Impact of PmrB mutations on clinical Klebsiella pneumoniae with variable colistin-susceptibilities: Structural insights and potent therapeutic solutions.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections continue to impose high morbidity threats to hospitalized patients worldwide, limiting therapeutic options to last-resort antibiotics like colistin. However, the dynamic genomic landscape of colistin-resistant K. pneumoniae (COLR-Kp) invoked ardent exploration of underlying molecular signatures for therapeutic propositions/designs. We unveiled the structural impact of the widespread and emerging PmrB mutations involved in colistin resistance (COLR) in K. pneumoniae. In the present study, clinical isolates of K. pneumoniae expressed variable susceptibilities to colistin (>0.5 µg/mL for resistant and ≤0.25 µg/mL for susceptible) despite mutations such as T157P, G207D and T246A. The protein sequences extracted from in-house sequenced genomes were used to model mutant PmrB proteins and analyze the underlying structural alterations. The mutations were contrasted based on molecular dynamics simulation trajectories, free-energy landscapes and structural flexibility profiles. The altered backbone flexibilities can be an essential factor for mutant selection by COLR K. pneumoniae and can provide clues to deal with emerging mutants. Furthermore, PmrB having high druggability confidence (>0.99), was explored as a potential target for 1396 virtually screened FDA-approved drug candidates. Among the top-10 compounds (scores >70), amphotericin B was found to be potential candidate with high affinity (Binding energy <-8 kcal/mol) and stable interactions (RMSF <0.7 Å) against PmrB druggable pockets, despite the mutations, which encourages future adjunct therapeutic research against COLR-Kp.

A novel FGF23 mutation in hyperphosphatemic familial tumoral calcinosis and its deleterious effect on protein O-glycosylation.

Background: Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare disease characterized by hyperphosphatemia and ectopic calcification, predominantly at periarticular locations. This study was performed to characterize the clinical profile of tumoral calcinosis and to identify gene mutations associated with HFTC and elucidated its pathogenic role. Methods: The three subjects (two male and one female) were aged 30, 25 and 15 years, respectively. The clinical features, histopathological findings, and outcomes of three subjects with HFTC were retrospectively reviewed. The three subjects were analyzed for FGF23, GALNT3 and KL mutations. Function of mutant gene was analyzed by western blotting and wheat germ agglutinin affinity chromatography. Results: All subjects had hyperphosphatemia and elevated calcium-phosphorus product. Calcinosis positions included the left shoulder, left index finger, and right hip. Bone and joint damage were present in two cases and multiple foci influenced body growth in one case. The histopathological features were firm, rubbery masses comprising multiple nodules of calcified material bordered by the proliferation of mononuclear or multinuclear macrophages, osteoclastic-like giant cells, fibroblasts, and chronic inflammatory cells. The novel mutation c.484A>G (p.N162D) in exon 3 of FGF23 was identified in one subject and his family members. Measurement of circulating FGF23 in the subject confirmed low intact FGF23 and increased C-terminal fragment. In vitro experiments showed that the mutant FGF23 proteins had defective O-glycosylation and impaired protein proteolysis protection. Conclusion: We identified a novel FGF23 missense mutation, and confirmed its damaging role in FGF23 protein O-glycosylation. Our findings expand the current spectrum of FGF23 variations that influence phosphorus metabolism.

Molecular insights on the conformational dynamics of a P76C mutant of human cytochrome c and the enhancement on its peroxidase activity.

In apoptotic pathway, the interaction of Cytochrome c (Cytc) with cardiolipin in vivo is a key process to induce peroxidase activity of Cytc and trigger the release of Cytc in the inner mitochondria into cytosol. The peroxidase active form of Cytc occurs due to local conformational changes that support the opening of the heme crevice and the loss of an axial ligand between Met80 and heme Fe. Structural adjustments at the Ω-loop segments of Cytc are required for such process. To study the role of the distal Ω-loop segments comprising residues 71-85 in human Cytc (hCytc), we investigated a cysteine mutation at Pro76, one of the highly conserved residues in this loop. The effect of P76C mutant was explored by the combination of experimental characterizations and molecular dynamics (MD) simulations. The peroxidase activity of the P76C mutant was found to be significantly increased by ∼13 folds relative to the wild type. Experimental data on global denaturation, alkaline transition, heme bleaching, and spin-labeling Electron Spin Resonance were in good agreement with the enhancement of peroxidase activity. The MD results of hCytc in the hexacoordinate form suggest the important changes in P76C mutant occurred due to the unfolding at the central Ω-loop (residues 40-57), and the weakening of H-bond between Tyr67 and Met80. Whereas the experimental data implied that the P76C mutant tend to be in equilibrium between the pentacoordinate and hexacoordinate forms, the MD and experimental information are complementary and were used to support the mechanisms of peroxidase active form of hCytc.

Therapeutic potential of ginsenoside Rg3 and Rf for Huntington's disease.

Ginseng is a popular herbal medicine and known to have protective and therapeutic effects in various diseases. Ginsenosides are active gradients representing the diverse pharmacological efficacy of ginseng. Huntington's disease (HD) is incurable genetic disorder associated with mutant huntingtin (mHtt) aggregation in the central nervous system. This study was conducted to investigate the effects of ginsenoside Rg3 and Rf on mHtt aggregation, cell viability, mitochondrial function, and apoptotic molecules on HD model. To investigate the effect of ginsenosides on HD, neural stem cells were isolated from the R6/2 mouse brain and used as a cellular model of HD. Nuclear aggregation of mHtt was measured by immunocytochemistry, and expressions of mitochondrial biogenesis and apoptotic molecules were investigated by western blot. As a result, the number of mHtt aggregates positive cells has decreased by ginsenoside Rg3 and Rf treatment in cellular model of HD. Mitochondrial biogenesis-related molecules such as PGC-1α and phosphorylated CREB were increased or showed increased tendency by ginsenoside Rg3 and Rf. Apoptotic molecules, p53, Bax, and cleaved caspase-3, were down-regulated by treatment of ginsenoside Rg3 and Rf. In addition, Lysotracker staining result showed that cellular lysosomal content was reduced by ginsenoside Rg3 and Rf. Given that ginsenoside Rg3 and Rf have the potential to reduce mHtt aggregation and cellular apoptosis, these ginsenosides can be possible therapeutic candidates for treating HD phenotypes.

An L-type lectin receptor-like kinase promotes starch accumulation during rice pollen maturation.

Starch accumulation is key for the maturity of rice pollen grains; however, the regulatory mechanism underlying this process remains unknown. Here, we have isolated a male-sterile rice mutant, abnormal pollen 1 (ap1), which produces nonviable pollen grains with defective starch accumulation. Functional analysis revealed that AP1 encodes an active L-type lectin receptor-like kinase (L-LecRLK). AP1 is localized to the plasma membrane and its transcript is highly accumulated in pollen during the starch synthesis phase. RNA-seq and phosphoproteomic analysis revealed that the expression/phosphorylation levels of numerous genes/proteins involved in starch and sucrose metabolism pathway were significantly altered in the mutant pollen, including a known rice UDP-glucose pyrophosphorylase (OsUGP2). We further found that AP1 physically interacts with OsUGP2 to elevate its enzymatic activity, likely through targeted phosphorylation. These findings revealed a novel role of L-LecRLK in controlling pollen maturity via modulating sucrose and starch metabolism.

Gene delivery corrects N-acetylglutamate synthase deficiency and enables insights in the physiological impact of L-arginine activation of N-acetylglutamate synthase.

The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to urea. N-acetylglutamate synthase (NAGS) catalyzes formation of N-acetylglutamate, an essential allosteric activator of carbamylphosphate synthetase 1. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine, but the physiological significance of NAGS activation by L-arginine has been unknown. The NAGS knockout (Nags-/-) mouse is an animal model of inducible hyperammonemia, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG + Cit). We used adeno associated virus (AAV) based gene transfer to correct NAGS deficiency in the Nags-/- mice, established the dose of the vector needed to rescue Nags-/- mice from hyperammonemia and measured expression levels of Nags mRNA and NAGS protein in the livers of rescued animals. This methodology was used to investigate the effect of L-arginine on ureagenesis in vivo by treating Nags-/- mice with AAV vectors encoding either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine. The Nags-/- mice expressing E354A mNAGS were viable but had elevated plasma ammonia concentration despite similar levels of the E354A and wild-type mNAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was identified in a patient with NAGS deficiency. Our results show that NAGS deficiency can be rescued by gene therapy, and suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.

An abundant biliary metabolite derived from dietary omega-3 polyunsaturated fatty acids regulates triglycerides.

Omega-3 fatty acids from fish oil reduce triglyceride levels in mammals, yet the mechanisms underlying this effect have not been fully clarified, despite the clinical use of omega-3 ethyl esters to treat severe hypertriglyceridemia and reduce cardiovascular disease risk in humans. Here, we identified in bile a class of hypotriglyceridemic omega-3 fatty acid-derived N-acyl taurines (NATs) that, after dietary omega-3 fatty acid supplementation, increased to concentrations similar to those of steroidal bile acids. The biliary docosahexaenoic acid-containing (DHA-containing) NAT C22:6 NAT was increased in human and mouse plasma after dietary omega-3 fatty acid supplementation and potently inhibited intestinal triacylglycerol hydrolysis and lipid absorption. Supporting this observation, genetic elevation of endogenous NAT levels in mice impaired lipid absorption, whereas selective augmentation of C22:6 NAT levels protected against hypertriglyceridemia and fatty liver. When administered pharmacologically, C22:6 NAT accumulated in bile and reduced high-fat diet-induced, but not sucrose-induced, hepatic lipid accumulation in mice, suggesting that C22:6 NAT is a negative feedback mediator that limits excess intestinal lipid absorption. Thus, biliary omega-3 NATs may contribute to the hypotriglyceridemic mechanism of action of fish oil and could influence the design of more potent omega-3 fatty acid-based therapeutics.

Pharmacological Characteristics of Porcine Orexin 2 Receptor and Mutants.

Orexin receptors (OXRs) play a critical regulatory role in central control of food intake, maintenance of sleeping states, energy metabolism, and neuroendocrine homeostasis. However, most previous studies have focused on the sleep-promoting functions of OXRs in human beings, while their potential value in enhancing food intake for livestock breeding has not been fully exploited. In this study, we successfully cloned porcine orexin 2 receptor (pOX2R) complementary DNA and constructed four pOX2R mutants (P10S, P11T, V308I, and T401I) by site-directed mutagenesis, and their functional expressions were further confirmed through Western blotting analysis. Pharmacological characteristics of pOX2R and their mutants were further investigated. These results showed that the P10S, P11T, and T401I mutants had decreased cAMP signaling with orexin A, whereas only the P11T mutant decreased under the stimulation of orexin B. Besides, only P10S displayed a decreased calcium release in response to both orexin ligands. Importantly, these mutants exhibited decreased phosphorylation levels of ERK1/2, p38, and CREB to some degree compared with wild-type pOX2R. Collectively, these findings highlight the critical role of these mutations in pOX2R signaling and expand our understanding of molecular and pharmacological characterization of pOX2R.

Ebselen as template for stabilization of A4V mutant dimer for motor neuron disease therapy.

Mutations to the gene encoding superoxide dismutase-1 (SOD1) were the first genetic elements discovered that cause motor neuron disease (MND). These mutations result in compromised SOD1 dimer stability, with one of the severest and most common mutations Ala4Val (A4V) displaying a propensity to monomerise and aggregate leading to neuronal death. We show that the clinically used ebselen and related analogues promote thermal stability of A4V SOD1 when binding to Cys111 only. We have developed a A4V SOD1 differential scanning fluorescence-based assay on a C6S mutation background that is effective in assessing suitability of compounds. Crystallographic data show that the selenium atom of these compounds binds covalently to A4V SOD1 at Cys111 at the dimer interface, resulting in stabilisation. This together with chemical amenability for hit expansion of ebselen and its on-target SOD1 pharmacological chaperone activity holds remarkable promise for structure-based therapeutics for MND using ebselen as a template.

Photobiomodulation Using a Low-Level Light-Emitting Diode Improves Cognitive Dysfunction in the 5XFAD Mouse Model of Alzheimer's Disease.

Photobiomodulation using low-level light-emitting diode can be rapidly applied in neurological and physiological disorders safely and noninvasively. Photobiomodulation is effective for chronic diseases because of fewer side effects than drugs. Here we investigated the effects of photobiomodulation using light-emitting diode on amyloid plaques, gliosis, and neuronal loss to prevent and/or recover cognitive impairment, and optimal timing of photobiomodulation initiation for recovering cognitive function in a mouse model of Alzheimer's disease. 5XFAD mice were used as an Alzheimer's disease model. Animals receiving photobiomodulation treatment were divided into two groups: an early group starting photobiomodulation at 2 months of age (5XFAD+Early), and a late group starting photobiomodulation at 6 months of age (5XFAD+Delay). Both groups received photobiomodulation 20 minutes per session three times per week for 14 weeks. The Morris water maze, passive avoidance, and elevated plus maze tests were performed at 10 months of age. Immunohistochemistry and Western blot were performed after behavioral evaluation. The results showed that photobiomodulation treatment at early stages reduced amyloid accumulation, neuronal loss, and microgliosis and alleviated the cognitive dysfunction in 5XFAD mice, possibly by increasing insulin degrading enzyme related to amyloid-beta degradation. Photobiomodulation may be an excellent candidate for advanced preclinical Alzheimer's disease research.

A small molecule chaperone rescues the stability and activity of a cancer-associated variant of NAD(P)H:quinone oxidoreductase 1 in vitro.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a human FAD-dependent enzyme that plays a crucial role in the antioxidant defense system. A naturally occurring single-nucleotide polymorphism (NQO1*2) in the NQO1 gene leads to an amino acid substitution (P187S), which severely compromises the activity and stability of the enzyme. The NQO1*2 genotype has been linked to a higher risk for several types of cancer and poor survival rate after anthracycline-based chemotherapy. In this study, we show that a small molecular chaperone (N-(2-bromophenyl)pyrrolidine-1-sulfonamide) repopulates the native wild-type conformation. As a consequence of the stabilizing effect, the enzymatic activity of the P187S variant protein is strongly improved in the presence of the molecular chaperone in vitro.

Root cultures of potato mutant lacking MSPI isoform, indispensable for photosynthetic light reactions, exhibit characteristics similar to intact plant roots.

Potato (Solanum tuberosum) mutant (ST) lacking one isoform of manganese-stabilizing protein (MSPI) of photosystem II exhibited besides spontaneous tuberization also growth changes with strongly impaired root system development. Previous studies revealed marked changes in carbohydrate levels and allocation within ST plant body. To verify causal relationship between changed carbohydrate balance and root growth restriction we engaged dark grown sucrose-supplied root organ-cultures of ST plants to exclude/confirm shoot effects. Unexpectedly, in ST root cultures we observed large alterations in growth and architecture as well as saccharide status similar to those found in the intact plant roots. The gene expression analysis, however, proved PsbO1 transcript (coding MSPI protein) neither in ST nor in WT root-organ cultures. Therefore, the results point to indirect effects of PsbO1 allele absence connected possibly with some epigenetic modulations.

Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds.

Accumulation of mutant proteins is a major cause of many diseases (collectively called proteopathies), and lowering the level of these proteins can be useful for treatment of these diseases. We hypothesized that compounds that interact with both the autophagosome protein microtubule-associated protein 1A/1B light chain 3 (LC3)1 and the disease-causing protein may target the latter for autophagic clearance. Mutant huntingtin protein (mHTT) contains an expanded polyglutamine (polyQ) tract and causes Huntington's disease, an incurable neurodegenerative disorder2. Here, using small-molecule-microarray-based screening, we identified four compounds that interact with both LC3 and mHTT, but not with the wild-type HTT protein. Some of these compounds targeted mHTT to autophagosomes, reduced mHTT levels in an allele-selective manner, and rescued disease-relevant phenotypes in cells and in vivo in fly and mouse models of Huntington's disease. We further show that these compounds interact with the expanded polyQ stretch and could lower the level of mutant ataxin-3 (ATXN3), another disease-causing protein with an expanded polyQ tract3. This study presents candidate compounds for lowering mHTT and potentially other disease-causing proteins with polyQ expansions, demonstrating the concept of lowering levels of disease-causing proteins using autophagosome-tethering compounds.

Genetic and tissue-specific RNA-sequencing analysis of self-compatible mutant TSC28 in Brassica rapa L. toward identification of a novel self-incompatibility factor.

Self-incompatibility (SI) is a sophisticated system for pollen selectivity to prevent pollination by genetically identical pollen. In Brassica, it is genetically controlled by a single, highly polymorphic S-locus, and the male and female S-determinant factors have been identified as S-locus protein 11 (SP11)/S-locus cysteine-rich protein (SCR) and S-locus receptor kinase (SRK), respectively. However, the overall molecular system and identity of factors in the downstream cascade of the SI reaction remain unclear. Previously, we identified a self-compatible B. rapa mutant line, TSC28, which has a disruption in an unidentified novel factor of the SI signaling cascade. Here, in a genetic analysis of TSC28, using an F2 population from a cross with the reference B. rapa SI line Chiifu-401, the causal gene was mapped to a genetic region of DNA containing markers BrSA64 and ACMP297 in B. rapa chromosome A1. By fine mapping using an F2 population of 1,034 plants, it was narrowed down to a genetic region between DNA markers ACMP297 and BrgMS4028, with physical length approximately 1.01 Mbp. In this genomic region, 113 genes are known to be located and, among these, we identified 55 genes that were expressed in the papilla cells. These are candidates for the gene responsible for the disruption of SI in TSC28. This list of candidate genes will contribute to the discovery of a novel downstream factor in the SP11-SRK signaling cascade in the Brassica SI system.

Inhibition of mutant Kras and p53-driven pancreatic carcinogenesis by atorvastatin: Mainly via targeting of the farnesylated DNAJA1 in chaperoning mutant p53.

Recent studies have indicated that using statins to inhibit the mevalonate pathway induces mutant p53 degradation by impairing the interaction of mutant p53 with DnaJ subfamily A member 1 (DNAJA1). However, the role of the C-terminus of DNAJA1 with a CAAX box for farnesylation in the binding, folding, and translocation of client proteins such as mutant p53 is not known. In the present study, we used a genetically engineered mouse model of pancreatic carcinoma and showed that atorvastatin significantly increased animal survival and inhibited pancreatic carcinogenesis. There was a dramatic decrease in mutant p53 protein accumulation in the pancreatic acini, pancreas intraepithelial neoplasia lesions, and adenocarcinoma. Supplementation with farnesyl pyrophosphate, a substrate for protein farnesylation, rescued atorvastatin-induced mutant p53 degradation in pancreatic cancer cells. Tipifarnib, a farnesyltransferase inhibitor, mirrored atorvastatin's effects on mutant p53, degraded mutant p53 in a dose-dependent manner, and converted farnesylated DNAJA1 into unfarnesylated DNAJA1. Farnesyltransferase gene knockdown also significantly promoted mutant p53 degradation. Coimmunoprecipitation either by an anti-DNAJA1 or p53 antibody confirmed the direct interaction of mutant p53 and DNAJA1 and higher doses of atorvastatin treatments converted more farnesylated DNAJA1 into unfarnesylated DNAJA1 with much less mutant p53 pulled down by DNAJA1. Strikingly, C394S mutant DNAJA1, in which the cysteine of the CAAX box was mutated to serine, was no longer able to be farnesylated and lost the ability to maintain mutant p53 stabilization. Our results show that farnesylated DNAJA1 is a crucial chaperone in maintaining mutant p53 stabilization and targeting farnesylated DNAJA1 by atorvastatin will be critical for inhibiting p53 mutant cancer.

Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation.

Limb-girdle muscular dystrophy type 2D (LGMD2D) is characterized by a progressive proximal muscle weakness. LGMD2D is caused by mutations in the gene encoding α-sarcoglycan (α-SG), a dystrophin-associated glycoprotein that plays a key role in the maintenance of sarcolemma integrity in striated muscles. We report here on the development of a new in vitro high-throughput screening assay that allows the monitoring of the proper localization of the most prevalent mutant form of α-SG (R77C substitution). Using this assay, we screened a library of 2560 FDA-approved drugs and bioactive compounds and identified thiostrepton, a cyclic antibiotic, as a potential drug to repurpose for LGMD2D treatment. Characterization of the thiostrepton effect revealed a positive impact on R77C-α-SG and other missense mutant protein localization (R34H, I124T, V247M) in fibroblasts overexpressing these proteins. Finally, further investigations of the molecular mechanisms of action of the compound revealed an inhibition of the chymotrypsin-like activity of the proteasome 24 h after thiostrepton treatment and a synergistic effect with bortezomib, an FDA-approved proteasome inhibitor. This study reports on the first in vitro model for LGMD2D that is compatible with high-throughput screening and proposes a new therapeutic option for LGMD2D caused by missense mutations of α-SG.

TP53 Status as a Determinant of Pro- vs Anti-Tumorigenic Effects of Estrogen Receptor-Beta in Breast Cancer.

BACKGROUND: Anti-tumorigenic vs pro-tumorigenic roles of estrogen receptor-beta (ESR2) in breast cancer remain unsettled. We investigated the potential of TP53 status to be a determinant of the bi-faceted role of ESR2 and associated therapeutic implications for triple negative breast cancer (TNBC). METHODS: ESR2-TP53 interaction was analyzed with multiple assays including the in situ proximity ligation assay. Transcriptional effects on TP53-target genes and cell proliferation in response to knocking down or overexpressing ESR2 were determined. Patient survival according to ESR2 expression levels and TP53 mutation status was analyzed in the basal-like TNBC subgroup in the Molecular Taxonomy of Breast Cancer International Consortium (n = 308) and Roswell Park Comprehensive Cancer Center (n = 46) patient cohorts by univariate Cox regression and log-rank test. All statistical tests are two-sided. RESULTS: ESR2 interaction with wild-type and mutant TP53 caused pro-proliferative and anti-proliferative effects, respectively. Depleting ESR2 in cells expressing wild-type TP53 resulted in increased expression of TP53-target genes CDKN1A (control group mean [SD] = 1 [0.13] vs ESR2 depletion group mean [SD] = 2.08 [0.24], P = .003) and BBC3 (control group mean [SD] = 1 [0.06] vs ESR2 depleted group mean [SD] = 1.92 [0.25], P = .003); however, expression of CDKN1A (control group mean [SD] = 1 [0.21] vs ESR2 depleted group mean [SD] = 0.56 [0.12], P = .02) and BBC3 (control group mean [SD] = 1 [0.03] vs ESR2 depleted group mean [SD] = 0.55 [0.09], P = .008) was decreased in cells expressing mutant TP53. Overexpressing ESR2 had opposite effects. Tamoxifen increased ESR2-mutant TP53 interaction, leading to reactivation of TP73 and apoptosis. High levels of ESR2 expression in mutant TP53-expressing basal-like tumors is associated with better prognosis (Molecular Taxonomy of Breast Cancer International Consortium cohort: log-rank P = .001; hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.84, univariate Cox P = .02). CONCLUSIONS: TP53 status is a determinant of the functional duality of ESR2. Our study suggests that ESR2-mutant TP53 combination prognosticates survival in TNBC revealing a novel strategy to stratify TNBC for therapeutic intervention potentially by repurposing tamoxifen.

Simulations of mutant p53 DNA binding domains reveal a novel druggable pocket.

The DNA binding domain (DBD) of the tumor suppressor p53 is the site of several oncogenic mutations. A subset of these mutations lowers the unfolding temperature of the DBD. Unfolding leads to the exposure of a hydrophobic ß-strand and nucleates aggregation which results in pathologies through loss of function and dominant negative/gain of function effects. Inspired by the hypothesis that structural changes that are associated with events initiating unfolding in DBD are likely to present opportunities for inhibition, we investigate the dynamics of the wild type (WT) and some aggregating mutants through extensive all atom explicit solvent MD simulations. Simulations reveal differential conformational sampling between the WT and the mutants of a turn region (S6-S7) that is contiguous to a known aggregation-prone region (APR). The conformational properties of the S6-S7 turn appear to be modulated by a network of interacting residues. We speculate that changes that take place in this network as a result of the mutational stress result in the events that destabilize the DBD and initiate unfolding. These perturbations also result in the emergence of a novel pocket that appears to have druggable characteristics. FDA approved drugs are computationally screened against this pocket.

PLX9486 shows anti-tumor efficacy in patient-derived, tyrosine kinase inhibitor-resistant KIT-mutant xenograft models of gastrointestinal stromal tumors.

The purpose of the present study was to investigate the in vitro and in vivo activity of PLX9486, a tyrosine kinase inhibitor (TKI) targeting both primary KIT exon 9 and 11 and secondary exon 17 and 18 mutations in gastrointestinal stromal tumors (GISTs). Imatinib, a potent inhibitor of mutated KIT, has revolutionized the clinical management of advanced, metastatic GIST. However, secondary resistance develops mainly through acquired mutations in KIT exons 13/14 or exons 17/18. Second-line sunitinib potently inhibits KIT exon 13/14 mutants but is ineffective against exon 17 mutations. In our study, PLX9486 demonstrated in vitro nanomolar potency in inhibiting the growth and KIT phosphorylation of engineered BaF3 cells transformed with KIT exon 17 mutations (p.D816V) and with the double KIT exon 11/17 mutations (p.V560G/D816V). The in vivo efficacy of PLX9486 was evaluated using two imatinib-resistant GIST patient-derived xenograft (PDX) models. In UZLX-GIST9 (KIT: p.P577del;W557LfsX5;D820G), PLX9486 100 mg/kg/day resulted in significant inhibition of proliferation. Pharmacodynamic analysis showed a pronounced reduction in mitogen-activated protein kinase (MAPK) activation and other downstream effects of the KIT signaling pathway but no significant effect on KIT Y703 and Y719 phosphorylation. Similarly, in MRL-GIST1 (KIT: p.W557_K558del;Y823D) PLX9486 treatment led to significant tumor regression and strong inhibition of MAPK activation. Interestingly, the inhibitory effect on MAPK activation was evident even after a single dose of PLX9486. In conclusion, PLX9486 showed anti-tumor efficacy in patient-derived imatinib-resistant GIST xenograft models, mainly through inhibition of KIT signaling. These preclinical efficacy data encourage further testing of PLX9486 in the clinical setting.

N-acetylcysteine and vitamin E rescue animal longevity and cellular oxidative stress in pre-clinical models of mitochondrial complex I disease.

Oxidative stress is a known contributing factor in mitochondrial respiratory chain (RC) disease pathogenesis. Yet, no efficient means exists to objectively evaluate the comparative therapeutic efficacy or toxicity of different antioxidant compounds empirically used in human RC disease. We postulated that pre-clinical comparative analysis of diverse antioxidant drugs having suggested utility in primary RC disease using animal and cellular models of RC dysfunction may improve understanding of their integrated effects and physiologic mechanisms, and enable prioritization of lead antioxidant molecules to pursue in human clinical trials. Here, lifespan effects of N-acetylcysteine (NAC), vitamin E, vitamin C, coenzyme Q10 (CoQ10), mitochondrial-targeted CoQ10 (MS010), lipoate, and orotate were evaluated as the primary outcome in a well-established, short-lived C. elegans gas-1(fc21) animal model of RC complex I disease. Healthspan effects were interrogated to assess potential reversal of their globally disrupted in vivo mitochondrial physiology, transcriptome profiles, and intermediary metabolic flux. NAC or vitamin E fully rescued, and coenzyme Q, lipoic acid, orotic acid, and vitamin C partially rescued gas-1(fc21) lifespan toward that of wild-type N2 Bristol worms. MS010 and CoQ10 largely reversed biochemical pathway expression changes in gas-1(fc21) worms. While nearly all drugs normalized the upregulated expression of the "cellular antioxidant pathway", they failed to rescue the mutant worms' increased in vivo mitochondrial oxidant burden. NAC and vitamin E therapeutic efficacy were validated in human fibroblast and/or zebrafish complex I disease models. Remarkably, rotenone-induced zebrafish brain death was preventable partially with NAC and fully with vitamin E. Overall, these pre-clinical model animal data demonstrate that several classical antioxidant drugs do yield significant benefit on viability and survival in primary mitochondrial disease, where their major therapeutic benefit appears to result from targeting global cellular, rather than intramitochondria-specific, oxidative stress. Clinical trials are needed to evaluate whether the two antioxidants, NAC and vitamin E, that show greatest efficacy in translational model animals significantly improve the survival, function, and feeling of human subjects with primary mitochondrial RC disease.