Catalytically distinct IDH1 mutants tune phenotype severity in tumor models.

Mutations in isocitrate dehydrogenase 1 (IDH1) impart a neomorphic reaction that produces the oncometabolite D-2-hydroxyglutarate (D2HG), which can inhibit DNA and histone demethylases to drive tumorigenesis via epigenetic changes. Though heterozygous point mutations in patients primarily affect residue R132, there are myriad D2HG-producing mutants that display unique catalytic efficiency of D2HG production. Here, we show that catalytic efficiency of D2HG production is greater in IDH1 R132Q than R132H mutants, and expression of IDH1 R132Q in cellular and mouse xenograft models leads to higher D2HG concentrations in cells, tumors, and sera compared to R132H-expressing models. Reduced representation bisulfite sequencing (RRBS) analysis of xenograft tumors shows expression of IDH1 R132Q relative to R132H leads to hypermethylation patterns in pathways associated with DNA damage. Transcriptome analysis indicates that the IDH1 R132Q mutation has a more aggressive pro-tumor phenotype, with members of EGFR, Wnt, and PI3K signaling pathways differentially expressed, perhaps through non-epigenetic routes. Together, these data suggest that the catalytic efficiency of IDH1 mutants modulate D2HG levels in cellular and in vivo models, resulting in unique epigenetic and transcriptomic consequences where higher D2HG levels appear to be associated with more aggressive tumors.

Spinach genomes reveal migration history and candidate genes for important crop traits.

Spinach (Spinacia oleracea) is an important leafy crop possessing notable economic value and health benefits. Current genomic resources include reference genomes and genome-wide association studies. However, the worldwide genetic relationships and the migration history of the crop remained uncertain, and genome-wide association studies have produced extensive gene lists related to agronomic traits. Here, we re-analysed the sequenced genomes of 305 cultivated and wild spinach accessions to unveil the phylogeny and history of cultivated spinach and to explore genetic variation in relation to phenotypes. In contrast to previous studies, we employed machine learning methods (based on Extreme Gradient Boosting, XGBoost) to detect variants that are collectively associated with agronomic traits. Variant-based cluster analyses revealed three primary spinach groups in the Middle East, Asia and Europe/US. Combining admixture analysis and allele-sharing statistics, migration routes of spinach from the Middle East to Europe and Asia are presented. Using XGBoost machine learning models we predict genomic variants influencing bolting time, flowering time, petiole color, and leaf surface texture and propose candidate genes for each trait. This study enhances our understanding of the history and phylogeny of domesticated spinach and provides valuable information on candidate genes for future genetic improvement of the crop.

T- and L-Type Calcium Channels Maintain Calcium Oscillations in the Murine Zona Glomerulosa.

BACKGROUND: The zona glomerulosa of the adrenal gland is responsible for the synthesis and release of the mineralocorticoid aldosterone. This steroid hormone regulates salt reabsorption in the kidney and blood pressure. The most important stimuli of aldosterone synthesis are the serum concentrations of angiotensin II and potassium. In response to these stimuli, voltage and intracellular calcium levels in the zona glomerulosa oscillate, providing the signal for aldosterone synthesis. It was proposed that the voltage-gated T-type calcium channel CaV3.2 is necessary for the generation of these oscillations. However, Cacna1h knock-out mice have normal plasma aldosterone levels, suggesting additional calcium entry pathways. METHODS: We used a combination of calcium imaging, patch clamp, and RNA sequencing to investigate calcium influx pathways in the murine zona glomerulosa. RESULTS: Cacna1h-/- glomerulosa cells still showed calcium oscillations with similar concentrations as wild-type mice. No calcium channels or transporters were upregulated to compensate for the loss of CaV3.2. The calcium oscillations observed were instead dependent on L-type voltage-gated calcium channels. Furthermore, we found that L-type channels can also partially compensate for an acute inhibition of CaV3.2 in wild-type mice. Only inhibition of both T- and L-type calcium channels abolished the increase of intracellular calcium caused by angiotensin II in wild-type. CONCLUSIONS: Our study demonstrates that T-type calcium channels are not strictly required to maintain glomerulosa calcium oscillations and aldosterone production. Pharmacological inhibition of T-type channels alone will likely not significantly impact aldosterone production in the long term.

Isradipine therapy in Cacna1dIle772Met/+ mice ameliorates primary aldosteronism and neurologic abnormalities.

Somatic gain-of-function mutations in the L-type calcium channel CaV1.3 (CACNA1D gene) cause adrenal aldosterone-producing adenomas and micronodules. De novo germline mutations are found in a syndrome of primary aldosteronism, seizures, and neurologic abnormalities (PASNA) as well as in autism spectrum disorder. Using CRISPR/Cas9, we here generated mice with a Cacna1d gain-of-function mutation found in both adenomas and PASNA syndrome (Cacna1dIle772Met/+). These mice show reduced body weight and increased mortality from weaning to approximately 100 days of age. Male mice do not breed, likely due to neuromotor impairment, and the offspring of female mice die perinatally, likely due to lack of maternal care. Mice generated by in vitro fertilization showed elevated intracellular calcium in the aldosterone-producing zona glomerulosa, an elevated aldosterone/renin ratio, and persistently elevated serum aldosterone on a high-salt diet as signs of primary aldosteronism. Anesthesia with ketamine and xylazine induced tonic-clonic seizures. Neurologic abnormalities included hyperlocomotion, impaired performance in the rotarod test, impaired nest building, and slight changes in social behavior. Intracellular calcium in the zona glomerulosa, aldosterone levels, and rotarod performance responded to treatment with the calcium channel blocker isradipine, with implications for the therapy of patients with aldosterone-producing lesions and with PASNA syndrome.

Genomic alterations involved in fluoroquinolone resistance development in Staphylococcus aureus.

AIM: Fluoroquinolone (FQ) is a potent antibiotic class. However, resistance to this class emerges quickly which hinders its application. In this study, mechanisms leading to the emergence of multidrug-resistant (MDR) Staphylococcus aureus (S. aureus) strains under FQ exposure were investigated. METHODOLOGY: S. aureus ATCC 29213 was serially exposed to ciprofloxacin (CIP), ofloxacin (OFL), or levofloxacin (LEV) at sub-minimum inhibitory concentrations (sub-MICs) for 12 days to obtain S. aureus -1 strains and antibiotic-free cultured for another 10 days to obtain S. aureus-2 strains. The whole genome (WGS) and target sequencing were applied to analyze genomic alterations; and RT-qPCR was used to access the expressions of efflux-related genes, alternative sigma factors, and genes involved in FQ resistance. RESULTS: A strong and irreversible increase of MICs was observed in all applied FQs (32 to 128 times) in all S. aureus-1 and remained 16 to 32 times in all S. aureus-2. WGS indicated 10 noticeable mutations occurring in all FQ-exposed S. aureus including 2 insdel mutations in SACOL0573 and rimI; a synonymous mutation in hslO; and 7 missense mutations located in an untranslated region. GrlA, was found mutated (R570H) in all S. aureus-1 and -2. Genes encoding for efflux pumps and their regulator (norA, norB, norC, and mgrA); alternative sigma factors (sigB and sigS); acetyltransferase (rimI); methicillin resistance (fmtB); and hypothetical protein BJI72_0645 were overexpressed in FQ-exposed strains. CONCLUSION: The emergence of MDR S. aureus was associated with the mutations in the FQ-target sequences and the overexpression of efflux pump systems and their regulators.

CaV3.2 (CACNA1H) in Primary Aldosteronism.

Aldosterone is a steroid hormone produced in the zona glomerulosa (ZG) of the adrenal cortex. The most prominent function of aldosterone is the control of electrolyte homeostasis and blood pressure via the kidneys. The primary factors regulating aldosterone synthesis are the serum concentrations of angiotensin II and potassium. The T-type voltage-gated calcium channel CaV3.2 (encoded by CACNA1H) is an important component of electrical as well as intracellular calcium oscillations, which govern aldosterone production in the ZG. Excessive aldosterone production that is (partially) uncoupled from physiological stimuli leads to primary aldosteronism, the most common cause of secondary hypertension. Germline gain-of-function mutations in CACNA1H were identified in familial hyperaldosteronism, whereas somatic mutations are a rare cause of aldosterone-producing adenomas. In this review, we summarize these findings, put them in perspective, and highlight missing knowledge.

Temporal quality degradation in AI models.

As AI models continue to advance into many real-life applications, their ability to maintain reliable quality over time becomes increasingly important. The principal challenge in this task stems from the very nature of current machine learning models, dependent on the data as it was at the time of training. In this study, we present the first analysis of AI "aging": the complex, multifaceted phenomenon of AI model quality degradation as more time passes since the last model training cycle. Using datasets from four different industries (healthcare operations, transportation, finance, and weather) and four standard machine learning models, we identify and describe the main temporal degradation patterns. We also demonstrate the principal differences between temporal model degradation and related concepts that have been explored previously, such as data concept drift and continuous learning. Finally, we indicate potential causes of temporal degradation, and suggest approaches to detecting aging and reducing its impact.

Health risks and perceptions of residents exposed to multiple sources of air pollutions: A cross-sectional study on landfill and stone mining in Danang city, Vietnam.

Health risks and perceptions of residents living nearby landfills or stone mines/quarries have been well documented; however, the multiple impacts of these two pollution sources have yet to be investigated. This study aims at giving insights into the impacts of residents exposed to two pollution sources in Danang city, Vietnam: Khanh Son landfill and Phuoc Tuong quarry. In this cross-sectional study, 7-point-Likert scale questionnaires were used to collect information from 314 respondents which were divided into three groups: i) Landfill-Stone mine (LS) exposed group within 1 km from both sources, ii) Stone mine (ST) exposed group within 1 km from the stone mine, and iii) the far-exposed group about 2-3 km from the two emission sites. Air pollutants (including H2S, CH4, and total suspended particles (TSP)) and noise levels were also sampled. In addition, we estimated the potential health risk assessment due to the exposure to hydrogen sulphide in the study areas. The findings have shown that 1) There is a statistical difference (p-value < 0.001) between the two exposed groups and far-exposed group regarding health risks and residents' perceptions; 2) Compared to the ST exposed group, the LS exposed group statistically showed a higher perception to bad odours and skin diseases; lower satisfaction with regards to the personal health condition and the operation of the landfill; and higher willingness to change current living locations; and 3) There is evidence to indicate that elevated concentrations of air pollutants are associated with the high self-reported health risks and residents' perceptions. Urgent actions should be focused on the LS exposed group, landfill and quarry operations to prevent further adverse health effects.

Convergence of case-specific epigenetic alterations identify a confluence of genetic vulnerabilities tied to opioid overdose.

Opioid use disorder is a highly heterogeneous disease driven by a variety of genetic and environmental risk factors which have yet to be fully elucidated. Opioid overdose, the most severe outcome of opioid use disorder, remains the leading cause of accidental death in the United States. We interrogated the effects of opioid overdose on the brain using ChIP-seq to quantify patterns of H3K27 acetylation in dorsolateral prefrontal cortical neurons isolated from 51 opioid-overdose cases and 51 accidental death controls. Among opioid cases, we observed global hypoacetylation and identified 388 putative enhancers consistently depleted for H3K27ac. Machine learning on H3K27ac patterns predicted case-control status with high accuracy. We focused on case-specific regulatory alterations, revealing 81,399 hypoacetylation events, uncovering vast inter-patient heterogeneity. We developed a strategy to decode this heterogeneity based on convergence analysis, which leveraged promoter-capture Hi-C to identify five genes over-burdened by alterations in their regulatory network or "plexus": ASTN2, KCNMA1, DUSP4, GABBR2, ENOX1. These convergent loci are enriched for opioid use disorder risk genes and heritability for generalized anxiety, number of sexual partners, and years of education. Overall, our multi-pronged approach uncovers neurobiological aspects of opioid use disorder and captures genetic and environmental factors perpetuating the opioid epidemic.

Factors influencing adverse events following immunization with AZD1222 in Vietnamese adults during first half of 2021.

BACKGROUND: COVID-19 vaccines have been speedily developed and deployed. The more vaccine doses are delivered to users, the more common adverse events following immunization (AEFI) are reported. This study aimed to identify factors affecting AEFI in Vietnamese people receiving the COVID-19 vaccine AZD1222 developed by AstraZeneca and Oxford University. METHODS: In July 2021, an online cross-sectional survey was conducted among Vietnamese who have been vaccinated with COVID-19 vaccines. The questionnaire collected demographic characteristics, medical history, types of injected vaccines, common AEFI, and post-vaccination activities from respondents. The effects of host-related factors on AEFI including 24 specific symptoms were also explored. RESULTS: After screening, 1028 participants who were Vietnamese, over 18 years old and received at least one dose of AZD1222, were included in the study. Only 40/1028 (3.9%) participants reported not having any AEFI, whereas 25/1028 (2.4%) reported to have severe symptoms. The most common AEFI were moderate fever (69.4%), muscle aches (68.6%), followed by fatigue/ sleepiness (62.5%), body aches (59.4%), headache (58.5%), pain at injection site (58.3%) and chills (45.7%). Data analysis showed that females complained about AEFI particularly gastrointestinal symptoms more frequently than males. Age of participants and number of doses were also important factors affecting AEFI as the increase of age or number of vaccine doses was associated with the decrease of self-reported AEFI frequency. CONCLUSIONS: This study provides a detailed assessment of risk factors associated with AEFI in Vietnamese people vaccinated with AZD1222. It seems that gender, age and vaccine doses are important factors affecting AEFI.

Enhanced Ca2+ signaling, mild primary aldosteronism, and hypertension in a familial hyperaldosteronism mouse model (Cacna1hM1560V/+ ).

Gain-of-function mutations in the CACNA1H gene (encoding the T-type calcium channel CaV3.2) cause autosomal-dominant familial hyperaldosteronism type IV (FH-IV) and early-onset hypertension in humans. We used CRISPR/Cas9 to generate Cacna1hM1560V/+ knockin mice as a model of the most common FH-IV mutation, along with corresponding knockout mice (Cacna1h-/- ). Adrenal morphology of both Cacna1hM1560V/+ and Cacna1h-/- mice was normal. Cacna1hM1560V/+ mice had elevated aldosterone:renin ratios (a screening parameter for primary aldosteronism). Their adrenal Cyp11b2 (aldosterone synthase) expression was increased and remained elevated on a high-salt diet (relative autonomy, characteristic of primary aldosteronism), but plasma aldosterone was only elevated in male animals. The systolic blood pressure of Cacna1hM1560V/+ mice was 8 mmHg higher than in wild-type littermates and remained elevated on a high-salt diet. Cacna1h-/- mice had elevated renal Ren1 (renin-1) expression but normal adrenal Cyp11b2 levels, suggesting that in the absence of CaV3.2, stimulation of the renin-angiotensin system activates alternative calcium entry pathways to maintain normal aldosterone production. On a cellular level, Cacna1hM1560V/+ adrenal slices showed increased baseline and peak intracellular calcium concentrations in the zona glomerulosa compared to controls, but the frequency of calcium spikes did not rise. We conclude that FH-IV, on a molecular level, is caused by elevated intracellular Ca2+ concentrations as a signal for aldosterone production in adrenal glomerulosa cells. We demonstrate that a germline Cacna1h gain-of-function mutation is sufficient to cause mild primary aldosteronism, whereas loss of CaV3.2 channel function can be compensated for in a chronic setting.

Prevalence and associated factors of asymptomatic leishmaniasis: a systematic review and meta-analysis.

Asymptomatic leishmaniasis is believed to play important role in maintaining the transmission of Leishmania spp. within endemic communities. Therefore, the efforts to eliminate leishmaniasis are daunting if we cannot manage asymptomatic leishmaniasis well. To clarify the global prevalence and factors associated with the asymptomatic Leishmania infection, we assessed the prevalence of asymptomatic leishmaniasis by a systematic review followed by meta-analyses. In addition, factors associated with the asymptomatic leishmaniasis versus symptomatic were also analyzed. We included all of the original articles alluding to the human asymptomatic leishmaniasis that was confirmed by at least one laboratory diagnosis method regardless of age, sex, race, and ethnicity of the patients, study design, publication date or languages. In total, 111 original articles were chosen for the data extraction. Based on our meta-analyses of the original articles reporting asymptomatic leishmaniasis mostly in endemic areas, the prevalence of asymptomatic leishmaniasis was 11.2% [95% confidence interval (CI) 8.6%-14.4%] in general population, 36.7% [95% CI 27.6%-46.8%] in inhabitants living in the same or neighboring household to the symptomatic patients, and 11.8% [95% CI 7.1%-19%] in HIV infected patients. Among individuals with leishmaniasis, 64.9% [95% CI 54.7%-73.9%] were asymptomatic and males were more susceptible to develop symptoms, with OR=1.88, 95% CI 1.19-2.99, P=0.007. Meta-regression analysis showed no significant change in the prevalence of asymptomatic leishmaniasis during the last 40 years.

Prevalence of smoking among health science students in Vietnam in 2018 and associated factors: A cross-sectional study.

Smoking is a global health problem among health science students. The objectives of this study were to estimate the prevalence of smoking and to examine associated factors among health science students from five universities in Vietnam. Among 5946 participants (95.0% response rate), the smoking prevalence was 19.2% (95% CI:17.4-21.0%) among male students and 2.9% (95% CI:2.4-3.5%) among female students. In the multivariable regression models, significant factors for smoking were the perceived financial burden, the respondent's year in university, a non-self-determined motivation profile, self-reported depression and/or anxiety, the level of vigorous physical activity, and alcohol drinking.

An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity.

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (αKG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for αKG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ∼12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

Cell Type-Specific Intralocus Interactions Reveal Oligodendrocyte Mechanisms in MS.

Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. VIDEO ABSTRACT.

SpinDoctor: A MATLAB toolbox for diffusion MRI simulation.

The complex transverse water proton magnetization subject to diffusion-encoding magnetic field gradient pulses in a heterogeneous medium can be modeled by the multiple compartment Bloch-Torrey partial differential equation. Under the assumption of negligible water exchange between compartments, the time-dependent apparent diffusion coefficient can be directly computed from the solution of a diffusion equation subject to a time-dependent Neumann boundary condition. This paper describes a publicly available MATLAB toolbox called SpinDoctor that can be used 1) to solve the Bloch-Torrey partial differential equation in order to simulate the diffusion magnetic resonance imaging signal; 2) to solve a diffusion partial differential equation to obtain directly the apparent diffusion coefficient; 3) to compare the simulated apparent diffusion coefficient with a short-time approximation formula. The partial differential equations are solved by P1 finite elements combined with built-in MATLAB routines for solving ordinary differential equations. The finite element mesh generation is performed using an external package called Tetgen. SpinDoctor provides built-in options of including 1) spherical cells with a nucleus; 2) cylindrical cells with a myelin layer; 3) an extra-cellular space enclosed either a) in a box or b) in a tight wrapping around the cells; 4) deformation of canonical cells by bending and twisting; 5) permeable membranes; Built-in diffusion-encoding pulse sequences include the Pulsed Gradient Spin Echo and the Oscillating Gradient Spin Echo. We describe in detail how to use the SpinDoctor toolbox. We validate SpinDoctor simulations using reference signals computed by the Matrix Formalism method. We compare the accuracy and computational time of SpinDoctor simulations with Monte-Carlo simulations and show significant speed-up of SpinDoctor over Monte-Carlo simulations in complex geometries. We also illustrate several extensions of SpinDoctor functionalities, including the incorporation of T2 relaxation, the simulation of non-standard diffusion-encoding sequences, as well as the use of externally generated geometrical meshes.

Anti-plasmodial Effects of Zanthoxylum zanthoxyloides.

Zanthoxylum zanthoxyloides, syn. Fagara zanthoxyloides, is a tree growing in West Africa and is used in traditional medicine against a variety of diseases, including malaria. In the work reported here, root bark and stem bark extracts of this tree, as well as compounds isolated from the extracts, have been investigated for activity in vitro against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. In addition, toxicity against nauplii of the brine shrimp Artemia salina has been studied. Dichloromethane extracts of the root bark and stem bark, and a methanol extract of the stem bark, showed anti-parasitic activity towards chloroquine-sensitive as well as chloroquine-resistant P. falciparum, with IC50 values between 1 and 10 µg/mL. Among the isolated compounds, bis-dihydrochelerythrinyl ether, buesgenine, chelerythrine, γ-fagarine, skimmianine, and pellitorine were the most active, with IC50 values of less than 5 µg/mL. The dichloromethane extracts were toxic to brine shrimp nauplii, with LC50 values of less than 1 µg/mL. Methanol extracts were much less toxic (LC50 between 50 and 100 µg/mL). Among the isolated substances, bis-dihydrochelethrinyl ether was the most toxic (LC50 ca. 2 µg/mL).

Inhibitor potency varies widely among tumor-relevant human isocitrate dehydrogenase 1 mutants.

Mutations in isocitrate dehydrogenase 1 (IDH1) drive most low-grade gliomas and secondary glioblastomas and many chondrosarcomas and acute myeloid leukemia cases. Most tumor-relevant IDH1 mutations are deficient in the normal oxidization of isocitrate to α-ketoglutarate (αKG), but gain the neomorphic activity of reducing αKG to D-2-hydroxyglutarate (D2HG), which drives tumorigenesis. We found previously that IDH1 mutants exhibit one of two reactivities: deficient αKG and moderate D2HG production (including commonly observed R132H and R132C) or moderate αKG and high D2HG production (R132Q). Here, we identify a third type of reactivity, deficient αKG and high D2HG production (R132L). We show that R132Q IDH1 has unique structural features and distinct reactivities towards mutant IDH1 inhibitors. Biochemical and cell-based assays demonstrate that while most tumor-relevant mutations were effectively inhibited by mutant IDH1 inhibitors, R132Q IDH1 had up to a 16 300-fold increase in IC50 versus R132H IDH1. Only compounds that inhibited wild-type (WT) IDH1 were effective against R132Q. This suggests that patients with a R132Q mutation may have a poor response to mutant IDH1 therapies. Molecular dynamics simulations revealed that near the NADP+/NADPH-binding site in R132Q IDH1, a pair of α-helices switches between conformations that are more wild-type-like or more mutant-like, highlighting mechanisms for preserved WT activity. Dihedral angle changes in the dimer interface and buried surface area charges highlight possible mechanisms for loss of inhibitor affinity against R132Q. This work provides a platform for predicting a patient's therapeutic response and identifies a potential resistance mutation that may arise upon treatment with mutant IDH inhibitors.

Molecular mechanisms of isocitrate dehydrogenase 1 (IDH1) mutations identified in tumors: The role of size and hydrophobicity at residue 132 on catalytic efficiency.

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate (ICT) to α-ketoglutarate (αKG) in the cytosol and peroxisomes. Mutations in IDH1 have been implicated in >80% of lower grade gliomas and secondary glioblastomas and primarily affect residue 132, which helps coordinate substrate binding. However, other mutations found in the active site have also been identified in tumors. IDH1 mutations typically result in a loss of catalytic activity, but many also can catalyze a new reaction, the NADPH-dependent reduction of αKG to d-2-hydroxyglutarate (D2HG). D2HG is a proposed oncometabolite that can competitively inhibit αKG-dependent enzymes. Some kinetic parameters have been reported for several IDH1 mutations, and there is evidence that mutant IDH1 enzymes vary widely in their ability to produce D2HG. We report that most IDH1 mutations identified in tumors are severely deficient in catalyzing the normal oxidation reaction, but that D2HG production efficiency varies among mutant enzymes up to ∼640-fold. Common IDH1 mutations have moderate catalytic efficiencies for D2HG production, whereas rarer mutations exhibit either very low or very high efficiencies. We then designed a series of experimental IDH1 mutants to understand the features that support D2HG production. We show that this new catalytic activity observed in tumors is supported by mutations at residue 132 that have a smaller van der Waals volume and are more hydrophobic. We report that one mutation can support both the normal and neomorphic reactions. These studies illuminate catalytic features of mutations found in the majority of patients with lower grade gliomas.