Epidermal Growth Factor Receptor Inhibitors in Glioblastoma: Current Status and Future Possibilities.

Glioblastoma, a grade 4 glioma as per the World Health Organization, poses a challenge in adult primary brain tumor management despite advanced surgical techniques and multimodal therapies. This review delves into the potential of targeting epidermal growth factor receptor (EGFR) with small-molecule inhibitors and antibodies as a treatment strategy. EGFR, a mutationally active receptor tyrosine kinase in over 50% of glioblastoma cases, features variants like EGFRvIII, EGFRvII and missense mutations, necessitating a deep understanding of their structures and signaling pathways. Although EGFR inhibitors have demonstrated efficacy in other cancers, their application in glioblastoma is hindered by blood-brain barrier penetration and intrinsic resistance. The evolving realm of nanodrugs and convection-enhanced delivery offers promise in ensuring precise drug delivery to the brain. Critical to success is the identification of glioblastoma patient populations that benefit from EGFR inhibitors. Tools like radiolabeled anti-EGFR antibody 806i facilitate the visualization of EGFR conformations, aiding in tailored treatment selection. Recognizing the synergistic potential of combination therapies with downstream targets like mTOR, PI3k, and HDACs is pivotal for enhancing EGFR inhibitor efficacy. In conclusion, the era of precision oncology holds promise for targeting EGFR in glioblastoma, contingent on tailored treatments, effective blood-brain barrier navigation, and the exploration of synergistic therapies.

Myocardial infarction elevates endoplasmic reticulum stress and protein aggregation in heart as well as brain.

Cardiovascular diseases, including myocardial infarction (MI), constitute the leading cause of morbidity and mortality worldwide. Protein-aggregate deposition is a hallmark of aging and neurodegeneration. Our previous study reported that aggregation is strikingly elevated in hearts of hypertensive and aged mice; however, no prior study has addressed MI effects on aggregation in heart or brain. Here, we present novel data on heart and brain aggregation in mice following experimental MI, induced by left coronary artery (LCA) ligation. Infarcted and peri-infarcted heart tissue, and whole cerebra, were isolated from mice at sacrifice, 7 days following LCA ligation. Sham-MI mice (identical surgery without ligation) served as controls. We purified detergent-insoluble aggregates from these tissues, and quantified key protein constituents by high-resolution mass spectrometry (LC-MS/MS). Infarct heart tissue had 2.5- to 10-fold more aggregates than non-infarct or sham-MI heart tissue (each P = 0.001). Protein constituents from MI cerebral aggregates overlapped substantially with those from human Alzheimer's disease brain. Prior injection of mice with mesenchymal stem cell (MSC) exosomes, shown to limit infarct size after LCA ligation, reduced cardiac aggregation ~ 60%, and attenuated markers of endoplasmic reticulum (ER) stress in heart and brain (GRP78, ATF6, P-PERK) by 50-75%. MI also elevated aggregate constituents enriched in Alzheimer's disease (AD) aggregates, such as proteasomal subunits, heat-shock proteins, complement C3, clusterin/ApoJ, and other apolipoproteins. These data provide novel evidence that aggregation is elevated in mouse hearts and brains after myocardial ischemia, leading to cognitive impairment resembling AD, but can be attenuated by exosomes or drug (CDN1163) interventions that oppose ER stress.

Intrinsically disordered proteins identified in the aggregate proteome serve as biomarkers of neurodegeneration.

A protein's structure is determined by its amino acid sequence and post-translational modifications, and provides the basis for its physiological functions. Across all organisms, roughly a third of the proteome comprises proteins that contain highly unstructured or intrinsically disordered regions. Proteins comprising or containing extensive unstructured regions are referred to as intrinsically disordered proteins (IDPs). IDPs are believed to participate in complex physiological processes through refolding of IDP regions, dependent on their binding to a diverse array of potential protein partners. They thus play critical roles in the assembly and function of protein complexes. Recent advances in experimental and computational analyses predicted multiple interacting partners for the disordered regions of proteins, implying critical roles in signal transduction and regulation of biological processes. Numerous disordered proteins are sequestered into aggregates in neurodegenerative diseases such as Alzheimer's disease (AD) where they are enriched even in serum, making them good candidates for serum biomarkers to enable early detection of AD.

Physiological Consequences of Targeting 14-3-3 and Its Interacting Partners in Neurodegenerative Diseases.

The mammalian 14-3-3 family comprises seven intrinsically unstructured, evolutionarily conserved proteins that bind >200 protein targets, thereby modulating cell-signaling pathways. The presence of 14-3-3 proteins in cerebrospinal fluid provides a sensitive and specific biomarker of neuronal damage associated with Alzheimer's disease (AD), Creutzfeldt−Jakob disease (CJD), spongiform encephalitis, brain cancers, and stroke. We observed significant enrichment of 14-3-3 paralogs G, S, and Z in human brain aggregates diagnostic of AD. We used intra-aggregate crosslinking to identify 14-3-3 interaction partners, all of which were significantly enriched in AD brain aggregates relative to controls. We screened FDA-approved drugs in silico for structures that could target the 14-3-3G/hexokinase interface, an interaction specific to aggregates and AD. C. elegans possesses only two 14-3-3 orthologs, which bind diverse proteins including DAF-16 (a FOXO transcription factor) and SIR-2.1 (a sensor of nutrients and stress), influencing lifespan. Top drug candidates were tested in C. elegans models of neurodegeneration-associated aggregation and in a human neuroblastoma cell-culture model of AD-like amyloidosis. Several drugs opposed aggregation in all models assessed and rescued behavioral deficits in C. elegans AD-like neuropathy models, suggesting that 14-3-3 proteins are instrumental in aggregate accrual and supporting the advancement of drugs targeting 14-3-3 protein complexes with their partners.

Computational and in vitro characterization of ICY-5: A potential candidate promoting mitochondrial apoptosis via the c-MET and STAT3 pathways.

Targeted chemotherapy remains the primary choice in controlling various forms of breast cancer (BC) due to its heterogenous gene expressions in various subtypes. In silico and in vitro evaluation of ICY-5, a novel arylidene analogue against c-MET, was performed. ICY-5 exhibited a docking score of -9.6 kcal/mol in inactive conformation and, - 8.6 kcal/mol in active conformation for c-MET. ICY-5 inhibited c-MET enzyme with an IC50 of 34.34 nM. The compound effectively inhibited MDA-MB 231 and MCF-7 cell proliferation, with GI50 values of 62.61 and 75.31 nM, respectively, and hepatocyte growth factor (HGF)/R c-MET phosphorylation with IC50 s of 71.41 and 83.77 nM, respectively. ICY-5 dose-dependently inhibited HGF-induced transmigration, cell scattering, invasion and altered cell cycle. An increase in apoptotic populations of these cells, with a dose-dependent decease in phosphorylation of STAT3 protein was observed. Furthermore, ICY-5 upregulated the caspase-3, caspase-9, Bcl-2-associated X and survivin, and downregulated Bcl-2, vascular endothelial growth factor, matrix metalloproteinase-2 (MMP-2), and MMP-9 in both BC cell lines. In summary, ICY-5 exhibited excellent efficacy in BC cells, targeting c-MET/SAT-3-mediated mitochondrial apoptosis. Further research will be required to ascertain ICY-5 suitability as a targeted chemotherapeutic against multiple forms of BC.

Novel hydroxybenzylamine-deoxyvasicinone hybrids as anticholinesterase therapeutics for Alzheimer's disease.

A series of novel 2-hydroxybenzylamine-deoxyvasicinone hybrid analogs (8a-8n) have been synthesized and evaluated as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and as inhibitors of amyloid peptide (Aß1-42) aggregation, for treatment of Alzheimer's disease (AD). These dual acting compounds exhibited good AChE inhibitory activities ranging from 0.34 to 6.35 µM. Analogs8g and 8n were found to be the most potent AChE inhibitors in the series with IC50values of 0.38 µM and 0.34 µM, respectively. All the analogs (8a-8n) exhibited weak BuChE inhibitory activities ranging from 14.60 to 21.65 µM. Analogs8g and 8n exhibited BuChE with IC50values of 15.38 µM and 14.60 µM, respectively, demonstrating that these analogs were greater than 40-fold more selective for inhibition of AChE over BuChE. Additionally, compounds8g and 8n were also found to be the best inhibitors of self-induced Aß1-42 peptide aggregation with IC50values of 3.91 µM and 3.22 µM, respectively; 8g and 8n also inhibited AChE-induced Aß1-42 peptide aggregation by 68.7% and 72.6%, respectively. Kinetic analysis and molecular docking studies indicate that analogs 8g and 8n bind to a new allosteric pocket (site B) on AChE. In addition, the observed inhibition of AChE-induced Aß1-42 peptide aggregation by 8n is likely due to allosteric inhibition of the binding of this peptide at the CAS site on AChE. Overall, these results indicate that 8g and 8n are examples of dual-acting lead compounds for the development of highly effective anti-AD drugs.

FCX-146, a potent allosteric inhibitor of Akt kinase in cancer cells: Lead optimization of the second-generation arylidene indanone scaffold.

Akt, a serine-threonine protein kinase, is regulated by class-I PI3K signaling. Akt regulates a wide variety of cell processes including cell proliferation, survival, and angiogenesis through serine/threonine phosphorylation of downstream targets including mTOR and glycogen-synthase-kinase-3-beta (GSK3ß). Targeting cancer-specific overexpression of Akt protein could be an efficient way to control cancer-cell proliferation. However, the ATP-competitive inhibitors are challenged by the highly conserved ATP binding site, and by competition with high cellular concentrations of ATP. We previously developed an allosteric inhibitor, 2-arylidene-4, 7-dimethyl indan-1-one (FXY-1) that showed promising activity against several lung cancer models. In this work, we designed a congeneric series of molecules based on FXY-1 and optimized lead based on computational, in vitro assays. Computational screening followed by enzyme-inhibition and cell-proliferation assays identified a derivative (FCX-146) as a new lead molecule with threefold greater potency than the parent compound. FCX-146 increased apoptosis in HL-60 cells, mediated in part through decreased expression of antiapoptotic Bcl-2 protein and increased levels of Bax-2 and Caspase-3. Molecular-dynamic simulations showed stable binding of FCX-146 to an allosteric (i.e., noncatalytic) pocket in Akt. Together, we propose FCX-146 as a potent second-generation arylidene indanone compound that binds to the allosteric pocket of Akt and potently inhibits its activation.

Interleukin-1ß drives NEDD8 nuclear-to-cytoplasmic translocation, fostering parkin activation via NEDD8 binding to the P-ubiquitin activating site.

BACKGROUND: Neuroinflammation, typified by elevated levels of interleukin-1 (IL-1) α and ß, and deficits in proteostasis, characterized by accumulation of polyubiquitinated proteins and other aggregates, are associated with neurodegenerative disease independently and through interactions of the two phenomena. We investigated the influence of IL-1ß on ubiquitination via its impact on activation of the E3 ligase parkin by either phosphorylated ubiquitin (P-Ub) or NEDD8. METHODS: Immunohistochemistry and Proximity Ligation Assay were used to assess colocalization of parkin with P-tau or NEDD8 in hippocampus from Alzheimer patients (AD) and controls. IL-1ß effects on PINK1, P-Ub, parkin, P-parkin, and GSK3ß-as well as phosphorylation of parkin by GSK3ß-were assessed in cell cultures by western immunoblot, using two inhibitors and siRNA knockdown to suppress GSK3ß. Computer modeling characterized the binding and the effects of P-Ub and NEDD8 on parkin. IL-1α, IL-1ß, and parkin gene expression was assessed by RT-PCR in brains of 2- and 17-month-old PD-APP mice and wild-type littermates. RESULTS: IL-1α, IL-1ß, and parkin mRNA levels were higher in PD-APP mice compared with wild-type littermates, and IL-1α-laden glia surrounded parkin- and P-tau-laden neurons in human AD. Such neurons showed a nuclear-to-cytoplasmic translocation of NEDD8 that was mimicked in IL-1ß-treated primary neuronal cultures. These cultures also showed higher parkin levels and GSK3ß-induced parkin phosphorylation; PINK1 levels were suppressed. In silico simulation predicted that binding of either P-Ub or NEDD8 at a singular position on parkin opens the UBL domain, exposing Ser65 for parkin activation. CONCLUSIONS: The promotion of parkin- and NEDD8-mediated ubiquitination by IL-1ß is consistent with an acute neuroprotective role. However, accumulations of P-tau and P-Ub and other elements of proteostasis, such as translocated NEDD8, in AD and in response to IL-1ß suggest either over-stimulation or a proteostatic failure that may result from chronic IL-1ß elevation, easily envisioned considering its early induction in Down's syndrome and mild cognitive impairment. The findings further link autophagy and neuroinflammation, two important aspects of AD pathogenesis, which have previously been only loosely related.

A novel tetrazole analogue of resveratrol is a potent anticancer agent.

A series of novel tetrazole analogues of resveratrol were synthesized and evaluated for their anti-leukemic activity against an extensive panel of human cancer cell lines and against the MV4-11 AML cell line. These molecules were designed as drug-like derivatives of the resveratrol analogue DMU-212 and its cyano derivatives. Four compounds 8g, 8h, 10a and 10b exhibited LD50 values of 4.60 µM, 0.02 µM, 1.46 µM, and 1.08 µM, respectively, against MV4-11 leukemia cells. The most potent compounds, 8h and 10b, were also found to be active against an extensive panel of human hematological and solid tumor cell lines; compound 8h was the most potent compound with GI50 values <10 nM against more than 90% of the human cancer cell lines in the 60-cell panel. Analogues 8g, 8h, 10a and 10b were also tested for their ability to inhibit the polymerization of tubulin, and compound 8h was found to be the most potent analogue. Molecular modeling studies demonstrated that 8h binds to the colchicine binding site on tubulin. Thus, compound 8h is considered to be a lead druglike molecule from this tetrazole series of compounds.

Apolipoprotein E4 inhibits autophagy gene products through direct, specific binding to CLEAR motifs.

INTRODUCTION: Alzheimer apolipoprotein E (APOE) ɛ4/ɛ4 carriers have earlier disease onset and more protein aggregates than patients with other APOE genotypes. Autophagy opposes aggregation, and important autophagy genes are coordinately regulated by transcription factor EB (TFEB) binding to "coordinated lysosomal expression and regulation" (CLEAR) DNA motifs. METHODS: Autophagic gene expression was assessed in brains of controls and Alzheimer's disease (AD) patients parsed by APOE genotype and in a glioblastoma cell line expressing either apoE3 or apoE4. Computational modeling assessed interactions between apoE and mutated apoE with CLEAR or modified DNA. RESULTS: Three TFEB-regulated mRNA transcripts-SQSTM, MAP1LC3B, and LAMP2-were lower in AD ɛ4/ɛ4 than in AD ɛ3/ɛ3 brains. Computational modeling predicted avid specific binding of apoE4 to CLEAR motifs. ApoE was found in cellular nuclei, and in vitro binding assays suggest competition between apoE4 and TFEB at CLEAR sites. CONCLUSION: ApoE4-CLEAR interactions may account for suppressed autophagy in APOE ɛ4/ɛ4 carriers and, in this way, contribute to earlier AD onset.

Leave-one-out-analysis (LOOA): web-based tool to predict influential proteins and interactions in aggregate-crosslinking proteomic data.

Many age-progressive diseases are accompanied by (and likely caused by) the presence of protein aggregation in affected tissues. Protein aggregates are conjoined by complex protein-protein interactions, which remain poorly understood. Knowledge of the proteins that comprise aggregates, and their adherent interfaces, can be useful to identify therapeutic targets to treat or prevent pathology, and to discover small molecules for disease interventions. We present web-based software to evaluate and rank influential proteins and protein-protein interactions based on graph modelling of the cross linked aggregate interactome. We have used two network-graph-based techniques: Leave-One-Vertex-Out (LOVO) and Leave-One-Edge-Out (LOEO), each followed by dimension reduction and calculation of influential vertices and edges using Principal Components Analysis (PCA) implemented as an R program. This method enables researchers to quickly and accurately determine influential proteins and protein-protein interactions present in their aggregate interactome data.

Alzheimer's-specific brain amyloid interactome: Neural-network analysis of intra-aggregate crosslinking identifies novel drug targets.

Alzheimer's disease (AD) is characterized by peri-neuronal amyloid plaque and intra-neuronal neurofibrillary tangles. These aggregates are identified by the immunodetection of "seed" proteins (Aß1-42 and hyperphosphorylated tau, respectively), but include many other proteins incorporated nonrandomly. Using click-chemistry intra-aggregate crosslinking, we previously modeled amyloid "contactomes" in SY5Y-APPSw neuroblastoma cells, revealing that aspirin impedes aggregate growth and complexity. By an analogous strategy, we now construct amyloid-specific aggregate interactomes of AD and age-matched-control hippocampi. Comparing these interactomes reveals AD-specific interactions, from which neural-network (NN) analyses predict proteins with the highest impact on pathogenic aggregate formation and/or stability. RNAi knockdowns of implicated proteins, in C. elegans and human-cell-culture models of AD, validated those predictions. Gene-Ontology meta-analysis of AD-enriched influential proteins highlighted the involvement of mitochondrial and cytoplasmic compartments in AD-specific aggregation. This approach derives dynamic consensus models of aggregate growth and architecture, implicating highly influential proteins as new targets to disrupt amyloid accrual in the AD brain.

Ezetimibe Lowers Risk of Alzheimer's and Related Dementias over Sevenfold, Reducing Aggregation in Model Systems by Inhibiting 14-3-3G::Hexokinase Interaction.

Numerous factors predispose to progression of cognitive impairment to Alzheimer's disease and related dementias (ADRD), most notably age, APOE(ε4) alleles, traumatic brain injury, heart disease, hypertension, obesity/diabetes, and Down's syndrome. Protein aggregation is diagnostic for neurodegenerative diseases, and may be causal through promotion of chronic neuroinflammation. We isolated aggregates from postmortem hippocampi of ADRD patients, heart-disease patients, and age-matched controls. Aggregates, characterized by high-resolution proteomics (with or without crosslinking), were significantly elevated in heart-disease and ADRD hippocampi. Hexokinase-1 (HK1) and 14-3-3G/γ proteins, previously implicated in neuronal signaling and neurodegeneration, are especially enriched in ADRD and heart-disease aggregates vs. controls (each P<0.008), and their interaction was implied by extensive crosslinking in both disease groups. Screening the hexokinase-1::14-3-3G interface with FDA-approved drug structures predicted strong affinity for ezetimibe, a benign cholesterol-lowering medication. Diverse cultured human-cell and whole-nematode models of ADRD aggregation showed that this drug potently disrupts HK1::14-3-3G adhesion, reduces disease-associated aggregation, and activates autophagy. Mining clinical databases supports drug reduction of ADRD risk, decreasing it to 0.14 overall (P<0.0001; 95% C.I. 0.06-0.34), and <0.12 in high-risk heart-disease subjects (P<0.006). These results suggest that drug disruption of the 14-3-3G::HK1 interface blocks an early "lynchpin" adhesion, prospectively reducing aggregate accrual and progression of ADRD.

Rescue of ApoE4-related lysosomal autophagic failure in Alzheimer's disease by targeted small molecules.

Homozygosity for the ε4 allele of APOE increases the odds of developing Alzheimer's by 12 to 15 times relative to the most common ε3;ε3 genotype, and its association with higher plaque loads comports with evidence that APOEε4 compromises autophagy. The ApoE4 protein specifically binds a cis element ("CLEAR") in the promoters of several autophagy genes to block their transcription. We used a multifaceted approach to identify a druggable site in ApoE4, and virtual screening of lead-like compounds identified small molecules that specifically bind to this site to impede ApoE4::DNA binding. We validated these molecules both in vitro and in vivo with models expressing ApoE4, including ApoE4 targeted-replacement mice. One compound was able to significantly restore transcription of several autophagy genes and protected against amyloid-like aggregation in a C. elegans AD model. Together, these findings provide proof-of-principle evidence for pharmacological remediation of lysosomal autophagy by ApoE4 via ApoE4-targeted lead molecules that represent a novel tack on neurodegenerative disorders.

In silico analysis of TUBA4A mutations in Amyotrophic Lateral Sclerosis to define mechanisms of microtubule disintegration.

Amyotrophic lateral sclerosis (ALS) is an inexorably progressive and degenerative disorder of motor neurons with no currently-known cure. Studies to determine the mechanism of neurotoxicity and the impact of ALS-linked mutations (SOD1, FUS, TARDP, C9ORF72, PFN1, TUBA4A and others) have greatly expanded our knowledge of ALS disease mechanisms and have helped to identify potential targets for ALS therapy. Cellular pathologies (e.g., aggregation of mutant forms of SOD1, TDP43, FUS, Ubiqulin2, PFN1, and C9ORF72), mitochondrial dysfunction, neuroinflammation, and oxidative damage are major pathways implicated in ALS. Nevertheless, the selective vulnerability of motor neurons remains unexplained. The importance of tubulins for long-axon infrastructure, and the special morphology and function of motor neurons, underscore the central role of the cytoskeleton. The recent linkage of mutations to the tubulin α chain, TUBA4A, to familial and sporadic cases of ALS provides a new investigative opportunity to shed light on both mechanisms of ALS and the vulnerability of motor neurons. In the current study we investigate TUBA4A, a structural microtubule protein with mutations causal to familial ALS, using molecular-dynamic (MD) modeling of protein structure to predict the effects of each mutation and its overall impact on GTP binding, chain stability, tubulin assembly, and aggregation propensity. These studies predict that each of the reported mutations will cause notable structural changes to the TUBA4A (α chain) tertiary protein structure, adversely affecting its physical properties and functions. Molecular docking and MD simulations indicate certain α chain mutations (e.g. K430N, R215C, and W407X) may cause structural deviations that impair GTP binding, and plausibly prevent or destabilize tubulin polymerization. Furthermore, several mutations (including R320C and K430N) confer a significant increase in predicted aggregation propensity of TUBA4A mutants relative to wild-type. Taken together, these in silico modeling studies predict structural perturbations and disruption of GTP binding, culminating in failure to form a stable tubulin heterocomplex, which may furnish an important pathogenic mechanism to trigger motor neuron degeneration in ALS.

Thiadiazolidinone (TDZD) Analogs Inhibit Aggregation-Mediated Pathology in Diverse Neurodegeneration Models, and Extend C. elegans Life- and Healthspan.

Chronic, low-grade inflammation has been implicated in aging and age-dependent conditions, including Alzheimer's disease, cardiomyopathy, and cancer. One of the age-associated processes underlying chronic inflammation is protein aggregation, which is implicated in neuroinflammation and a broad spectrum of neurodegenerative diseases such as Alzheimer's, Huntington's, and Parkinson's diseases. We screened a panel of bioactive thiadiazolidinones (TDZDs) from our in-house library for rescue of protein aggregation in human-cell and C. elegans models of neurodegeneration. Among the tested TDZD analogs, PNR886 and PNR962 were most effective, significantly reducing both the number and intensity of Alzheimer-like tau and amyloid aggregates in human cell-culture models of pathogenic aggregation. A C. elegans strain expressing human Aß1-42 in muscle, leading to AD-like amyloidopathy, developed fewer and smaller aggregates after PNR886 or PNR962 treatment. Moreover, age-progressive paralysis was reduced 90% by PNR886 and 75% by PNR962, and "healthspan" (the median duration of spontaneous motility) was extended 29% and 62%, respectively. These TDZD analogs also extended wild-type C. elegans lifespan by 15-30% (p < 0.001), placing them among the most effective life-extension drugs. Because the lead drug in this family, TDZD-8, inhibits GSK3ß, we used molecular-dynamic tools to assess whether these analogs may also target GSK3ß. In silico modeling predicted that PNR886 or PNR962 would bind to the same allosteric pocket of inactive GSK3ß as TDZD-8, employing the same pharmacophore but attaching with greater avidity. PNR886 and PNR962 are thus compelling candidate drugs for treatment of tau- and amyloid-associated neurodegenerative diseases such as AD, potentially also reducing all-cause mortality.

Machine-learning analysis of intrinsically disordered proteins identifies key factors that contribute to neurodegeneration-related aggregation.

Protein structure is determined by the amino acid sequence and a variety of post-translational modifications, and provides the basis for physiological properties. Not all proteins in the proteome attain a stable conformation; roughly one third of human proteins are unstructured or contain intrinsically disordered regions exceeding 40% of their length. Proteins comprising or containing extensive unstructured regions are termed intrinsically disordered proteins (IDPs). IDPs are known to be overrepresented in protein aggregates of diverse neurodegenerative diseases. We evaluated the importance of disordered proteins in the nematode Caenorhabditis elegans, by RNAi-mediated knockdown of IDPs in disease-model strains that mimic aggregation associated with neurodegenerative pathologies. Not all disordered proteins are sequestered into aggregates, and most of the tested aggregate-protein IDPs contribute to important physiological functions such as stress resistance or reproduction. Despite decades of research, we still do not understand what properties of a disordered protein determine its entry into aggregates. We have employed machine-learning models to identify factors that predict whether a disordered protein is found in sarkosyl-insoluble aggregates isolated from neurodegenerative-disease brains (both AD and PD). Machine-learning predictions, coupled with principal component analysis (PCA), enabled us to identify the physiochemical properties that determine whether a disordered protein will be enriched in neuropathic aggregates.

Glial Fibrillary Acidic Protein: A Biomarker and Drug Target for Alzheimer's Disease.

Glial fibrillary acidic protein (GFAP) is an intermediate filament structural protein involved in cytoskeleton assembly and integrity, expressed in high abundance in activated glial cells. GFAP is neuroprotective, as knockout mice are hypersensitive to traumatic brain injury. GFAP in cerebrospinal fluid is a biomarker of Alzheimer's disease (AD), dementia with Lewy bodies, and frontotemporal dementia (FTD). Here, we present novel evidence that GFAP is markedly overexpressed and differentially phosphorylated in AD hippocampus, especially in AD with the apolipoprotein E [ε4, ε4] genotype, relative to age-matched controls (AMCs). Kinases that phosphorylate GFAP are upregulated in AD relative to AMC. A knockdown of these kinases in SH-SY5Y-APPSw human neuroblastoma cells reduced amyloid accrual and lowered protein aggregation and associated behavioral traits in C. elegans models of polyglutamine aggregation (as observed in Huntington's disease) and of Alzheimer's-like amyloid formation. In silico screening of the ChemBridge structural library identified a small molecule, MSR1, with stable and specific binding to GFAP. Both MSR1 exposure and GF AP-specific RNAi knockdown reduce aggregation with remarkably high concordance of aggregate proteins depleted. These data imply that GFAP and its phosphorylation play key roles in neuropathic aggregate accrual and provide valuable new biomarkers, as well as novel therapeutic targets to alleviate, delay, or prevent AD.

"Protein aggregates" contain RNA and DNA, entrapped by misfolded proteins but largely rescued by slowing translational elongation.

All neurodegenerative diseases feature aggregates, which usually contain disease-specific diagnostic proteins; non-protein constituents, however, have rarely been explored. Aggregates from SY5Y-APPSw neuroblastoma, a cell model of familial Alzheimer's disease, were crosslinked and sequences of linked peptides identified. We constructed a normalized "contactome" comprising 11 subnetworks, centered on 24 high-connectivity hubs. Remarkably, all 24 are nucleic acid-binding proteins. This led us to isolate and sequence RNA and DNA from Alzheimer's and control aggregates. RNA fragments were mapped to the human genome by RNA-seq and DNA by ChIP-seq. Nearly all aggregate RNA sequences mapped to specific genes, whereas DNA fragments were predominantly intergenic. These nucleic acid mappings are all significantly nonrandom, making an artifactual origin extremely unlikely. RNA (mostly cytoplasmic) exceeded DNA (chiefly nuclear) by twofold to fivefold. RNA fragments recovered from AD tissue were ~1.5-to 2.5-fold more abundant than those recovered from control tissue, similar to the increase in protein. Aggregate abundances of specific RNA sequences were strikingly differential between cultured SY5Y-APPSw glioblastoma cells expressing APOE3 vs. APOE4, consistent with APOE4 competition for E-box/CLEAR motifs. We identified many G-quadruplex and viral sequences within RNA and DNA of aggregates, suggesting that sequestration of viral genomes may have driven the evolution of disordered nucleic acid-binding proteins. After RNA-interference knockdown of the translational-procession factor EEF2 to suppress translation in SY5Y-APPSw cells, the RNA content of aggregates declined by >90%, while reducing protein content by only 30% and altering DNA content by ≤10%. This implies that cotranslational misfolding of nascent proteins may ensnare polysomes into aggregates, accounting for most of their RNA content.

Antitumor properties of novel sesquiterpene lactone analogs as NFκB inhibitors that bind to the IKKß ubiquitin-like domain (ULD).

Melampomagnolide B (MMB, 3) is a parthenolide (PTL, 1) based sesquiterpene lactone that has been used as a template for the synthesis of a plethora of lead anticancer agents owing to its reactive C-10 primary hydroxyl group. Such compounds have been shown to inhibit the IKKß subunit, preventing phosphorylation of the cytoplasmic IκB inhibitory complex. The present study focuses on the synthesis and in vitro antitumor properties of novel benzyl and phenethyl carbamates of MMB (7a-7k). Screening of these MMB carbamates identified analogs with potent growth inhibition properties against a panel of 60 human cancer cell lines (71% of the molecules screened had GI50 values < 2 µM). Two analogs, the benzyl carbamate 7b and the phenethyl carbamate7k, were the most active compounds. Lead compound 7b inhibited cell proliferation in M9 ENL AML cells, and in TMD-231, OV-MD-231 and SUM149 breast cancer cell lines. Interestingly, mechanistic studies showed that 7b did not inhibit p65 phosphorylation in M9 ENL AML and OV-MD-231 cells, but did inhibit phophorylation of both p65 and IκBα in SUM149 cells. 7b also reduced NFκB binding to DNA in both OV-MD-231 and SUM149 cells. Molecular docking studies indicated that 7b and 7k are both predicted to interact with the ubiquitin-like domain (ULD) of the IKKß subunit. These data suggest that in SUM149 cells, 7b is likely acting as an allosteric inhibitor of IKKß, whereas in M9 ENL AML and OV-MD-231 cells 7b is able to inhibit an event after IκB/p65/p50 phosphorylation by IKKß that leads to inhibition of NFκB activation and reduction in NFκB-DNA binding. Analog 7b was by far the most potent compound in either carbamate series, and was considered an important lead compound for further optimization and development as an anticancer agent.