The Derivatives of Cromolyn Ameliorate the Abnormal Misfolding of Amyloid Proteins and Neuroinflammation in the Neural Cells.

BACKGROUND: The representative symptom of Alzheimer's Disease (AD) has mainly been mentioned to be misfolding of amyloid proteins, such as amyloid-beta (Aß) and tau protein. In addition, the neurological pathology related to neuroinflammatory signaling has recently been raised as an important feature in AD. Currently, numerous drug candidates continue to be investigated to reduce symptoms of AD, including amyloid proteins misfolding and neuroinflammation. OBJECTIVE: Our research aimed to identify the anti-AD effects of two chemical derivatives modified from cromoglicic acid, CNU 010 and CNU 011. METHODS: CNU 010 and CNU 011 derived from cromoglicic acid were synthesized. The inhibitory effects of Aß and tau were identified by thioflavin T assay. Moreover, western blots were conducted with derivates CNU 010 and CNU 011 to confirm the effects on inflammation. RESULTS: CNU 010 and CNU 011 significantly inhibited the aggregation of Aß and tau proteins. Moreover, they reduced the expression levels of mitogen-activated protein (MAP) kinase and nuclear factor kappa-light-chain-enhancer of activated B cells (NF- κB) signaling proteins, which are representative early inflammatory signaling markers. Also, the inhibitory effects on the lipopolysaccharide (LPS)-induced cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) expression referring to late inflammation were confirmed. CONCLUSION: Our results showing multiple beneficial effects of cromolyn derivatives against abnormal aggregation of amyloid proteins and neuroinflammatory signaling provide evidence that CNU 010 and CNU 011 could be further developed as potential drug candidates for AD treatment.

Comparative genomic analysis of plasmids encoding metallo-ß-lactamase NDM-5 in Enterobacterales Korean isolates from companion dogs.

Carbapenems are broad-spectrum antibiotics widely used for the treatment of human infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However, emerging carbapenemase-producing Enterobacterales (CPE) are rising as a public threat to human and animal health. We screened clinical bacterial isolates from 241 dogs and 18 cats hospitalized at Veterinary Medical Teaching Hospital, Seoul National University, from 2018 to 2020 for carbapenemase production. In our study, 5 strains of metallo-ß-lactamase NDM-5-producing Escherichia coli and Klebsiella pneumoniae were isolated from 4 different dogs. Multilocus sequence typing (MLST) results showed that all E. coli strains were ST410 and all K. pneumoniae strains were ST378. Whole genome analysis of the plasmid showed that blaNDM-5 is carried on a IncX3 plasmid, showing a high concordance rate with plasmids detected worldwide in human and animal isolates. The blaNDM gene was associated with the bleMBL gene and the ISAba125 element, truncated with the IS5 element. The results of this study show that CPE has already become as a threat to both animals and humans in our society, posing the necessity to solve it in terms of "One Health". Therefore, preventive strategies should be developed to prevent the spread of CPE in animal and human societies.

MicroRNA profiling in bovine serum according to the stage of Mycobacterium avium subsp. paratuberculosis infection.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), and it causes diarrhea and weakness in cattle. During a long subclinical stage, infected animals without clinical signs shed pathogens through feces. For this reason, the diagnosis of JD during the subclinical stage is very important. Circulating miRNAs are attracting attention as useful biomarkers in various veterinary diseases because of their expression changes depending on the state of the disease. Based on current knowledge, circulating miRNAs extracted from bovine serum were used to develop a diagnostic tool for JD. In this study, the animals were divided into 4 groups according to fecal shedding, the presence of antibodies, and clinical signs. Gene expression was analyzed by performing miRNA sequencing for each group, and it was identified that the miRNA expression changed more as the MAP infection progressed. The eight miRNAs that were differentially expressed in all infected groups were selected as biomarker candidates based on their significant differences compared to the control group. These biomarker candidates were validated by qRT-PCR. Considering the sequencing data, two upregulated miRNAs and two downregulated miRNAs showed the same trend in the validation results. Network analysis was also conducted and the results showed that mRNAs (IL-10, TGF-ß1) associated with regulatory T cells were predicted to be activated in the subclinical stage. Taken together, our data suggest that two miRNAs (bta-miR-374b, bta-miR-2887) may play major roles in the immune response to MAP infection during the subclinical stage.

Cromolyn platform suppresses fibrosis and inflammation, promotes microglial phagocytosis and neurite outgrowth.

Neurodegenerative diseases are characterized by chronic neuroinflammation and may perpetuate ongoing fibrotic reactions within the central nervous system. Unfortunately, there is no therapeutic available that treats neurodegenerative inflammation and its sequelae. Here we utilize cromolyn, a mast cell inhibitor with anti-inflammatory capabilities, and its fluorinated analogue F-cromolyn to study fibrosis-related protein regulation and secretion downstream of neuroinflammation and their ability to promote microglial phagocytosis and neurite outgrowth. In this report, RNA-seq analysis shows that administration of the pro-inflammatory cytokine TNF-α to HMC3 human microglia results in a robust upregulation of fibrosis-associated genes. Subsequent treatment with cromolyn and F-cromolyn resulted in reduced secretion of collagen XVIII, fibronectin, and tenascin-c. Additionally, we show that cromolyn and F-cromolyn reduce pro-inflammatory proteins PLP1, PELP1, HSP90, IL-2, GRO-α, Eotaxin, and VEGF-Α, while promoting secretion of anti-inflammatory IL-4 in HMC3 microglia. Furthermore, cromolyn and F-cromolyn augment neurite outgrowth in PC12 neuronal cells in concert with nerve growth factor. Treatment also differentially altered secretion of neurogenesis-related proteins TTL, PROX1, Rab35, and CSDE1 in HMC3 microglia. Finally, iPSC-derived human microglia more readily phagocytose Aß42 with cromolyn and F-cromolyn relative to controls. We propose the cromolyn platform targets multiple proteins upstream of PI3K/Akt/mTOR, NF-κB, and GSK-3ß signaling pathways to affect cytokine, chemokine, and fibrosis-related protein expression.

Genome-wide association study and genomic predictions for exterior traits in Yorkshire pigs1.

The objectives of this study were to identify informative genomic regions that affect the exterior traits of purebred Korean Yorkshire pigs and to investigate and compare the accuracy of genomic prediction for response variables. Phenotypic data on body height (BH), body length (BL), and total teat number (TTN) from 2,432 Yorkshire pigs were used to obtain breeding values including as response variable the estimated breeding value (EBV) and 2 types of deregressed EBVs-one including the parent average (DEBVincPA) and the other excluding it (DEBVexcPA). A final genotype panel comprising 46,199 SNP markers was retained for analysis after quality control for common SNPs. The BayesB and BayesC methods-with various π and weighted response variables (EBV, DEBVincPA, or DEBVexcPA)-were used to estimate SNP effects, through the genome-wide association study. The significance of genomic windows (1 Mb) was obtained at 1.0% additive genetic variance and was subsequently used to identify informative genomic regions. Furthermore, SNPs with a high model frequency (≥0.90) were considered informative. The accuracy of genomic prediction was estimated using a 5-fold cross-validation with the K-means clustering method. Genomic accuracy was measured as the genomic correlation between the molecular breeding value and the individual weighted response variables (EBV, DEBVincPA, or DEBVexcPA). The number of identified informative windows (1 Mb) for BH, BL, and TTN was 4, 3, and 4, respectively. The number of significant SNPs for BH, BL, and TTN was 6, 4, and 5, respectively. Diversity π did not influence the accuracy of genomic prediction. The BayesB method showed slightly higher genomic accuracy for exterior traits than BayesC method in this study. In addition, the genomic accuracy using DEBVincPA as response variable was higher than that using other response variables. Therefore, the genomic accuracy using BayesB (π = 0.90) with DEBVinPA as a response variable was the most effective in this study. The genomic accuracy values for BH, BL, and TTN were calculated to be 0.52, 0.60, and 0.51, respectively.

Molecular Characterization of Fluoroquinolone Resistance Mechanisms of Campylobacter Isolates from Duck Meats.

The purpose of this study was to identify the molecular basis of quinolone resistance of Campylobacter isolates recovered from duck meats. Sixty-one isolates from duck meat samples were studied using sequence analysis of the gyrA gene, and PCR assays were used to identify the presence of the CmeABC efflux pump and its restored sensitivity in the presence of efflux-pump inhibitors. High-level resistance to nalidixic acid and ciprofloxacin was attributed to amino acid substitutions Thr-86-Ile in some isolates. The PCR assay confirmed the presence of the cmeB gene in 29 (47.5%) of the 61 Campylobacter isolates. Phenylalanine arginine ß-naphthylamide reduced the MICs of ciprofloxacin and nalidixic acid in 16 (55.2%) and 26 (89.7%) isolates, respectively. The Thr-86-Ile substitution in the gyrA was the primary contributor to the high-level quinolone resistance in Campylobacter isolates from duck meats.

Optimization of a series of heterocycles as survival motor neuron gene transcription enhancers.

Spinal muscular atrophy (SMA) is a neurodegenerative disorder that results from mutations in the SMN1 gene, leading to survival motor neuron (SMN) protein deficiency. One therapeutic strategy for SMA is to identify compounds that enhance the expression of the SMN2 gene, which normally only is a minor contributor to functional SMN protein production, but which is unaffected in SMA. A recent high-throughput screening campaign identified a 3,4-dihydro-4-phenyl-2(1H)-quinolinone derivative (2) that increases the expression of SMN2 by 2-fold with an EC50 = 8.3 µM. A structure-activity relationship (SAR) study revealed that the array of tolerated substituents, on either the benzo portion of the quinolinone or the 4-phenyl, was very narrow. However, the lactam ring of the quinolinone was more amenable to modifications. For example, the quinazolinone (9a) and the benzoxazepin-2(3H)-one (19) demonstrated improved potency and efficacy for increase in SMN2 expression as compared to 2.

Large-scale machine learning of media outlets for understanding public reactions to nation-wide viral infection outbreaks.

From May to July 2015, there was a nation-wide outbreak of Middle East respiratory syndrome (MERS) in Korea. MERS is caused by MERS-CoV, an enveloped, positive-sense, single-stranded RNA virus belonging to the family Coronaviridae. Despite expert opinions that the danger of MERS might be exaggerated, there was an overreaction by the public according to the Korean mass media, which led to a noticeable reduction in social and economic activities during the outbreak. To explain this phenomenon, we presumed that machine learning-based analysis of media outlets would be helpful and collected a number of Korean mass media articles and short-text comments produced during the 10-week outbreak. To process and analyze the collected data (over 86 million words in total) effectively, we created a methodology composed of machine-learning and information-theoretic approaches. Our proposal included techniques for extracting emotions from emoticons and Internet slang, which allowed us to significantly (approximately 73%) increase the number of emotion-bearing texts needed for robust sentiment analysis of social media. As a result, we discovered a plausible explanation for the public overreaction to MERS in terms of the interplay between the disease, mass media, and public emotions.

Discovery of a Small Molecule Probe That Post-Translationally Stabilizes the Survival Motor Neuron Protein for the Treatment of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant death. We previously developed a high-throughput assay that employs an SMN2-luciferase reporter allowing identification of compounds that act transcriptionally, enhance exon recognition, or stabilize the SMN protein. We describe optimization and characterization of an analog suitable for in vivo testing. Initially, we identified analog 4m that had good in vitro properties but low plasma and brain exposure in a mouse PK experiment due to short plasma stability; this was overcome by reversing the amide bond and changing the heterocycle. Thiazole 27 showed excellent in vitro properties and a promising mouse PK profile, making it suitable for in vivo testing. This series post-translationally stabilizes the SMN protein, unrelated to global proteasome or autophagy inhibition, revealing a novel therapeutic mechanism that should complement other modalities for treatment of SMA.

Structure-activity relationship of 3,5-diaryl-2-aminopyridine ALK2 inhibitors reveals unaltered binding affinity for fibrodysplasia ossificans progressiva causing mutants.

There are currently no effective therapies for fibrodysplasia ossificans progressiva (FOP), a debilitating and progressive heterotopic ossification disease caused by activating mutations of ACVR1 encoding the BMP type I receptor kinase ALK2. Recently, a subset of these same mutations of ACVR1 have been identified in diffuse intrinsic pontine glioma (DIPG) tumors. Here we describe the structure-activity relationship for a series of novel ALK2 inhibitors based on the 2-aminopyridine compound K02288. Several modifications increased potency in kinase, thermal shift, or cell-based assays of BMP signaling and transcription, as well as selectivity for ALK2 versus closely related BMP and TGF-ß type I receptor kinases. Compounds in this series exhibited a wide range of in vitro cytotoxicity that was not correlated with potency or selectivity, suggesting mechanisms independent of BMP or TGF-ß inhibition. The study also highlights a potent 2-methylpyridine derivative 10 (LDN-214117) with a high degree of selectivity for ALK2 and low cytotoxicity that could provide a template for preclinical development. Contrary to the notion that activating mutations of ALK2 might alter inhibitor efficacy due to potential conformational changes in the ATP-binding site, the compounds demonstrated consistent binding to a panel of mutant and wild-type ALK2 proteins. Thus, BMP inhibitors identified via activity against wild-type ALK2 signaling are likely to be of clinical relevance for the diverse ALK2 mutant proteins associated with FOP and DIPG.

Enhancement of SMN protein levels in a mouse model of spinal muscular atrophy using novel drug-like compounds.

Spinal muscular atrophy (SMA) is a neurodegenerative disease that causes progressive muscle weakness, which primarily targets proximal muscles. About 95% of SMA cases are caused by the loss of both copies of the SMN1 gene. SMN2 is a nearly identical copy of SMN1, which expresses much less functional SMN protein. SMN2 is unable to fully compensate for the loss of SMN1 in motor neurons but does provide an excellent target for therapeutic intervention. Increased expression of functional full-length SMN protein from the endogenous SMN2 gene should lessen disease severity. We have developed and implemented a new high-throughput screening assay to identify small molecules that increase the expression of full-length SMN from a SMN2 reporter gene. Here, we characterize two novel compounds that increased SMN protein levels in both reporter cells and SMA fibroblasts and show that one increases lifespan, motor function, and SMN protein levels in a severe mouse model of SMA.

Optimization of tricyclic Nec-3 necroptosis inhibitors for in vitro liver microsomal stability.

Necroptosis is a regulated caspase-independent cell death pathway with morphological features resembling passive non-regulated necrosis. Several diverse structure classes of necroptosis inhibitors have been reported to date, including a series of 3,3a,4,5-tetrahydro-2H-benz[g]indazoles (referred to as the Nec-3 series) displaying potent activity in cellular assays. However, evaluation of the tricyclic necroptosis inhibitor's stability in mouse liver microsomes indicated that they were rapidly degraded. A structure-activity relationship (SAR) study of this compound series revealed that increased liver microsomal stability could be accomplished by modification of the pendent phenyl ring and by introduction of a hydrophilic substituent (i.e., α-hydroxyl) to the acetamide at the 2-position of the tricyclic ring without significantly compromising necroptosis inhibitory activity. Further increases in microsomal stability could be achieved by utilizing the 5,5-dioxo-3-phenyl-2,3,3a,4-tetrahydro-[1]benzothiopyrano[4,3-c]pyrazoles. However, in this case necroptosis inhibitory activity was not maintained. Overall, these results provide a strategy for generating potent and metabolically stable tricyclic necrostatin analogs (e.g., 33, LDN-193191) potentially suitable for in vivo studies.

Pharmacologic inhibition of the anaphase-promoting complex induces a spindle checkpoint-dependent mitotic arrest in the absence of spindle damage.

Microtubule inhibitors are important cancer drugs that induce mitotic arrest by activating the spindle assembly checkpoint (SAC), which, in turn, inhibits the ubiquitin ligase activity of the anaphase-promoting complex (APC). Here, we report a small molecule, tosyl-L-arginine methyl ester (TAME), which binds to the APC and prevents its activation by Cdc20 and Cdh1. A prodrug of TAME arrests cells in metaphase without perturbing the spindle, but nonetheless the arrest is dependent on the SAC. Metaphase arrest induced by a proteasome inhibitor is also SAC dependent, suggesting that APC-dependent proteolysis is required to inactivate the SAC. We propose that mutual antagonism between the APC and the SAC yields a positive feedback loop that amplifies the ability of TAME to induce mitotic arrest.

Designed inhibitors of insulin-degrading enzyme regulate the catabolism and activity of insulin.

BACKGROUND: Insulin is a vital peptide hormone that is a central regulator of glucose homeostasis, and impairments in insulin signaling cause diabetes mellitus. In principle, it should be possible to enhance the activity of insulin by inhibiting its catabolism, which is mediated primarily by insulin-degrading enzyme (IDE), a structurally and evolutionarily distinctive zinc-metalloprotease. Despite interest in pharmacological inhibition of IDE as an attractive anti-diabetic approach dating to the 1950s, potent and selective inhibitors of IDE have not yet emerged. METHODOLOGY/PRINCIPAL FINDINGS: We used a rational design approach based on analysis of combinatorial peptide mixtures and focused compound libraries to develop novel peptide hydroxamic acid inhibitors of IDE. The resulting compounds are approximately 10(6) times more potent than existing inhibitors, non-toxic, and surprisingly selective for IDE vis-à-vis conventional zinc-metalloproteases. Crystallographic analysis of an IDE-inhibitor complex reveals a novel mode of inhibition based on stabilization of IDE's "closed," inactive conformation. We show further that pharmacological inhibition of IDE potentiates insulin signaling by a mechanism involving reduced catabolism of internalized insulin. CONCLUSIONS/SIGNIFICANCE: The inhibitors we describe are the first to potently and selectively inhibit IDE or indeed any member of this atypical zinc-metalloprotease superfamily. The distinctive structure of IDE's active site, and the mode of action of our inhibitors, suggests that it may be possible to develop inhibitors that cross-react minimally with conventional zinc-metalloproteases. Significantly, our results reveal that insulin signaling is normally regulated by IDE activity not only extracellularly but also within cells, supporting the longstanding view that IDE inhibitors could hold therapeutic value for the treatment of diabetes.

Improving binding specificity of pharmacological chaperones that target mutant superoxide dismutase-1 linked to familial amyotrophic lateral sclerosis using computational methods.

We recently described a set of drug-like molecules obtained from an in silico screen that stabilize mutant superoxide dismutase-1 (SOD-1) linked to familial amyotrophic lateral sclerosis (ALS) against unfolding and aggregation but exhibited poor binding specificity toward SOD-1 in presence of blood plasma. A reasonable but not a conclusive model for the binding of these molecules was proposed on the basis of restricted docking calculations and site-directed mutagenesis of key residues at the dimer interface. A set of hydrogen bonding constraints obtained from these experiments were used to guide docking calculations with compound library around the dimer interface. A series of chemically unrelated hits were predicted, which were experimentally tested for their ability to block aggregation. At least six of the new molecules exhibited high specificity of binding toward SOD-1 in the presence of blood plasma. These molecules represent a new class of molecules for further development into clinical candidates.

Structure-activity relationship study of EphB3 receptor tyrosine kinase inhibitors.

A structure-activity relationship study for a 2-chloroanilide derivative of pyrazolo[1,5-a]pyridine revealed that increased EphB3 kinase inhibitory activity could be accomplished by retaining the 2-chloroanilide and introducing a phenyl or small electron donating substituents to the 5-position of the pyrazolo[1,5-a]pyridine. In addition, replacement of the pyrazolo[1,5-a]pyridine with imidazo[1,2-a]pyridine was well tolerated and resulted in enhanced mouse liver microsome stability. The structure-activity relationship for EphB3 inhibition of both heterocyclic series was similar. Kinase inhibitory activity was also demonstrated for representative analogs in cell culture. An analog (32, LDN-211904) was also profiled for inhibitory activity against a panel of 288 kinases and found to be quite selective for tyrosine kinases. Overall, these studies provide useful molecular probes for examining the in vitro, cellular and potentially in vivo kinase-dependent function of EphB3 receptor.

Kinetic studies of Cdk5/p25 kinase: phosphorylation of tau and complex inhibition by two prototype inhibitors.

Cdk5/p25 is a member of the family of cyclin-dependent, Ser/Thr kinases and is thought to play a causal role in Alzheimer's disease (AD) due to its ability to phosphorylate the protein tau, and thus promote the latter's aggregation into intraneuronal tangles. Given this, we and others are seeking inhibitors of cdk5/p25 as possible disease-modifying therapeutics for AD. In this paper, we first report the kinetic mechanism for the cdk5/p25-catalyzed phosphorylation of tau and histone H-1-derived peptide (H1P). These studies served as a necessary kinetic backdrop for investigations of the mechanism of inhibition by prototype inhibitors N4-(6-aminopyrimidin-4-yl)-sulfanilamide (APS) and 1-(5-cyclobutyl-thiazol-2-yl)-3-isoquinolin-5-yl-urea (CTIU). We found that the cdk5/p25-catalyzed phosphorylation of tau follows a rapid equilibrium, random kinetic mechanism, as evidenced by initial velocity analysis indicating sequential addition of tau and ATP, and studies of the mechanism of inhibition by substrate analogue AMP, product ADP, and analogues of peptide substrate H1P. Identical mechanistic conclusions were drawn when H1P was the phosphoryl acceptor. Subsequent studies of inhibition by APS and CTIU revealed that both compounds can bind to all four steady-state forms of the enzyme, to form the complexes E:I, E:I:tau, E:I:ATP, and E:I:tau:ATP. These results contrast with reported claims that APS and CTIU are competitive inhibitors of the binding of ATP.

Structure-activity relationship study of tricyclic necroptosis inhibitors.

Necroptosis is a regulated caspase-independent cell death mechanism that can be induced in multiple cell types and is characterized by morphological features resembling necrosis. Here we describe a series of tricyclic heterocycles (i.e., 3-phenyl-3,3a,4,5-tetrahydro-2H-benz[g]indazoles, 3-phenyl-2,3,3a,4-tetrahydro[1]benzopyrano[4,3-c]pyrazoles, 3-phenyl-2,3,3a,4-tetrahydro[1]benzothiopyrano[4,3-c]pyrazoles, and 5,5-dioxo-3-phenyl-2,3,3a,4-tetrahydro[1]benzothiopyrano[4,3-c]pyrazoles], collectively termed Nec-3, that can potently inhibit necroptosis. For example, compounds 8, 22, 41, 53, and 55 inhibit necroptosis in an FADD-deficient variant of human Jurkat T cells treated for 24 h with TNF-alpha with EC50 values in the range 0.15-0.29 microM. Distinct from the previously described series of hydantoin-containing indole derivatives (Nec-1), the Nec-3 series exhibits specificity in inhibiting TNF-alpha-induced necroptosis. A structure-activity relationship (SAR) study revealed that the (3R,3aR)-rel-diastereomers were more active than the (3R,3aS)-rel-diastereomers for all four ring systems. Introduction of fluorine or methoxy to the 8-position of the tricyclic ring and a methoxy to the 4-position of the pendent phenyl ring increased activity. Amides at the 2-position of the tricyclic ring were best. The Nec-3 series provides new tools for elucidating caspase-independent cell death pathways and potentially lead compounds for therapeutic development.

Fully degradable hydrophilic polyals for protein modification.

Modification of proteins with hydrophilic polymers is an effective strategy for regulation of protein pharmacokinetics. However, conjugates of slowly or non-biodegradable materials, such as poly(ethylene glycol), are known to cause long-lasting cell vacuolization, in particular in renal epithelium. Conjugates of more degradable polymers, e.g., polysaccharides, have a significant risk of immunotoxicity. Polymers that combine complete degradability, long circulation in vivo, and low immuno and chemical toxicity would be most beneficial as protein conjugate components. This study explores new fully biodegradable hydrophilic polymers, hydrophilic polyals. They are nontoxic, stable at physiological conditions, and undergo proton-catalyzed hydrolysis at lysosomal pH. The model enzyme-polyal conjugates were prepared with 61-98% yield using conventional and novel conjugation techniques and retained 90-95% of specific activity. The model conjugates showed a significant prolongation of protein circulation in rodents, with a 5-fold reduction in the renal accumulation. The data suggests that hydrophilic polyals may be useful in designing protein conjugates with improved properties.

Structure-activity relationship study of novel necroptosis inhibitors.

Necroptosis is a regulated caspase-independent cell death mechanism that results in morphological features resembling necrosis. It can be induced in a FADD-deficient variant of human Jurkat T cells treated with TNF-alpha. 5-(1H-Indol-3-ylmethyl)-2-thiohydantoins and 5-(1H-indol-3-ylmethyl)hydantoins were found to be potent necroptosis inhibitors (called necrostatins). A SAR study revealed that several positions of the indole were intolerant of substitution, while small substituents at the 7-position resulted in increased inhibitory activity. The hydantoin ring was also quite sensitive to structural modifications. A representative member of this compound class demonstrated moderate pharmacokinetic characteristics and readily entered the central nervous system upon intravenous administration.