Premature termination codon readthrough upregulates progranulin expression and improves lysosomal function in preclinical models of GRN deficiency.

BACKGROUND: Frontotemporal lobar degeneration (FTLD) is a devastating and progressive disorder, and a common cause of early onset dementia. Progranulin (PGRN) haploinsufficiency due to autosomal dominant mutations in the progranulin gene (GRN) is an important cause of FTLD (FTLD-GRN), and nearly a quarter of these genetic cases are due to a nonsense mutation. Premature termination codons (PTC) can be therapeutically targeted by compounds allowing readthrough, and aminoglycoside antibiotics are known to be potent PTC readthrough drugs. Restoring endogenous PGRN through PTC readthrough has not previously been explored as a therapeutic intervention in FTLD. METHODS: We studied whether the aminoglycoside G418 could increase PGRN expression in HEK293 and human induced pluripotent stem cell (hiPSC)-derived neurons bearing the heterozygous S116X, R418X, and R493X pathogenic GRN nonsense mutations. We further tested a novel substituted phthalimide PTC readthrough enhancer in combination with G418 in our cellular models. We next generated a homozygous R493X knock-in hiPSC isogenic line (R493X-/- KI), assessing whether combination treatment in hiPSC-derived neurons and astrocytes could increase PGRN and ameliorate lysosomal dysfunction relevant to FTLD-GRN. To provide in vivo proof-of-concept of our approach, we measured brain PGRN after intracerebroventricular administration of G418 in mice expressing the V5-tagged GRN nonsense mutation R493X. RESULTS: The R418X and R493X mutant GRN cell lines responded to PTC readthrough with G418, and treatments increased PGRN levels in R493X-/- KI hiPSC-derived neurons and astrocytes. Combining G418 with a PTC readthrough enhancer increased PGRN levels over G418 treatment alone in vitro. PGRN deficiency has been shown to impair lysosomal function, and the mature form of the lysosomal protease cathepsin D is overexpressed in R493X-/- KI neurons. Increasing PGRN through G418-mediated PTC readthrough normalized this abnormal lysosomal phenotype in R493X-/- KI neuronal cultures. A single intracerebroventricular injection of G418 induced GRN PTC readthrough in 6-week-old AAV-GRN-R493X-V5 mice. CONCLUSIONS: Taken together, our findings suggest that PTC readthrough may be a potential therapeutic strategy for FTLD caused by GRN nonsense mutations.

Directed evolution of a remarkably efficient Kdnase from a bacterial neuraminidase.

N-acetylneuraminic acid (5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid), which is the principal sialic acid family member of the non-2-ulosonic acids and their various derivatives, is often found at the terminal position on the glycan chains that adorn all vertebrate cells. This terminal position combined with subtle variations in structure and linkage to the underlying glycan chains between humans and other mammals points to the importance of this diverse group of nine-carbon sugars as indicators of the unique aspects of human evolution and is relevant to understanding an array of human conditions. Enzymes that catalyze the removal N-acetylneuraminic acid from glycoconjugates are called neuraminidases. However, despite their documented role in numerous diseases, due to the promiscuous activity of many neuraminidases, our knowledge of the functions and metabolism of many sialic acids and the effect of the attachment to cellular glycans is limited. To this end, through a concerted effort of generation of random and site-directed mutagenesis libraries, subsequent screens and positive and negative evolutionary selection protocols, we succeeded in identifying three enzyme variants of the neuraminidase from the soil bacterium Micromonospora viridifaciens with markedly altered specificity for the hydrolysis of natural Kdn (3-deoxy-d-glycero-d-galacto-non-2-ulosonic acid) glycosidic linkages compared to those of N-acetylneuraminic acid. These variants catalyze the hydrolysis of Kdn-containing disaccharides with catalytic efficiencies (second-order rate constants: kcat/Km) of greater than 105 M-1 s-1; the best variant displayed an efficiency of >106 M-1 s-1 at its optimal pH.

Both Chemical and Non-Chemical Steps Limit the Catalytic Efficiency of Family 4 Glycoside Hydrolases.

The glycoside hydrolase family 4 (GH4) α-galactosidase from Citrobacter freundii (MelA) catalyzes the hydrolysis of fluoro-substituted phenyl α-d-galactopyranosides by utilizing two cofactors, NAD+ and a metal cation, under reducing conditions. In order to refine the mechanistic understanding of this GH4 enzyme, leaving group effects were measured with various metal cations. The derived ßlg value on V/ K for strontium activation is indistinguishable from zero (0.05 ± 0.12). Deuterium kinetic isotope effects (KIEs) were measured for the activated substrates 2-fluorophenyl and 4-fluorophenyl α-d-galactopyranosides in the presence of Sr2+, Y3+, and Mn2+, where the isotopic substitution was on the carbohydrate at C-2 and/or C-3. To determine the contributing factors to the virtual transition state (TS) on which the KIEs report, kinetic isotope effects on isotope effects were measured on these KIEs using doubly deuterated substrates. The measured D V/ K KIEs for MelA-catalyzed hydrolysis of 2-fluorophenyl α-d-galactopyranoside are closer to unity than the measured effects on 4-fluorophenyl α-d-galactopyranoside, irrespective of the site of isotopic substitution and of the metal cation activator. These observations are consistent with hydride transfer at C-3 to the on-board NAD+, deprotonation at C-2, and a non-chemical step contributing to the virtual TS for V/ K.

C2-Oxyanion Neighboring Group Participation: Transition State Structure for the Hydroxide-Promoted Hydrolysis of 4-Nitrophenyl α-d-Mannopyranoside.

The hydroxide-catalyzed hydrolysis of aryl 1,2-trans-glycosides proceeds through a mechanism involving neighboring group participation by a C2-oxyanion and rate-limiting formation of a 1,2-anhydro sugar (oxirane) intermediate. The transition state for the hydroxide-catalyzed hydrolysis of 4-nitrophenyl α-d-mannopyranoside in aqueous media has been studied by the use of multiple kinetic isotope effect (KIE) measurements in conjunction with ab initio theoretical methods. The experimental KIEs are C1-2H (1.112 ± 0.004), C2-2H (1.045 ± 0.005), anomeric 1-13C (1.026 ± 0.006), C2-13C (0.999 ± 0.005), leaving group oxygen 2-18O (1.040 ± 0.012), and C2-18O (1.044 ± 0.006). The transition state for the hydrolysis reaction was modeled computationally using the experimental KIE values as constraints. Taken together, the reported kinetic isotope effects and computational modeling are consistent with the reaction mechanism involving rate-limiting formation of a transient oxirane intermediate that opens in water to give α-d-mannopyranose. The transition state has significant nucleophilic participation by the C2-alkoxide, an essentially cleaved glycosidic bond, and a slight shortening of the endocyclic C1-O5 bond. The TS is late, consistent with the large, normal C2-18O isotope effect.

Novel small molecules potentiate premature termination codon readthrough by aminoglycosides.

Nonsense mutations introduce premature termination codons and underlie 11% of genetic disease cases. High concentrations of aminoglycosides can restore gene function by eliciting premature termination codon readthrough but with low efficiency. Using a high-throughput screen, we identified compounds that potentiate readthrough by aminoglycosides at multiple nonsense alleles in yeast. Chemical optimization generated phthalimide derivative CDX5-1 with activity in human cells. Alone, CDX5-1 did not induce readthrough or increase TP53 mRNA levels in HDQ-P1 cancer cells with a homozygous TP53 nonsense mutation. However, in combination with aminoglycoside G418, it enhanced readthrough up to 180-fold over G418 alone. The combination also increased readthrough at all three nonsense codons in cancer cells with other TP53 nonsense mutations, as well as in cells from rare genetic disease patients with nonsense mutations in the CLN2, SMARCAL1 and DMD genes. These findings open up the possibility of treating patients across a spectrum of genetic diseases caused by nonsense mutations.

Novel spirothiazamenthane inhibitors of the influenza A M2 proton channel.

The development of treatments for influenza that inhibit the M2 proton channel without being susceptible to the widespread resistance mechanisms associated with the adamantanes is an ongoing challenge. Using a yeast high-throughput yeast growth restoration assay designed to identify M2 channel inhibitors, a single screening hit was uncovered. This compound (3), whose structure was incorrectly identified in the literature, is an inhibitor with similar potency to amantadine against WT M2. A library of derivatives of 3 was prepared and activity against WT M2 and the two principal mutant strains (V27A and S31N) was assessed in the yeast assay. The best compounds were further evaluated in an antiviral plaque reduction assay using engineered WT, V27A and S31N M2 influenza A strains with otherwise identical genetic background. Compound 63 was found to inhibit all three virus strains in this cell based antiviral assay at micromolar concentrations, possibly through a mechanism other than M2 inhibition.

Neuraminidase substrate promiscuity permits a mutant Micromonospora viridifaciens enzyme to synthesize artificial carbohydrates.

Mutation of the nucleophilic amino acid residue tyrosine to the small nonpolar residue glycine (Y370G) in the active site of Micromonospora viridifaciens neuraminidase (MvNA) produces an efficient catalyst for the transfer of N-acetylneuraminic acid from an artificial substrate (i.e., phenyl N-acetyl-ß-D-neuraminide) to a sugar acceptor (e.g., D-lactose, D-glucose, D-mannose, D-raffinose, D-allose, or D-fructose) to give N-acetyl-α-neuraminide coupled carbohydrate products. In addition, this mutant enzyme (MvNA Y370G) catalyzes the transfer of a sugar residue from the artificial substrate 2-fluorophenyl N-acetyl-ß-D-neuraminide to methyl glycopyranoside acceptors. Interestingly, when trans-glycosylation reactions are conducted in aqueous solutions containing 30% (v/v) acetonitrile, the α-anomeric acceptors of methyl glucopyranoside and galactopyranoside generate higher product yields than do their corresponding ß-anomers. Specifically, a 64 h reaction with 2-fluorophenyl N-acetyl-ß-D-neuraminide as the limiting reagent and the acceptors methyl α-d-galactopyranoside, methyl α-D-glucopyranoside, or methyl α-D-mannopyranoside gives trans-glycosylation product yields of 22%, 31%, or 34%, respectively. With methyl α-D-galactopyranoside as the acceptor, trans-glycosylations catalyzed by both MvNA Y370G and a 2,6-sialyltransferase yield identical products, which we identified as methyl N-acetyl-α-D-neuraminyl-(2 → 6)-α-D-galactopyranoside. The MvNA Y370G-catalyzed coupling of N-acetylneuraminic acid to these three methyl α-d-glycopyranoside acceptors is favored by factors of 18­27-fold over the competing hydrolysis reaction. These coupling efficiencies likely arise from nonselective interactions between the acceptor glycopyranoside and MvNA Y370G, which preferentially places a carbohydrate hydroxyl group rather than water in close proximity to the active site where this functionality intercepts the nascent neuraminyl oxacarbenium ion that is formed during cleavage of the glycosidic bond in the aryl N-acetyl-ß-D-neuraminide donor. The ability to transfer N-acetylneuraminic acid from a stable and readily accessible donor to acceptor carbohydrates that are not substrates for sialyltransferases is one step on the path for the production of pseudohuman glycoproteins from nonmammalian cell lines.

Kinetic and structural evaluation of selected active site mutants of the Aspergillus fumigatus KDNase (sialidase).

Aspergillus fumigatus is an airborne fungal pathogen. We previously cloned and characterized an exo-sialidase from A. fumigatus and showed that it preferred 2-keto-3-deoxynononic acid (KDN) as a substrate to N-acetylneuraminic acid (Neu5Ac). The purpose of this study was to investigate the structure-function relationships of critical catalytic site residues. Site-directed mutagenesis was used to create three mutant recombinant enzymes: the catalytic nucleophile (Y358H), the general acid/base catalyst (D84A), and an enlargement of the binding pocket to attempt to accommodate the N-acetyl group of Neu5Ac (R171L). Crystal structures for all enzymes were determined. The D84A mutation had an effect in decreasing the activity of AfKDNase that was stronger than that of the same mutation in the structurally similar sialidase from the bacterium Micromonospora viridifaciens. These data suggest that the catalytic acid is more important in the reaction of AfKDNase and that catalysis is less dependent on nucleophilic or electrostatic stabilization of the developing positive charge at the transition state for hydrolysis. Removal of the catalytic nucleophile (Y358H) significantly lowered the activity of the enzyme, but this mutant remained a retaining glycosidase as demonstrated by nuclear magnetic resonance spectroscopic analysis. This is a novel finding that has not been shown with other sialidases. Kinetic activity measured at pH 5.2 revealed that R171L had higher activity on a Neu5Ac-based substrate than wild-type KDNase; hence, leucine in place of arginine in the binding pocket improved catalysis toward Neu5Ac substrates. Hence, whether a sialidase is primarily a KDNase or a neuraminidase is due in part to the presence of an amino acid that creates a steric clash with the N-acetyl group.

Chemical insight into the emergence of influenza virus strains that are resistant to Relenza.

A reagent panel containing ten 4-substituted 4-nitrophenyl α-D-sialosides and a second panel of the corresponding sialic acid glycals were synthesized and used to probe the inhibition mechanism for two neuraminidases, the N2 enzyme from influenza type A virus and the enzyme from Micromonospora viridifaciens. For the viral enzyme the logarithm of the inhibition constant (Ki) correlated with neither the logarithm of the catalytic efficiency (kcat/Km) nor catalytic proficiency (kcat/Km kun). These linear free energy relationship data support the notion that these inhibitors, which include the therapeutic agent Relenza, are not transition state mimics for the enzyme-catalyzed hydrolysis reaction. Moreover, for the influenza enzyme, a correlation (slope, 0.80 ± 0.08) is observed between the logarithms of the inhibition (Ki) and Michaelis (Km) constants. We conclude that the free energy for Relenza binding to the influenza enzyme mimics the enzyme-substrate interactions at the Michaelis complex. Thus, an influenza mutational response to a 4-substituted sialic acid glycal inhibitor can weaken the interactions between the inhibitor and the viral neuraminidase without a concomitant decrease in free energy of binding for the substrate at the enzyme-catalyzed hydrolysis transition state. The current findings make it clear that new structural motifs and/or substitution patterns need to be developed in the search for a bona fide influenza viral neuraminidase transition state analogue inhibitor.

Brønsted analysis of an enzyme-catalyzed pseudo-deglycosylation reaction: mechanism of desialylation in sialidases.

The Micromonospora viridifaciens Y370G inverting mutant sialidase has been found to possess beta-sialidase activity with various fluoro-substituted phenyl beta-sialosides. A reagent panel of seven mono- and difluorophenyl beta-d-sialosides was synthesized, and these compounds were used, in conjunction with the parent phenyl beta-d-sialoside, to probe the mechanism of M. viridifaciens Y370G mutant sialidase-catalyzed hydrolyses. These hydrolysis reactions mimic the deglycosylation reaction step of the crucial tyrosinyl enzyme-bound intermediate that is formed during the corresponding wild-type sialidase reactions. The derived Brønsted parameter (beta(lg)) on k(cat)/K(m) is -0.46 +/- 0.02 for the four substrates that display significant activity, and these span a range of leaving group abilities (as judged by the pK(a) of their conjugate acids being between 7.09 and 9.87). The 4-fluoro, 2,3- and 2,5-difluorosubstrates display a diminished activity, whereas the 3,5-difluoro compound undergoes catalyzed hydrolysis exceedingly slowly. These observations, taken with solvent deuterium kinetic isotope effects (k(H(2))(O)/k(D(2))(O)) on the catalyzed hydrolysis of the 2-fluorophenyl substrate of 0.88 +/- 0.24 (k(cat)/K(m)) and 1.16 +/- 0.12 (k(cat)) and the poor inhibition shown by phenol (IC(50) > 1 mM), are consistent with glycosidic C-O cleavage being rate determining for both k(cat)/K(m) and k(cat) with little or no protonation of the departing aryloxide leaving group. The kinetic data reported herein are consistent with rate-limiting glycoside hydrolysis occurring via two distinct transition states that incorporates a nonproductive binding component for the tighter binding substrates.