the extent of RNFL loss with phenytoin compared with placebo. Treatment was well tolerated, with five (12%) of 42 patients having a serious adverse event in the phenytoin group (only one, severe rash, was attributable to phenytoin) compared with two (5%) of 44 in the placebo group. INTERPRETATION: These findings support the concept of neuroprotection with phenytoin in patients with acute optic neuritis at concentrations at which it blocks voltage-gated sodium channels selectively. Further investigation in larger clinical trials in optic neuritis and in relapsing multiple sclerosis is warranted. FUNDING: US National Multiple Sclerosis Society, Multiple Sclerosis Society of Great Britain and Northern Ireland, Novartis, UK National Institute for Health Research (NIHR), and NIHR UCLH/UCL Biomedical Research Centre.
Assuntos
Fármacos Neuroprotetores/farmacologia , Neurite Óptica/tratamento farmacológico , Avaliação de Resultados em Cuidados de Saúde , Fenitoína/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Adulto , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fármacos Neuroprotetores/administração & dosagem , Fenitoína/administração & dosagem , Fenitoína/efeitos adversos , Bloqueadores do Canal de Sódio Disparado por Voltagem/administração & dosagem , Bloqueadores do Canal de Sódio Disparado por Voltagem/efeitos adversosRESUMO
The biosynthetic gene cluster for the angiogenesis inhibitor borrelidin has been cloned from Streptomyces parvulus Tü4055. Sequence analysis indicates that the macrolide ring of borrelidin is formed by a modular polyketide synthase (PKS) (borA1-A6), a result that was confirmed by disruption of borA3. The borrelidin PKS is striking because only seven rather than the nine modules expected for a nonaketide product are encoded by borA1-A6. The starter unit of the PKS has been verified as trans-cyclopentane-1,2-dicarboxylic acid (trans-1,2-CPDA), and the genes involved in its biosynthesis identified. Other genes responsible for biosynthesis of the nitrile moiety, regulation, and self-resistance were also identified.
Assuntos
Inibidores da Angiogênese/biossíntese , Álcoois Graxos/metabolismo , Genes Bacterianos , Família Multigênica , Streptomyces/genética , Inibidores da Angiogênese/química , Clonagem Molecular , Ciclopentanos/síntese química , Ácidos Dicarboxílicos/síntese química , Álcoois Graxos/química , Modelos Químicos , Dados de Sequência Molecular , Estrutura Molecular , Complexos Multienzimáticos/genética , Análise de Sequência de DNA , Streptomyces/enzimologia , Streptomyces/metabolismoRESUMO
Novel spinosyns have been prepared by biotransformation, using a genetically engineered strain of Saccharopolyspora erythraea, in which the beta-D-forosamine moiety in glycosidic linkage to the hydroxy group at C17 is replaced by alpha-L-mycarose.
Assuntos
Antibacterianos/biossíntese , Desoxiaçúcares/metabolismo , Engenharia Genética , Biotransformação , Fermentação , Glicosiltransferases/genética , Hexoses/metabolismo , Macrolídeos , Saccharopolyspora/genéticaRESUMO
The acyltransferase (AT) domain in module 4 of the erythromycin polyketide synthase (PKS) was substituted with an AT domain from the rapamycin PKS module 2 in order to alter the substrate specificity from methylmalonyl-CoA to malonyl-CoA. The resulting strain produced 6-desmethyl erythromycin D as the predominant product. This AT domain swap completes the library of malonyl-CoA AT swaps on the erythromycin PKS and reinforces PKS engineering as a robust and generic tool.
Assuntos
Aciltransferases , Antibacterianos , Eritromicina , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Sequência de Bases , Eritromicina/análogos & derivados , Eritromicina/isolamento & purificação , Eritromicina/farmacologia , Fermentação , Testes de Sensibilidade Microbiana , Complexos Multienzimáticos , Relação Estrutura-Atividade , Especificidade por SubstratoAssuntos
Sirolimo/química , Sirolimo/metabolismo , Streptomyces/química , Clonagem Molecular , Cromatografia Gasosa-Espectrometria de Massas , Genes Bacterianos/genética , Imunossupressores/química , Imunossupressores/metabolismo , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Sirolimo/análogos & derivados , Sirolimo/isolamento & purificação , Estereoisomerismo , Streptomyces/genética , Streptomyces/metabolismoRESUMO
A biosynthetic medicinal chemistry approach was applied to the optimization of the natural product Hsp90 inhibitor macbecin. By genetic engineering, mutants have been created to produce novel macbecin analogues including a nonquinone compound (5) that has significantly improved binding affinity to Hsp90 (Kd 3 nM vs 240 nM for macbecin) and reduced toxicity (MTD > or = 250 mg/kg). Structural flexibility may contribute to the preorganization of 5 to exist in solution in the Hsp90-bound conformation.
Assuntos
Benzoquinonas/farmacologia , Produtos Biológicos/farmacologia , Engenharia Genética , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Benzoquinonas/química , Benzoquinonas/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/metabolismo , Dados de Sequência Molecular , Estrutura MolecularRESUMO
Rapamycin is an important macrocyclic polyketide produced by Streptomyces hygroscopicus and showing immunosuppressive, antifungal, and antitumor activities as well as displaying anti-inflammatory and neuroregenerative properties. The immense pharmacological potential of rapamycin has led to the production of an array of analogues, including through genetic engineering of the rapamycin biosynthetic gene cluster. This cluster contains several putative regulatory genes. Based on DNA sequence analysis, the products of genes rapH and rapG showed high similarities with two different families of transcriptional activators, LAL and AraC, respectively. Overexpression of either gene resulted in a substantial increase in rapamycin biosynthesis, confirming their positive regulatory role, while deletion of both from the chromosome of S. hygroscopicus resulted in a complete loss of antibiotic production. Complementation studies indicated an essential role of the RapG regulator for rapamycin biosynthesis and a supportive role of RapH. A direct effect of rapH and rapG gene products on the promoter of the rapamycin polyketide synthase operon, rapA-rapB, was observed using the chalcone synthase gene rppA as a reporter system.
Assuntos
Proteínas de Bactérias/fisiologia , Sirolimo/metabolismo , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Mutação , Óperon , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Streptomyces/genéticaRESUMO
A set of novel borrelidin analogues have been prepared by precursor-directed biosynthesis. Structure-activity relationship analysis suggests that steric structural arrangement within the C17 side chain is important for differentiating cytotoxic and anti-angiogenic activities. A C17-cyclobutyl analogue 3 was found to have markedly increased selectivity for in vitro angiogenesis inhibition over cytotoxicity and is therefore potentially useful as an anticancer agent.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Ciclobutanos/química , Inibidores da Angiogênese/síntese química , Antineoplásicos/síntese química , Linhagem Celular Tumoral/efeitos dos fármacos , Álcoois Graxos/síntese química , Álcoois Graxos/farmacologia , Humanos , Concentração Inibidora 50 , Relação Estrutura-AtividadeRESUMO
The spinosyns are a family of potent and highly selective insect control agents that display a favorable environmental profile. As some regions of the spinosyn molecule are recalcitrant to chemical modification, a targeted genetic approach was carried out to generate new analogues. The polyketide synthase (PKS) loading modules from the avermectin PKS of Streptomyces avermitilis and the erythromcyin PKS of Saccharopolyspora erythraea were each used to replace the spinosyn PKS loading module. Both of the resulting strains containing hybrid PKS pathways produced the anticipated spinosyn analogues. Supplementation of the culture media with a range of exogenous carboxylic acids led to the successful incorporation of these novel elements to yield further novel spinosyn molecules, some of which demonstrated potent and new insecticidal activities. Furthermore, it has been demonstrated that semisynthesis of such novel metabolites can then be used to generate active analogues, demonstrating the effectiveness of utilizing these complementary methods to search the chemical space around this template.