Identification of potential pharmacological chaperones that selectively stabilize mutated Aspartoacylases in Canavan disease.

Canavan disease is caused by mutations in the ASPA gene, leading to diminished catalytic activity of aspartoacylase in the brain. Clinical missense mutations are found throughout the enzyme structure, with many of these mutated enzymes having not only decreased activity but also compromised stability. High-throughput screening of a small molecule library has identified several compounds that significantly increase the thermal stability of the E285A mutant enzyme, the most predominant clinical mutation in Canavan disease, while having a negligible effect on the native enzyme.

A growth-based assay using fluorescent protein emission to screen for S-adenosylmethionine synthetase inhibitors.

The use of cell growth-based assays to identify inhibitory compounds is straightforward and inexpensive, but is also inherently insensitive and somewhat nonspecific. To overcome these limitations and develop a sensitive, specific cell-based assay, two different approaches were combined. To address the sensitivity limitation, different fluorescent proteins have been introduced into a bacterial expression system to serve as growth reporters.

Dual-Acting Peptides Target EZH2 and AR: A New Paradigm for Effective Treatment of Castration-Resistant Prostate Cancer.

Prostate cancer starts as a treatable hormone-dependent disease, but often ends in a drug-resistant form called castration-resistant prostate cancer (CRPC). Despite the development of the antiandrogens enzalutamide and abiraterone for CRPC, which target the androgen receptor (AR), drug resistance usually develops within 6 months and metastatic CRPC (mCRPC) leads to lethality. EZH2, found with SUZ12, EED, and RbAP48 in Polycomb repressive complex 2 (PRC2), has emerged as an alternative target for the treatment of deadly mCRPC.

A Comprehensive Biological and Synthetic Perspective on 2-Deoxy-d-Glucose (2-DG), A Sweet Molecule with Therapeutic and Diagnostic Potentials.

Glucose, the primary substrate for ATP synthesis, is catabolized during glycolysis to generate ATP and precursors for the synthesis of other vital biomolecules. Opportunistic viruses and cancer cells often hijack this metabolic machinery to obtain energy and components needed for their replication and proliferation. One way to halt such energy-dependent processes is by interfering with the glycolytic pathway.

Design and testing of selective inactivators against an antifungal enzyme target.

Systemic infections from fungal organisms are becoming increasingly difficult to treat as drug resistance continues to emerge. To substantially expand the antifungal drug landscape new compounds must be identified and developed with novel modes of action against previously untested drug targets. Most drugs block the activity of their targets through reversible, noncovalent interactions.

Engineering of a critical membrane-anchored enzyme for high solubility and catalytic activity.

Membrane-associated proteins carry out a wide range of essential cellular functions but the structural characterization needed to understand these functions is dramatically underrepresented in the Protein Data Bank. Producing a soluble, stable and active form of a membrane-associated protein presents formidable challenges, as evidenced by the variety of approaches that have been attempted with a multitude of different membrane proteins to achieve this goal. Aspartate N-acetyltransferase (ANAT) is a membrane-anchored enzyme that performs a critical function, the synthesis of N-acetyl-l-aspartate (NAA), the second most abundant amino acid in the brain.

Natural Products: A Rich Source of Antiviral Drug Lead Candidates for the Management of COVID-19.

Today, the world is suffering from the pandemic of a novel coronavirus disease (COVID-19), a respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This pandemic is the third fatal coronavirus outbreak that has already occurred in the 21st century. Even six months after its emergence, hundreds of thousands of people are still being infected with SARS-CoV-2, and thousands of lives are lost every day across the world.

Discovery of Novel Inhibitors of a Critical Brain Enzyme Using a Homology Model and a Deep Convolutional Neural Network.

Rare neglected diseases may be neglected but are hardly rare, affecting hundreds of millions of people around the world. Here, we present a hit identification approach using AtomNet, the world's first deep convolutional neural network for structure-based drug discovery, to identify inhibitors targeting aspartate -acetyltransferase (ANAT), a promising target for the treatment of patients suffering from Canavan disease. Despite the lack of a protein structure or high sequence identity homologous templates, the approach successfully identified five low-micromolar inhibitors with drug-like properties.

Aspartate semialdehyde dehydrogenase inhibition suppresses the growth of the pathogenic fungus Candida albicans.

Potent inhibitors of an essential microbial enzyme have been shown to be effective growth inhibitors of Candida albicans, a pathogenic fungus. C. albicans is the main cause of oropharyngeal candidiasis, and also causes invasive fungal infections, including systemic sepsis, leading to serious complications in immunocompromised patients.

The ammonia-lyases: enzymes that use a wide range of approaches to catalyze the same type of reaction.

The paradigm that protein structure determines protein function has been clearly established. What is less clear is whether a specific protein structure is always required to carry out a specific function. Numerous cases are now known where there is no apparent connection between the biological function of a protein and the other members of its structural class, and where functionally related proteins can have quite diverse structures.

Development of bisubstrate analog inhibitors of aspartate N-acetyltransferase, a critical brain enzyme.

Canavan disease (CD) is a fatal leukodystrophy caused by mutations in the aspA gene coding for the enzyme aspartoacylase. Insufficient catalytic activity by this enzyme leads to the accumulation of its substrate, N-acetyl-l-aspartate (NAA), and diminished production of acetate in brain oligodendrocytes of patients with CD. There is growing evidence that this accumulation of NAA is the cause of many of the developmental defects observed in these patients.

Structure of a critical metabolic enzyme: S-adenosylmethionine synthetase from Cryptosporidium parvum.

S-Adenosyl-L-methionine (AdoMet), the primary methyl donor in most biological methylation reactions, is produced from ATP and methionine in a multistep reaction catalyzed by AdoMet synthetase. The diversity of group-transfer reactions that involve AdoMet places this compound at a key crossroads in amino-acid, nucleic acid and lipid metabolism, and disruption of its synthesis has adverse consequences for all forms of life. The family of AdoMet synthetases is highly conserved, and structures of this enzyme have been determined from organisms ranging from bacteria to humans.

Structural insights into inhibitor binding to a fungal ortholog of aspartate semialdehyde dehydrogenase.

The aspartate pathway, uniquely found in plants and microorganisms, offers novel potential targets for the development of new antimicrobial drugs. Aspartate semialdehyde dehydrogenase (ASADH) catalyzes production of a key intermediate at the first branch point in this pathway. Several fungal ASADH structures have been determined, but the prior crystallization conditions had precluded complex formation with enzyme inhibitors.

A Fragment Library Screening Approach to Identify Selective Inhibitors against an Essential Fungal Enzyme.

Pathogenic fungi represent a growing threat to human health, with an increase in the frequency of drug-resistant fungal infections. Identifying targets from among the selected metabolic pathways that are unique to microbial species presents an opportunity to develop new antifungal agents against new and untested targets to combat this growth threat. Aspartate semialdehyde dehydrogenase (ASADH) catalyzes a key step in a uniquely microbial amino acid biosynthetic pathway and is essential for microbial viability.

Complementation of a metK-deficient E. coli strain with heterologous AdoMet synthetase genes.

S-adenosyl-l-methionine (AdoMet) is an essential metabolite, playing a wide variety of metabolic roles. The enzyme that produces AdoMet from l-methionine and ATP (methionine adenosyltransferase, MAT) is thus an attractive target for anti-cancer and antimicrobial agents. It would be very useful to have a system that allows rapid identification of species-specific inhibitors of this essential enzyme.

Structure of a fungal form of aspartate-semialdehyde dehydrogenase from Aspergillus fumigatus.

Aspartate-semialdehyde dehydrogenase (ASADH) functions at a critical junction in the aspartate biosynthetic pathway and represents a validated target for antimicrobial drug design. This enzyme catalyzes the NADPH-dependent reductive dephosphorylation of β-aspartyl phosphate to produce the key intermediate aspartate semialdehyde. The absence of this entire pathway in humans and other mammals will allow the selective targeting of pathogenic microorganisms for antimicrobial development.

Design and optimization of aspartate N-acetyltransferase inhibitors for the potential treatment of Canavan disease.

Canavan disease is a fatal neurological disorder caused by defects in the metabolism of N-acetyl-l-aspartate (NAA). Recent work has shown that the devastating symptoms of this disorder are correlated with the elevated levels of NAA observed in these patients, caused as a consequence of the inability of mutated forms of aspartoacylase to adequately catalyze its breakdown. The membrane-associated enzyme responsible for the synthesis of NAA, aspartate N-acetyltransferase (ANAT), has recently been purified and examined (Wang et al.

Effects of Extracellular Polymeric Substance Composition on Bacteria Disinfection by Monochloramine: Application of MALDI-TOF/TOF-MS and Multivariate Analysis.

In our previous study, we reported that the transport of monochloramine is affected by the extracellular polymeric substance (EPS) composition, which in turn affects the cell viability of both biofilm and detached clusters.11 However, although the transport and reaction of monochloramine in biofilm could be observed, the specific biomolecules reacting with the disinfectant and the mechanism of disinfection remains elusive. In this study, the impact of EPS composition on bacteria disinfection by monochloramine was qualitatively determined using both wild-type and isogenic mutant Pseudomonas strains with different EPS-secretion capacity and composition.

Structural Insights into the Tetrameric State of Aspartate-β-semialdehyde Dehydrogenases from Fungal Species.

Aspartate-β-semialdehyde dehydrogenase (ASADH) catalyzes the second reaction in the aspartate pathway, a pathway required for the biosynthesis of one fifth of the essential amino acids in plants and microorganisms. Microarray analysis of a fungal pathogen T. rubrum responsible for most human dermatophytoses identified the upregulation of ASADH (trASADH) expression when the fungus is exposed to human skin, underscoring its potential as a drug target.

Purification and characterization of aspartate N-acetyltransferase: A critical enzyme in brain metabolism.

Canavan disease (CD) is a neurological disorder caused by an interruption in the metabolism of N-acetylaspartate (NAA). Numerous mutations have been found in the enzyme that hydrolyzes NAA, and the catalytic activity of aspartoacylase is significantly impaired in CD patients. Recent studies have also supported an important role in CD for the enzyme that catalyzes the synthesis of NAA in the brain.

Structure of a fungal form of aspartate semialdehyde dehydrogenase from Cryptococcus neoformans.

Aspartate semialdehyde dehydrogenase (ASADH) functions at a critical junction in the aspartate-biosynthetic pathway and represents a valid target for antimicrobial drug design. This enzyme catalyzes the NADPH-dependent reductive dephosphorylation of β-aspartyl phosphate to produce the key intermediate aspartate semialdehyde. Production of this intermediate represents the first committed step in the biosynthesis of the essential amino acids methionine, isoleucine and threonine in fungi, and also the amino acid lysine in bacteria.

Elaboration of a fragment library hit produces potent and selective aspartate semialdehyde dehydrogenase inhibitors.

Aspartate-β-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the aspartate metabolic pathway which leads to the biosynthesis of several essential amino acids and some important metabolites. This pathway is crucial for many metabolic processes in plants and microbes like bacteria and fungi, but is absent in mammals. Therefore, the key microbial enzymes involved in this pathway are attractive potential targets for development of new antibiotics with novel modes of action.

A surprising range of modified-methionyl S-adenosylmethionine analogues support bacterial growth.

S-Adenosyl-l-methionine (AdoMet) is an essential metabolite, serving in a very wide variety of metabolic reactions. The enzyme that produces AdoMet from l-methionine and ATP (methionine adenosyltransferase, MAT) is thus an attractive target for antimicrobial agents. We previously showed that a variety of methionine analogues are MAT substrates, yielding AdoMet analogues that function in specific methyltransfer reactions.

A cautionary tale of structure-guided inhibitor development against an essential enzyme in the aspartate-biosynthetic pathway.

The aspartate pathway is essential for the production of the amino acids required for protein synthesis and of the metabolites needed in bacterial development. This pathway also leads to the production of several classes of quorum-sensing molecules that can trigger virulence in certain microorganisms. The second enzyme in this pathway, aspartate β-semialdehyde dehydrogenase (ASADH), is absolutely required for bacterial survival and has been targeted for the design of selective inhibitors.

Effect of Body Habitus on Radiation Dose During CT Fluoroscopy-Guided Spine Injections.

This study investigated the degree to which body habitus influences radiation dose during CT fluoroscopy (CTF)-guided lumbar epidural steroid injections (ESI). An anthropomorphic phantom containing metal oxide semiconductor field effect transistor (MOSFET) detectors was scanned at two transverse levels to simulate upper and lower lumbar CTF-guided ESI. Circumferential layers of adipose-equivalent material were sequentially added to model patients of three sizes: small (cross-sectional dimensions 25×30 cm), average (34×39 cm), and oversize (43×48 cm).