Brain penetrant structurally targeted allosteric regulators for treating GLB1-related disorders

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor Pi. Molecular ‘crowding’renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

Download Full-text

Coupled structural transitions enable highly cooperative regulation of human CTPS2 filaments

10.1101/770594 ◽

2019 ◽

Author(s):

Eric M. Lynch ◽

Justin M. Kollman

Keyword(s):

Enzyme Activity ◽

Active Sites ◽

Large Scale ◽

Conformational State ◽

Ctp Synthase ◽

Widespread Phenomenon ◽

And Control ◽

Allosteric Regulators ◽

Low Activity

Many enzymes assemble into defined oligomers, providing a mechanism for cooperatively regulating enzyme activity. Recent studies in tissues, cells, and in vitro have described a mode of regulation in which enzyme activity is modulated by polymerization into large-scale filaments1–5. Enzyme polymerization is often driven by binding to substrates, products, or allosteric regulators, and tunes enzyme activity by locking the enzyme in high or low activity states1–5. Here, we describe a unique, ultrasensitive form of polymerization-based regulation employed by human CTP synthase 2 (CTPS2). High-resolution cryoEM structures of active and inhibited CTPS2 filaments reveal the molecular basis of this regulation. Rather than selectively stabilizing a single conformational state, CTPS2 filaments dynamically switch between active and inactive filament forms in response to changes in substrate and product levels. Linking the conformational state of many CTPS2 subunits in a filament results in highly cooperative regulation, greatly exceeding the limits of cooperativity for the CTPS2 tetramer alone. The structures also reveal a link between conformational state and control of ammonia channeling between the enzyme’s two active sites. This filament-based mechanism of enhanced cooperativity demonstrates how the widespread phenomenon of enzyme polymerization can be adapted to achieve different regulatory outcomes.

Download Full-text

The 14-3-3 Proteins as Important Allosteric Regulators of Protein Kinases

International Journal of Molecular Sciences ◽

10.3390/ijms21228824 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8824

Author(s):

Veronika Obsilova ◽

Tomas Obsil

Keyword(s):

Protein Interactions ◽

Cell Cycle Progression ◽

State Of The Art ◽

Review Article ◽

Protein Protein Interactions ◽

Cycle Progression ◽

Kinase Regulation ◽

Functional Consequences ◽

Allosteric Regulators ◽

Structural Aspects

Phosphorylation by kinases governs many key cellular and extracellular processes, such as transcription, cell cycle progression, differentiation, secretion and apoptosis. Unsurprisingly, tight and precise kinase regulation is a prerequisite for normal cell functioning, whereas kinase dysregulation often leads to disease. Moreover, the functions of many kinases are regulated through protein–protein interactions, which in turn are mediated by phosphorylated motifs and often involve associations with the scaffolding and chaperon protein 14-3-3. Therefore, the aim of this review article is to provide an overview of the state of the art on 14-3-3-mediated kinase regulation, focusing on the most recent mechanistic insights into these important protein–protein interactions and discussing in detail both their structural aspects and functional consequences.

Download Full-text

P.078 Clinical trials in children with Down Syndrome: now and future

Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques ◽

10.1017/cjn.2019.176 ◽

2019 ◽

Vol 46 (s1) ◽

pp. S35

Author(s):

C Cieuta-Walti ◽

C Mircher ◽

I Marey ◽

J Toulas ◽

E Prioux ◽

...

Keyword(s):

Clinical Trials ◽

Down Syndrome ◽

Kinase Inhibitors ◽

Phenotypic Variability ◽

Cognitive Stimulation ◽

Cognitive Improvement ◽

Epigenetic Effect ◽

Therapeutic Trials ◽

Future Strategy ◽

Allosteric Regulators

Background: Down syndrome (DS) is the most common genetic cause of intellectual disability.Although progress in managing co-morbidities has improved life expectancy, no therapeutic options have showed to significantly improve intellectual deficiencies.The current focus of the pharmacological treatment of DS is on the improvement of the cognitive impairment that is probably due to neurodevelopmental alterations,neurotransmitter alterations and neurodegeneration,and is also targeted to the overexpression of selected genes on HSA21. Methods: We review the clinical trials of the last 5 years focusing on the cognitive improvement of children with DS. Results: We report the results of therapeutic trials concerning selective negative allosteric regulators of the GABAAa5 receptor, NMDA antagonists, Kinase inhibitors of DYRK1A, folinic acid and thyroid hormone supplementation, activators of serotonergic and cholinergic system. Conclusions: The incomplete understanding of individual phenotypic variability, natural history, lack of biomarkers, no adapted neuropsychological tests, placebo effect, epigenetic effect have limited our capacity to succeed, even when promising drugs are tested.We need new tools and models will allow a better understanding of the pathophysiology. We also need to create more sensitive and realistic outcome mesures to quantify disease and therapeutic efficacy. The association of different therapeutic agents (epidrugs included) with cognitive stimulation could be a future strategy.

Download Full-text