Jeyakumar Kandasamy

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.

Increased Selectivity toward Cytoplasmic versus Mitochondrial Ribosome Confers Improved Efficiency of Synthetic Aminoglycosides in Fixing Damaged Genes: A Strategy for Treatment of Genetic Diseases Caused by Nonsense Mutations

Compelling evidence is now available that gentamicin and Geneticin (G418) can induce the mammalian ribosome to suppress disease-causing nonsense mutations and partially restore the expression of functional proteins. However, toxicity and relative lack of efficacy at subtoxic doses limit the use of gentamicin for suppression therapy. Although G418 exhibits the strongest activity, it is very cytotoxic even at low doses. We describe here the first systematic development of the novel aminoglycoside (S)-11 exhibiting similar in vitro and ex vivo activity to that of G418, while its cell toxicity is significantly lower than those of gentamicin and G418. Using a series of biochemical assays, we provide proof of principle that antibacterial activity and toxicity of aminoglycosides can be dissected from their suppression activity. The data further indicate that the increased specificity toward cytoplasmic ribosome correlates with the increased activity and that the decreased specificity toward mitochondrial ribosome confers the lowered cytotoxicity.

Long-Term Nonsense Suppression Therapy Moderates MPS I-H Disease Progression

Nonsense suppression therapy is a therapeutic approach aimed at treating genetic diseases caused by in-frame premature termination codons (PTCs; also commonly known as nonsense mutations). This approach utilizes compounds that suppress translation termination at PTCs, which allows translation to continue and partial levels of deficient protein function to be restored. We hypothesize that suppression therapy can attenuate the lysosomal storage disease mucopolysaccharidosis type I-Hurler (MPS I-H), the severe form of α-L-iduronidase deficiency. α-L-iduronidase participates in glycosaminoglycan (GAG) catabolism and its insufficiency causes progressive GAG accumulation and onset of the MPS I-H phenotype, which consists of multiple somatic and neurological defects. 60-80% of MPS I-H patients carry a nonsense mutation in the IDUA gene. We previously showed that 2-week treatment with the designer aminoglycoside NB84 restored enough α-L-iduronidase function via PTC suppression to reduce tissue GAG accumulation in the Idua(tm1Kmke) MPS I-H mouse model, which carries a PTC homologous to the human IDUA-W402X nonsense mutation. Here we report that long-term NB84 administration maintains α-L-iduronidase activity and GAG reduction in Idua(tm1Kmke) mice throughout a 28-week treatment period. An examination of more complex MPS I-H phenotypes in Idua(tm1Kmke) mice following 28-week NB84 treatment revealed significant moderation of the disease in multiple tissues, including the brain, heart and bone, that are resistant to current MPS I-H therapies. This study represents the first demonstration that long-term nonsense suppression therapy can moderate progression of a genetic disease.

Automated solid phase synthesis of oligoarabinofuranosides

Automated solid phase synthesis enables rapid access to the linear and branched arabinofuranoside oligosaccharides. A simple purification step is sufficient to provide the conjugation ready oligosaccharides in good yield.

An efficient synthesis of N-nitrosamines under solvent, metal and acid free conditions using tert-butyl nitrite

Synthesis of various N-nitroso compounds from secondary amines is reported using tert-butyl nitrite (TBN) under solvent free conditions. Broad substrate scope, metal and acid free conditions, easy isolation procedure and excellent yields are few important features of this methodology. The acid labile protecting groups such as tert-butyldimethylsilyl (TBDMS) and tert-butyloxycarbonyl (Boc) as well as sensitive functional groups such as phenols, olefins and alkynes are found to be stable under the standard reaction conditions. Besides N-nitrosation, TBN is also found to be an efficient reagent in few other transformations including aryl hydrazines to aryl azides and primary amides to carboxylic acids under mild conditions.

Aminoglycosides: Redesign Strategies for Improved Antibiotics and Compounds for Treatment of Human Genetic Diseases

Aminoglycosides are highly potent, broad-spectrum antibiotics that kill bacteria by binding to the ribosomal decoding site and reducing the fidelity of protein synthesis. The emergence of bacterial strains resistant to these drugs, as well as their relative toxicity, have inspired extensive searches toward the goal of obtaining novel molecular designs with improved antibacterial activity and reduced toxicity. In recent years, a new therapeutic approach that employs the ability of certain aminoglycosides to induce mammalian ribosomes to readthrough premature stop codon mutations has emerged. This new and challenging task has introduced fresh research avenues in the field of aminoglycosides research. In this chapter, our recent observations and current challenges in the design of aminoglycosides with improved antibacterial activity and the treatment of human genetic diseases are discussed.

Modular automated solid phase synthesis of dermatan sulfate oligosaccharides

Dermatan sulfates are glycosaminoglycan polysaccharides that serve a multitude of biological roles as part of the extracellular matrix. Orthogonally protected D-galactosamine and L-iduronic acid building blocks and a photo-cleavable linker are instrumental for the automated synthesis of dermatan sulfate oligosaccharides. Conjugation-ready oligosaccharides were obtained in good yield.

Synthetic di-sulfated iduronic acid attenuates asthmatic response by blocking T-cell recruitment to inflammatory sites

Significance Asthmatic inflammation is orchestrated by T-lymphocyte cell trafficking network within lungs, blood circulation, secondary lymphoid organ, and peripheral tissue. Here, we demonstrated that T cell and following eosinophil recruitment was substantially reduced in our recently generated mouse model, where heparan sulfate synthase exostoses-1 (Ext1) is knockout in an inducible manner. Moreover, we discovered that even a monosaccharide, 2,4-disulfated iduronic acid (Di-S-IdoA), bound to chemokine CCL20 and significantly inhibited CCL20 binding to heparan sulfate and to endothelial cell surface. We found that Di-S-IdoA attenuated asthmatic reaction, measured by T cell, eosinophil, and CCL20 recruitment in asthmatic mice. These findings show for the first time (to our knowledge) that sulfate monosaccharide can be developed into a potent therapeutic agent for treating asthma. Identification of carbohydrate sequences that determine affinity to specific chemokines is a critical step for strategies to interfere with chemokine-mediated leukocyte trafficking. Here, we first characterized the development of allergic asthma in Tie2-dependent and inducible Ext1-knockout (Tie2-Ext1iKO) mice. We showed that heparan sulfate is essential for leukocyte recruitment in the peribronchial region and bronchoalveolar lavage fluid (BALF), and is crucial for induction of airway hyperresponsiveness. Our glycan microarray showed a unique affinity profile of chemokine CCL20 to substructures of heparin and heparin-like oligo/di/monosaccharides. Among them, we identified a synthetic and not naturally occurring monosaccharide, 2,4-O-di-sulfated iduronic acid (Di-S-IdoA), as a potential inhibitor for CCL20–heparan sulfate interaction. Mice injected with Di-S-IdoA via tail vain or nasal inhalation showed attenuated leukocyte recruitment into inflammatory sites and BALF. These results demonstrate a critical role of chemokine–heparan sulfate interaction in the asthma development and Di-S-IdoA as a potential drug for asthma treatment.

Bio-based green solvent for the catalyst free oxidation of arylboronic acids into phenols

A bio-based green solvent, lactic acid, is found to be an efficient reaction medium for the catalyst free oxidation of arylboronic acids into phenols with aqueous hydrogen peroxide. Various substituted arylboronic acids have undergone ipso-hydroxylation smoothly at room temperature to provide corresponding phenols in excellent yields. Remarkably, the oxidation susceptible functional groups such as sulphide, ketone, aldehyde and olefin are tolerated under the reaction conditions. Over all, lactic acid showed higher efficiency as a solvent medium when compared with conventional acetic acid.