PPA2-associated sudden cardiac death: extending the clinical and allelic spectrum in 20 new families.

Purpose: Biallelic hypomorphic variants in PPA2, encoding the mitochondrial inorganic pyrophosphatase 2 protein, have been recently identified in individuals presenting with sudden cardiac death, occasionally triggered by alcohol intake or a viral infection. Here we report 20 new families harboring PPA2 variants.

Methods: Synthesis of clinical and molecular data concerning 34 individuals harboring five previously reported PPA2 variants and 12 novel variants, 11 of which were functionally characterized.

Variants in GNAI1 cause a syndrome associated with variable features including developmental delay, seizures, and hypotonia.

Purpose: Neurodevelopmental disorders (NDDs) encompass a spectrum of genetically heterogeneous disorders with features that commonly include developmental delay, intellectual disability, and autism spectrum disorders. We sought to delineate the molecular and phenotypic spectrum of a novel neurodevelopmental disorder caused by variants in the GNAI1 gene.

Methods: Through large cohort trio-based exome sequencing and international data-sharing, we identified 24 unrelated individuals with NDD phenotypes and a variant in GNAI1, which encodes the inhibitory Gαi1 subunit of heterotrimeric G-proteins.

Mechanistic Insights into the Chaperoning of Human Lysosomal-Galactosidase Activity: Highly Functionalized Aminocyclopentanes and -5a-Substituted Derivatives of 4--Isofagomine.

Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid β-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent β-d-galactosidase inhibitor 4--isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a "strategic" hydroxyl group.

A Peroxodiiron(III/III) Intermediate Mediating Both -Hydroxylation Steps in Biosynthesis of the -Nitrosourea Pharmacophore of Streptozotocin by the Multi-domain Metalloenzyme SznF.

The alkylating warhead of the pancreatic cancer drug streptozotocin (SZN) contains an -nitrosourea moiety constructed from -methyl-l-arginine (l-NMA) by the multi-domain metalloenzyme SznF. The enzyme's central heme-oxygenase-like (HO-like) domain sequentially hydroxylates N and N' of l-NMA. Its C-terminal cupin domain then rearranges the triply modified arginine to -hydroxy-methyl--nitroso-l-citrulline, the proposed donor of the functional pharmacophore.

Late-onset Pseudoxanthoma Elasticum Associated with a Hypomorphic ABCC6 Variant.

Purpose: To describe patients with late-onset pseudoxanthoma elasticum (PXE) associated with a likely hypomorphic ABCC6 variant.

Design: Retrospective observational case series.

Methods: Clinical evaluation, multimodal retinal imaging, genetic testing, and molecular modeling.

Synthesis of montbretin A analogues yields potent competitive inhibitors of human pancreatic α-amylase.

Simplified analogues of the potent human amylase inhibitor montbretin A were synthesised and shown to bind tightly, = 60 and 70 nM, with improved specificity over medically relevant glycosidases, making them promising candidates for controlling blood glucose. Crystallographic analysis confirmed similar binding modes and identified new active site interactions.

Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism.

Recent studies have identified both recessive and dominant forms of mitochondrial disease that result from ATAD3A variants. The recessive form includes subjects with biallelic deletions mediated by non-allelic homologous recombination. We report five unrelated neonates with a lethal metabolic disorder characterized by cardiomyopathy, corneal opacities, encephalopathy, hypotonia, and seizures in whom a monoallelic reciprocal duplication at the ATAD3 locus was identified.

Substrate-Triggered Formation of a Peroxo-Fe(III/III) Intermediate during Fatty Acid Decarboxylation by UndA.

The iron-dependent oxidase UndA cleaves one C3-H bond and the C1-C2 bond of dodecanoic acid to produce 1-undecene and CO. A published X-ray crystal structure showed that UndA has a heme-oxygenase-like fold, thus associating it with a structural superfamily that includes known and postulated non-heme diiron proteins, but revealed only a single iron ion in the active site. Mechanisms proposed for initiation of decarboxylation by cleavage of the C3-H bond using a monoiron cofactor to activate O necessarily invoked unusual or potentially unfeasible steps.

Folding Then Binding vs Folding Through Binding in Macrocyclic Peptide Inhibitors of Human Pancreatic α-Amylase.

macrocyclic peptides, derived using selection technologies such as phage and mRNA display, present unique and unexpected solutions to challenging biological problems. This is due in part to their unusual folds, which are able to present side chains in ways not available to canonical structures such as α-helices and β-sheets. Despite much recent interest in these molecules, their folding and binding behavior remains poorly characterized.

Utility of systematic TSHR gene testing in adults with hyperthyroidism lacking overt autoimmunity and diffuse uptake on thyroid scintigraphy.

Objective: Patients with hyperthyroidism lacking autoimmune features but showing diffuse uptake on thyroid scintigram can have either Graves' disease or germline activating TSH receptor (TSHR) mutation. It is important to identify patients with activating TSHR mutation due to treatment implication, but the overlapping clinical features with Graves' disease make it difficult to discriminate these two conditions without genetic testing. Our study aimed to assess the potential of systematic TSHR mutation screening in adults with hyperthyroidism, showing diffuse uptake on thyroid scintigraphy but absence of TSH receptor antibodies (TRAb) and clinical signs of autoimmunity.

Structural Dissection of Helianthamide Reveals the Basis of Its Potent Inhibition of Human Pancreatic α-Amylase.

Helianthamide is a potent inhibitor of human pancreatic α-amylase (HPA) ( K = 0.01 nM) produced by the Caribbean sea anemone Stichodactyla helianthus. Helianthamide was previously shown to be structurally homologous to the β-defensins and represents a new structural class of protein inhibitors of α-amylase.

Diagnosis of lethal or prenatal-onset autosomal recessive disorders by parental exome sequencing.

Objective: Rare genetic disorders resulting in prenatal or neonatal death are genetically heterogeneous, but testing is often limited by the availability of fetal DNA, leaving couples without a potential prenatal test for future pregnancies. We describe our novel strategy of exome sequencing parental DNA samples to diagnose recessive monogenic disorders in an audit of the first 50 couples referred.

Method: Exome sequencing was carried out in a consecutive series of 50 couples who had 1 or more pregnancies affected with a lethal or prenatal-onset disorder.

A new type of pharmacological chaperone for G-gangliosidosis related human lysosomal β-galactosidase: N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols.

N-Functionalized amino(hydroxymethyl)cyclopentanetriols are potent inhibitors of β-d-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for G-gangliosidosis-associated lysosomal acid β-galactosidase thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.

N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols: A new family of activity promotors for a G-gangliosidosis related human lysosomal β-galactosidase mutant.

From 1,2;3,4-di-O-isopropylidene-α-D-galactopyranose, a series of highly functionalized (hydroxymethyl)cyclopentanes was easily available. In line with reports by Reymond and Jäger on similar structures, these amine containing basic carbasugars are potent inhibitors of β-D-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for G-gangliosidosis-associated lysosomal acid β-galactosidase mutant R201C, thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.

A Morita-Baylis-Hillman based route to C-5a-chain-extended 4-epi-isofagomine type glycosidase inhibitors.

By Morita-Baylis-Hillman reaction of 2,3-O-isopropylidene-D-glyceraldehyde with α,β-unsaturated carbonyl as well as hetero analogous carbonyl compounds such as acrylonitrile, suitable precursors of isofagomine and of 4-epi-isofagomine are available. Elaboration of the structures by amine introduction, followed by intramolecular ring closure and subsequent hydroboration of the double bond provides 4-epi-isofagomine derivatives featuring chain extensions at C-5a which are determined by the structures of the carbonyl compounds employed. As an example, the synthesis of C-(5aR)- and C-(5aS)-5a-C-pentyl-4-epi-isofagomines, powerful inhibitors of β-galactosidases, is outlined.

Rapid Discovery of Potent and Selective Glycosidase-Inhibiting De Novo Peptides.

Human pancreatic α-amylase (HPA) is responsible for degrading starch to malto-oligosaccharides, thence to glucose, and is therefore an attractive therapeutic target for the treatment of diabetes and obesity. Here we report the discovery of a unique lariat nonapeptide, by means of the RaPID (Random non-standard Peptides Integrated Discovery) system, composed of five amino acids in a head-to-side-chain thioether macrocycle and a further four amino acids in a 3 helical C terminus. This is a potent inhibitor of HPA (K = 7 nM) yet exhibits selectivity for the target over other glycosidases tested.

C-5a-substituted validamine type glycosidase inhibitors.

A series of N-alkyl derivatives of the D-galactosidase inhibitor 1,4-di-epi-validamine featuring lipophilic substituents at position C-5a was prepared and screened for their glycosidase inhibitory properties. Products turned out selective for β-galactosidases as well as β-glucosidases.

Fungal Glycolipid Hydrolase Inhibitors and Their Effect on Cryptococcus neoformans.

Pathogenic fungi kill an estimated 1.3 million people each year. This number is predicted to rise as drug resistance spreads, thus antifungal drugs with novel modes of action are urgently required.

Glycosynthase mediated synthesis of psychosine.

Globoid cell leukodystrophy (GCL), or Krabbe disease, is a lysosomal storage disorder characterized by a deficiency in galactosylceramidase (GALC), which hydrolyses galactosylceramide and galactosylsphingosine (psychosine). Early detection of GCL in newborns is essential for timely therapeutic intervention and could be achieved by testing infant blood samples with isotopically labeled lysosmal enzyme substrates and mass spectrometry. While isotopically labeled psychosine would be a useful tool for the early diagnosis of GCL, its synthesis is lengthy and expensive.

The Staudinger/aza-Wittig/Grignard reaction as key step for the concise synthesis of 1-C-Alkyl-iminoalditol glycomimetics.

The scope of a one-pot tandem approach for the synthesis of C-1 alkyl iminoalditol derivatives with a Staudinger/aza-Wittig/Grignard cascade has been evaluated. The reaction conditions have been optimized for two azidodeoxy aldose substrates and a range of Grignard reagents. The nature of both, substrate as well as nucleophile, was found to control the stereoselectivity of the alkyl addition to the cyclic iminium intermediate at position C-1.

Potent Human α-Amylase Inhibition by the β-Defensin-like Protein Helianthamide.

Selective inhibitors of human pancreatic α-amylase (HPA) are an effective means of controlling blood sugar levels in the management of diabetes. A high-throughput screen of marine natural product extracts led to the identification of a potent ( = 10 pM) peptidic HPA inhibitor, helianthamide, from the Caribbean sea anemone Active helianthamide was produced in via secretion as a barnase fusion protein. X-ray crystallographic analysis of the complex of helianthamide with porcine pancreatic α-amylase revealed that helianthamide adopts a β-defensin fold and binds into and across the amylase active site, utilizing a contiguous YIYH inhibitory motif.

Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable β-galactosidase inhibitors and activity promotors of GM1-gangliosidosis related human lysosomal β-galactosidase mutant R201C.

From an easily available partially protected analog of 1-deoxy-L-gulo-nojirimycin, by chain-branching at C-4 and suitable modification, lipophilic analogs of the powerful β-D-galactosidase inhibitor 4-epi-isofagomine have been prepared. New compounds exhibit considerably improved inhibitory activities when compared with the unsubstituted parent compound and may serve as leads toward new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.

The amylase inhibitor montbretin A reveals a new glycosidase inhibition motif.

The complex plant flavonol glycoside montbretin A is a potent (Ki = 8 nM) and specific inhibitor of human pancreatic α-amylase with potential as a therapeutic for diabetes and obesity. Controlled degradation studies on montbretin A, coupled with inhibition analyses, identified an essential high-affinity core structure comprising the myricetin and caffeic acid moieties linked via a disaccharide. X-ray structural analyses of the montbretin A-human α-amylase complex confirmed the importance of this core structure and revealed a novel mode of glycosidase inhibition wherein internal π-stacking interactions between the myricetin and caffeic acid organize their ring hydroxyls for optimal hydrogen bonding to the α-amylase catalytic residues D197 and E233.

Functional and in silico assessment of MAX variants of unknown significance.

Unlabelled: The presence of germline mutations affecting the MYC-associated protein X (MAX) gene has recently been identified as one of the now 11 major genetic predisposition factors for the development of hereditary pheochromocytoma and/or paraganglioma. Little is known regarding how missense variants of unknown significance (VUS) in MAX affect its pivotal role in the regulation of the MYC/MAX/MXD axis. In the present study, we propose a consensus computational prediction based on five "state-of-the-art" algorithms.