Valery Belakhov

Development of novel aminoglycoside (NB54) with reduced toxicity and enhanced suppression of disease-causing premature stop mutations

Nonsense mutations promote premature translational termination and represent the underlying cause of a large number of human genetic diseases. The aminoglycoside antibiotic gentamicin has the ability to allow the mammalian ribosome to read past a false-stop signal and generate full-length functional proteins. However, severe toxic side effects along with the reduced suppression efficiency at subtoxic doses limit the use of gentamicin for suppression therapy. We describe here the first systematic development of the novel aminoglycoside 2 (NB54) exhibiting superior in vitro readthrough efficiency to that of gentamicin in seven different DNA fragments derived from mutant genes carrying nonsense mutations representing the genetic diseases Usher syndrome, cystic fibrosis, Duchenne muscular dystrophy, and Hurler syndrome. Comparative acute lethal toxicity in mice, cell toxicity, and the assessment of hair cell toxicity in cochlear explants further indicated that 2 exhibits far lower toxicity than that of gentamicin.

Crystal structure and snapshots along the reaction pathway of a family 51 α‐l‐arabinofuranosidase

High‐resolution crystal structures of α‐L‐arabinofuranosidase from Geobacillus stearothermophilus T‐6, a family 51 glycosidase, are described. The enzyme is a hexamer, and each monomer is organized into two domains: a (β/α)8‐barrel and a 12‐stranded β sandwich with jelly‐roll topology. The structures of the Michaelis complexes with natural and synthetic substrates, and of the transient covalent arabinofuranosyl—enzyme intermediate represent two stable states in the double displacement mechanism, and allow thorough examination of the catalytic mechanism. The arabinofuranose sugar is tightly bound and distorted by an extensive network of hydrogen bonds. The two catalytic residues are 4.7 Å apart, and together with other conserved residues contribute to the stabilization of the oxocarbenium ion‐like transition state via charge delocalization and specific protein—substrate interactions. The enzyme is an anti‐protonator, and a 1.7 Å electrophilic migration of the anomeric carbon takes place during the hydrolysis.

Repairing faulty genes by aminoglycosides: Development of new derivatives of geneticin (G418) with enhanced suppression of diseases-causing nonsense mutations

New pseudo-di- and pseudo-trisaccharide derivatives of the aminoglycoside drug G418 were designed, synthesized and their ability to readthrough nonsense mutations was examined in both in vitro and ex vivo systems, along with the toxicity tests. Two novel lead structures, NB74 and NB84, exhibiting significantly reduced cell toxicity and superior readthrough efficiency than those of gentamicin, were discovered. The superiority of new leads was demonstrated in six different nonsense DNA-constructs underling the genetic diseases cystic fibrosis, Duchenne muscular dystrophy, Usher syndrome and Hurler syndrome.

Readthrough of nonsense mutations in Rett syndrome: evaluation of novel aminoglycosides and generation of a new mouse model

Thirty-five percent of patients with Rett syndrome carry nonsense mutations in the MECP2 gene. We have recently shown in transfected HeLa cells that readthrough of nonsense mutations in the MECP2 gene can be achieved by treatment with gentamicin and geneticin. This study was performed to test if readthrough can also be achieved in cells endogenously expressing mutant MeCP2 and to evaluate potentially more effective readthrough compounds. A mouse model was generated carrying the R168X mutation in the MECP2 gene. Transfected HeLa cells expressing mutated MeCP2 fusion proteins and mouse ear fibroblasts isolated from the new mouse model were treated with gentamicin and the novel aminoglycosides NB30, NB54, and NB84. The localization of the readthrough product was tested by immunofluorescence. Readthrough of the R168X mutation in mouse ear fibroblasts using gentamicin was detected but at lower level than in HeLa cells. As expected, the readthrough product, full-length Mecp2 protein, was located in the nucleus. NB54 and NB84 induced readthrough more effectively than gentamicin, while NB30 was less effective. Readthrough of nonsense mutations can be achieved not only in transfected HeLa cells but also in fibroblasts of the newly generated Mecp2R168X mouse model. NB54 and NB84 were more effective than gentamicin and are therefore promising candidates for readthrough therapy in Rett syndrome patients.

Design, synthesis, and evaluation of novel fluoroquinolone-aminoglycoside hybrid antibiotics.

A series of new hybrid structures containing fluoroquinolone (ciprofloxacin) and aminoglycoside (neomycin) antibiotics linked via 1,2,3-triazole moiety were designed and synthesized, and their antibacterial activities were determined against both Gram-negative and Gram-positive bacteria, including resistant strains. The nature of spacers in both the ciprofloxacin and neomycin parts greatly influenced the antibacterial activity. The majority of hybrids was significantly more potent than the parent neomycin and overcame most prevalent types of resistance associated with aminoglycosides. Selected hybrids inhibited bacterial protein synthesis with the potencies similar to or better than that of neomycin and were up to 32-fold more potent inhibitors than ciprofloxacin for the fluoroquinolone targets, DNA gyrase and toposiomerase IV, indicating a balanced dual mode of action. Significant delay of resistance formation was observed in both E. coli and B. subtilis to the treatment with ciprofloxacin-neomycin hybrid in comparison to that of each drug separately or their 1:1 mixture.

The identification of the acid–base catalyst of α-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase

The α‐L‐arabinofuranosidase from Geobacillus stearothermophilus T‐6 (AbfA T‐6) belongs to the retaining family 51 glycoside hydrolases. The conserved Glu175 was proposed to be the acid–base catalytic residue. AbfA T‐6 exhibits residual activity towards aryl β‐D‐xylopyranosides. This phenomenon was used to examine the catalytic properties of the putative acid–base mutant E175A. Data from kinetic experiments, pH profiles, azide rescue, and the identification of the xylopyranosyl azide product provide firm support to the assignment of Glu175 as the acid–base catalyst of AbfA T‐6.

Synthetic Aminoglycosides Efficiently Suppress Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations and Are Enhanced by Ivacaftor

New drugs are needed to enhance premature termination codon (PTC) suppression to treat the underlying cause of cystic fibrosis (CF) and other diseases caused by nonsense mutations. We tested new synthetic aminoglycoside derivatives expressly developed for PTC suppression in a series of complementary CF models. Using a dual-luciferase reporter system containing the four most prevalent CF transmembrane conductance regulator (CFTR) nonsense mutations (G542X, R553X, R1162X, and W1282X) within their local sequence contexts (the three codons on either side of the PTC), we found that NB124 promoted the most readthrough of G542X, R1162X, and W1282X PTCs. NB124 also restored full-length CFTR expression and chloride transport in Fischer rat thyroid cells stably transduced with a CFTR-G542XcDNA transgene, and was superior to gentamicin and other aminoglycosides tested. NB124 restored CFTR function to roughly 7% of wild-type activity in primary human bronchial epithelial (HBE) CF cells (G542X/delF508), a highly relevant preclinical model with endogenous CFTR expression. Efficacy was further enhanced by addition of the CFTR potentiator, ivacaftor (VX-770), to airway cells expressing CFTR PTCs. NB124 treatment rescued CFTR function in a CF mouse model expressing a human CFTR-G542X transgene; efficacy was superior to gentamicin and exhibited favorable pharmacokinetic properties, suggesting that in vitro results translated to clinical benefit in vivo. NB124 was also less cytotoxic than gentamicin in a tissue-based model for ototoxicity. These results provide evidence that NB124 and other synthetic aminoglycosides provide a 10-fold improvement in therapeutic index over gentamicin and other first-generation aminoglycosides, providing a promising treatment for a wide array of CFTR nonsense mutations.

A comparative evaluation of NB30, NB54 and PTC124 in translational read-through efficacy for treatment of an USH1C nonsense mutation

Translational read‐through‐inducing drugs (TRIDs) promote read‐through of nonsense mutations, placing them in the spotlight of current gene‐based therapeutic research. Here, we compare for the first time the relative efficacies of new‐generation aminoglycosides NB30, NB54 and the chemical compound PTC124 on retinal toxicity and read‐through efficacy of a nonsense mutation in the USH1C gene, which encodes the scaffold protein harmonin. This mutation causes the human Usher syndrome, the most common form of inherited deaf‐blindness. We quantify read‐through efficacy of the TRIDs in cell culture and show the restoration of harmonin function. We do not observe significant differences in the read‐through efficacy of the TRIDs in retinal cultures; however, we show an excellent biocompatibility in retinal cultures with read‐through versus toxicity evidently superior for NB54 and PTC124. In addition, in vivo administration of NB54 and PTC124 induced recovery of the full‐length harmonin a1 with the same efficacy. The high biocompatibilities combined with the sustained read‐through efficacies of these drugs emphasize the potential of NB54 and PTC124 in treating nonsense mutation‐based retinal disorders.

The designer aminoglycoside NB84 significantly reduces glycosaminoglycan accumulation associated with MPS I-H in the Idua-W392X mouse.

Suppression therapy utilizes compounds that suppress translation termination at in-frame premature termination codons (PTCs) to restore full-length, functional protein. This approach may provide a treatment for diseases caused by nonsense mutations such as mucopolysaccharidosis type I-Hurler (MPS I-H). MPS I-H is a lysosomal storage disease caused by severe α-L-iduronidase deficiency and subsequent lysosomal glycosaminoglycan (GAG) accumulation. MPS I-H represents a good target for suppression therapy because the majority of MPS I-H patients carry nonsense mutations, and restoration of even a small amount of functional α-L-iduronidase may attenuate the MPS I-H phenotype. In this study, we investigated the efficiency of suppression therapy agents to suppress the Idua-W392X nonsense mutation in an MPS I-H mouse model. The drugs tested included the conventional aminoglycosides gentamicin, G418, amikacin, and paromomycin. In addition, the designer aminoglycosides NB54 and NB84, two compounds previously designed to mediate efficient PTC suppression with reduced toxicity, were also examined. Overall, NB84 suppressed the Idua-W392X nonsense mutation much more efficiently than any of the other compounds tested. NB84 treatment restored enough functional α-L-iduronidase activity to partially reverse abnormal GAG accumulation and lysosomal abundance in mouse embryonic fibroblasts derived from the Idua-W392X mouse. Finally, in vivo administration of NB84 to Idua-W392X mice significantly reduced urine GAG excretion and tissue GAG storage. Together, these results suggest that NB84-mediated suppression therapy has the potential to attenuate the MPS I-H disease phenotype.

Attenuation of Nonsense-Mediated mRNA Decay Enhances In Vivo Nonsense Suppression

Nonsense suppression therapy is an approach to treat genetic diseases caused by nonsense mutations. This therapeutic strategy pharmacologically suppresses translation termination at Premature Termination Codons (PTCs) in order to restore expression of functional protein. However, the process of Nonsense-Mediated mRNA Decay (NMD), which reduces the abundance of mRNAs containing PTCs, frequently limits this approach. Here, we used a mouse model of the lysosomal storage disease mucopolysaccharidosis I-Hurler (MPS I-H) that carries a PTC in the Idua locus to test whether NMD attenuation can enhance PTC suppression in vivo. Idua encodes alpha-L-iduronidase, an enzyme required for degradation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. We found that the NMD attenuator NMDI-1 increased the abundance of the PTC-containing Idua transcript. Furthermore, co-administration of NMDI-1 with the PTC suppression drug gentamicin enhanced alpha-L-iduronidase activity compared to gentamicin alone, leading to a greater reduction of GAG storage in mouse tissues, including the brain. These results demonstrate that NMD attenuation significantly enhances suppression therapy in vivo.