Discovery of a CK2α'-Biased ATP-Competitive Inhibitor from a High-Throughput Screen of an Allosteric-Inhibitor-Like Compound Library.

Protein kinase CK2 is a holoenzyme composed of two regulatory subunits (CK2ß) and two catalytic subunits (CK2α and CK2α'). CK2 controls several cellular processes, including proliferation, inflammation, and cell death. However, CK2α and CK2α' possess different expression patterns and substrates and therefore impact each of these processes differently. Elevated CK2α participates in the development of cancer, while increased CK2α' has been associated with neurodegeneration, especially Huntington's disease (HD). HD is a fatal disease for which no effective therapies are available. Genetic deletion of CK2α' in HD mouse models has ameliorated neurodegeneration. Therefore, pharmacological inhibition of CK2α' presents a promising therapeutic strategy for treating HD. However, current CK2 inhibitors are unable to discriminate between CK2α and CK2α' due to their high structural homology, especially in the targeted ATP-binding site. Using computational analyses, we found a potential type IV ("D" pocket) allosteric site that contained different residues between CK2α and CK2α' and was distal from the ATP-binding pocket featured in both kinases. We decided to look for allosteric modulators that might interact in a biased fashion with the type IV pocket on both CK2α and CK2α'. We screened a commercial library containing ∼29,000 allosteric-kinase-inhibitor-like compounds using a CK2α' activity-dependent ADP-Glo Kinase assay. Obtained hits were counter-screened against CK2α using the ADP-Glo Kinase assay, revealing two CK2α'-biased compounds. These two compounds might serve as the basis for further medicinal chemistry optimization for the potential treatment of HD.

Development of Allosteric Small Molecule APOBEC3B Inhibitors from In Silico Screening.

APOBEC3B cytosine deaminase contributes to the mutational burdens of tumors, resulting in tumor progression and therapy resistance. Small molecule APOBEC3B inhibitors have potential to slow or mitigate these detrimental outcomes. Through molecular dynamics (MD) simulations and computational solvent mapping analysis, we identified a novel putative allosteric pocket on the C-terminal domain of APOBEC3B (A3Bctd), and virtually screened the ChemBridge Diversity Set (N~110,000) against both the active and potential allosteric sites. Selected high-scoring compounds were subsequently purchased, characterized for purity and composition, and tested in biochemical assays, which yielded 13 hit compounds. Orthogonal NMR assays verified binding to the target protein. Initial selectivity studies suggest these compounds preferentially target A3Bctd over related deaminase APOBEC3A (A3A), and MD simulations indicate this selectivity may be due to the steric repulsion from H56 that is unique to A3A. Taken together, our studies represent the first virtual screening effort against A3Bctd that has yielded candidate inhibitors suitable for further development.

Identification of CK2α' selective inhibitors by the screening of an allosteric-kinase-inhibitor-like compound library.

Protein Kinase CK2 is a holoenzyme composed of two regulatory subunits (CK2ß) and two catalytic subunits (CK2α and CK2α'). CK2 controls several cellular processes including proliferation, inflammation, and cell death. However, CK2α and CK2α' possess different expression patterns and substrates and therefore impact each of these processes differently. Elevated CK2α participates in the development of cancer, while increased CK2α' has been associated with neurodegeneration, especially Huntington's disease (HD). HD is a fatal disease for which no effective therapies are available. Genetic deletion of CK2α' in HD mouse models has ameliorated neurodegeneration. Therefore, pharmacological inhibition of CK2α' presents a promising therapeutic strategy for treating HD. However, current CK2 inhibitors are unable to discriminate between CK2α and CK2α' due to their high structural homology, especially in the targeted ATP binding site. Using computational analyses, we found a potential Type IV ("D" pocket) allosteric site on CK2α' that contained different residues than CK2α and was distal from the ATP binding pocket featured in both kinases. With this potential allosteric site in mind, we screened a commercial library containing ~29,000 allosteric-kinase-inhibitor-like compounds using a CK2α' activity-dependent ADP-Glo™ Kinase assay. Obtained hits were counter-screened against CK2α revealing two CK2α' selective compounds. These two compounds might serve as the basis for further medicinal chemistry optimization for the potential treatment of HD.

Synthesis of Guaianolide Analogues with a Tunable α-Methylene-γ-lactam Electrophile and Correlating Bioactivity with Thiol Reactivity.

α-Methylene-γ-lactones are present in ∼3% of known natural products, and compounds comprising this motif display a range of biological activities. However, this reactive lactone limits informed structure-activity relationships for these bioactive molecules. Herein, we describe chemically tuning the electrophilicity of the α-methylene-γ-lactone by replacement with an α-methylene-γ-lactam. Guaianolide analogues having α-methylene-γ-lactams are synthesized using the allenic Pauson-Khand reaction. Substitution of the lactam nitrogen with electronically different groups affords diverse thiol reactivity. Cellular NF-κB inhibition assays for these lactams were benchmarked against parthenolide and a synthetic α-methylene-γ-lactone showing a positive correlation between thiol reactivity and bioactivity. Cytotoxicity assays show good correlation at the outer limits of thiol reactivity but less so for compounds with intermediate reactivity. A La assay to detect reactive molecules by nuclear magnetic resonance and mass spectrometry peptide sequencing assays with the La antigen protein demonstrate that lactam analogues with muted nonspecific thiol reactivities constitute a better electrophile for rational chemical probe and therapeutic molecule design.

Discovery of Acylsulfonohydrazide-Derived Inhibitors of the Lysine Acetyltransferase, KAT6A, as Potent Senescence-Inducing Anti-Cancer Agents.

A high-throughput screen designed to discover new inhibitors of histone acetyltransferase KAT6A uncovered CTX-0124143 (1), a unique aryl acylsulfonohydrazide with an IC50 of 1.0 µM. Using this acylsulfonohydrazide as a template, we herein disclose the results of our extensive structure-activity relationship investigations, which resulted in the discovery of advanced compounds such as 55 and 80. These two compounds represent significant improvements on our recently reported prototypical lead WM-8014 (3) as they are not only equivalently potent as inhibitors of KAT6A but are less lipophilic and significantly more stable to microsomal degradation. Furthermore, during this process, we discovered a distinct structural subclass that contains key 2-fluorobenzenesulfonyl and phenylpyridine motifs, culminating in the discovery of WM-1119 (4). This compound is a highly potent KAT6A inhibitor (IC50 = 6.3 nM; KD = 0.002 µM), competes with Ac-CoA by binding to the Ac-CoA binding site, and has an oral bioavailability of 56% in rats.

Development of Benzenesulfonamide Derivatives as Potent Glutathione Transferase Omega-1 Inhibitors.

Glutathione transferase omega-1 (GSTO1-1) is an enzyme whose function supports the activation of interleukin (IL)-1ß and IL-18 that are implicated in a variety of inflammatory disease states for which small-molecule inhibitors are sought. The potent reactivity of the active-site cysteine has resulted in reported inhibitors that act by covalent labeling. In this study, structure-activity relationship (SAR) elaboration of the reported GSTO1-1 inhibitor C1-27 was undertaken. Compounds were evaluated for inhibitory activity toward purified recombinant GSTO1-1 and for indicators of target engagement in cell-based assays. As covalent inhibitors, the kinact/KI values of selected compounds were determined, as well as in vivo pharmacokinetics analysis. Cocrystal structures of key novel compounds in complex with GSTO1-1 were also solved. This study represents the first application of a biochemical assay for GSTO1-1 to determine kinact/KI values for tested inhibitors and the most extensive set of cell-based data for a GSTO1-1 inhibitor SAR series reported to date. Our research culminated in the discovery of 25, which we propose as the preferred biochemical tool to interrogate cellular responses to GSTO1-1 inhibition.

Anastrozole has an Association between Degree of Estrogen Suppression and Outcomes in Early Breast Cancer and is a Ligand for Estrogen Receptor α.

PURPOSE: To determine if the degree of estrogen suppression with aromatase inhibitors (AI: anastrozole, exemestane, letrozole) is associated with efficacy in early-stage breast cancer, and to examine for differences in the mechanism of action between the three AIs. EXPERIMENTAL DESIGN: Matched case-control studies [247 matched sets from MA.27 (anastrozole vs. exemestane) and PreFace (letrozole) trials] were undertaken to assess whether estrone (E1) or estradiol (E2) concentrations after 6 months of adjuvant therapy were associated with risk of an early breast cancer event (EBCE). Preclinical laboratory studies included luciferase activity, cell proliferation, radio-labeled ligand estrogen receptor binding, surface plasmon resonance ligand receptor binding, and nuclear magnetic resonance assays. RESULTS: Women with E1 ≥1.3 pg/mL and E2 ≥0.5 pg/mL after 6 months of AI treatment had a 2.2-fold increase in risk (P = 0.0005) of an EBCE, and in the anastrozole subgroup, the increase in risk of an EBCE was 3.0-fold (P = 0.001). Preclinical laboratory studies examined mechanisms of action in addition to aromatase inhibition and showed that only anastrozole could directly bind to estrogen receptor α (ERα), activate estrogen response element-dependent transcription, and stimulate growth of an aromatase-deficient CYP19A1-/- T47D breast cancer cell line. CONCLUSIONS: This matched case-control clinical study revealed that levels of estrone and estradiol above identified thresholds after 6 months of adjuvant anastrozole treatment were associated with increased risk of an EBCE. Preclinical laboratory studies revealed that anastrozole, but not exemestane or letrozole, is a ligand for ERα. These findings represent potential steps towards individualized anastrozole therapy.

Cytotoxic unsaturated electrophilic compounds commonly target the ubiquitin proteasome system.

A large number of natural products have been advocated as anticancer agents. Many of these compounds contain functional groups characterized by chemical reactivity. It is not clear whether distinct mechanisms of action can be attributed to such compounds. We used a chemical library screening approach to demonstrate that a substantial fraction (~20%) of cytotoxic synthetic compounds containing Michael acceptor groups inhibit proteasome substrate processing and induce a cellular response characteristic of proteasome inhibition. Biochemical and structural analyses showed binding to and inhibition of proteasome-associated cysteine deubiquitinases, in particular ubiquitin specific peptidase 14 (USP14). The results suggested that compounds bind to a crevice close to the USP14 active site with modest affinity, followed by covalent binding. A subset of compounds was identified where cell death induction was closely associated with proteasome inhibition and that showed significant antineoplastic activity in a zebrafish embryo model. These findings suggest that proteasome inhibition is a relatively common mode of action by cytotoxic compounds containing Michael acceptor groups and help to explain previous reports on the antineoplastic effects of natural products containing such functional groups.

Reduction of protein kinase A-mediated phosphorylation of ATXN1-S776 in Purkinje cells delays onset of Ataxia in a SCA1 mouse model.

Spinocerebellar ataxia type 1 (SCA1) is a polyglutamine (polyQ) repeat neurodegenerative disease in which a primary site of pathogenesis are cerebellar Purkinje cells. In addition to polyQ expansion of ataxin-1 protein (ATXN1), phosphorylation of ATXN1 at the serine 776 residue (ATXN1-pS776) plays a significant role in protein toxicity. Utilizing a biochemical approach, pharmacological agents and cell-based assays, including SCA1 patient iPSC-derived neurons, we examine the role of Protein Kinase A (PKA) as an effector of ATXN1-S776 phosphorylation. We further examine the implications of PKA-mediated phosphorylation at ATXN1-S776 on SCA1 through genetic manipulation of the PKA catalytic subunit Cα in Pcp2-ATXN1[82Q] mice. Here we show that pharmacologic inhibition of S776 phosphorylation in transfected cells and SCA1 patient iPSC-derived neuronal cells lead to a decrease in ATXN1. In vivo, reduction of PKA-mediated ATXN1-pS776 results in enhanced degradation of ATXN1 and improved cerebellar-dependent motor performance. These results provide evidence that PKA is a biologically important kinase for ATXN1-pS776 in cerebellar Purkinje cells.

Assay interference and off-target liabilities of reported histone acetyltransferase inhibitors.

Many compounds with potentially reactive chemical motifs and poor physicochemical properties are published as selective modulators of biomolecules without sufficient validation and then propagated in the scientific literature as useful chemical probes. Several histone acetyltransferase (HAT) inhibitors with these liabilities are now routinely used to probe epigenetic pathways. We profile the most commonly used HAT inhibitors and confirm that the majority of them are nonselective interference compounds. Most (15 out of 23, 65%) of the inhibitors are flagged by ALARM NMR, an industry-developed counter-screen for promiscuous compounds. Biochemical counter-screens confirm that most of these compounds are either thiol-reactive or aggregators. Selectivity panels show many of these compounds modulate unrelated targets in vitro, while several also demonstrate nonspecific effects in cell assays. These data demonstrate the usefulness of performing counter-screens for bioassay promiscuity and assay interference, and raise caution about the utility of many widely used, but insufficiently validated, compounds employed in chemical biology.

Uncoupling Catalytic and Binding Functions in the Cyclic AMP-Dependent Protein Kinase A.

The canonical function of kinases is to transfer a phosphoryl group to substrates, initiating a signaling cascade; while their non-canonical role is to bind other kinases or substrates, acting as scaffolds, competitors, and signal integrators. Here, we show how to uncouple kinases' dual function by tuning the binding cooperativity between nucleotide (or inhibitors) and substrate allosterically. We demonstrate this new concept for the C subunit of protein kinase A (PKA-C). Using thermocalorimetry and nuclear magnetic resonance, we found a linear correlation between the degree of cooperativity and the population of the closed state of PKA-C. The non-hydrolyzable ATP analog (ATPγC) does not follow this correlation, suggesting that changing the chemical groups around the phosphoester bond can uncouple kinases' dual function. Remarkably, this uncoupling was also found for two ATP-competitive inhibitors, H89 and balanol. Since the mechanism for allosteric cooperativity is not conserved in different kinases, these results may suggest new approaches for designing selective kinase inhibitors.

Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast.

Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast "knock-out/knock-in" screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival.

PAINS in the assay: chemical mechanisms of assay interference and promiscuous enzymatic inhibition observed during a sulfhydryl-scavenging HTS.

Significant resources in early drug discovery are spent unknowingly pursuing artifacts and promiscuous bioactive compounds, while understanding the chemical basis for these adverse behaviors often goes unexplored in pursuit of lead compounds. Nearly all the hits from our recent sulfhydryl-scavenging high-throughput screen (HTS) targeting the histone acetyltransferase Rtt109 were such compounds. Herein, we characterize the chemical basis for assay interference and promiscuous enzymatic inhibition for several prominent chemotypes identified by this HTS, including some pan-assay interference compounds (PAINS). Protein mass spectrometry and ALARM NMR confirmed these compounds react covalently with cysteines on multiple proteins. Unfortunately, compounds containing these chemotypes have been published as screening actives in reputable journals and even touted as chemical probes or preclinical candidates. Our detailed characterization and identification of such thiol-reactive chemotypes should accelerate triage of nuisance compounds, guide screening library design, and prevent follow-up on undesirable chemical matter.

Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases.

During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies.

Anthrax toxin lethal factor domain 3 is highly mobile and responsive to ligand binding.

The secreted anthrax toxin consists of three components: the protective antigen (PA), edema factor (EF) and lethal factor (LF). LF, a zinc metalloproteinase, compromises the host immune system primarily by targeting mitogen-activated protein kinase kinases in macrophages. Peptide substrates and small-molecule inhibitors bind LF in the space between domains 3 and 4 of the hydrolase. Domain 3 is attached on a hinge to domain 2 via residues Ile300 and Pro385, and can move through an angular arc of greater than 35° in response to the binding of different ligands. Here, multiple LF structures including five new complexes with co-crystallized inhibitors are compared and three frequently populated LF conformational states termed `bioactive', `open' and `tight' are identified. The bioactive position is observed with large substrate peptides and leaves all peptide-recognition subsites open and accessible. The tight state is seen in unliganded and small-molecule complex structures. In this state, domain 3 is clamped over certain substrate subsites, blocking access. The open position appears to be an intermediate state between these extremes and is observed owing to steric constraints imposed by specific bound ligands. The tight conformation may be the lowest-energy conformation among the reported structures, as it is the position observed with no bound ligand, while the open and bioactive conformations are likely to be ligand-induced.

Specific inhibition of p97/VCP ATPase and kinetic analysis demonstrate interaction between D1 and D2 ATPase domains.

The p97 AAA (ATPase associated with diverse cellular activities), also called VCP (valosin-containing protein), is an important therapeutic target for cancer and neurodegenerative diseases. p97 forms a hexamer composed of two AAA domains (D1 and D2) that form two stacked rings and an N-terminal domain that binds numerous cofactor proteins. The interplay between the three domains in p97 is complex, and a deeper biochemical understanding is needed in order to design selective p97 inhibitors as therapeutic agents. It is clear that the D2 ATPase domain hydrolyzes ATP in vitro, but whether D1 contributes to ATPase activity is controversial. Here, we use Walker A and B mutants to demonstrate that D1 is capable of hydrolyzing ATP and show for the first time that nucleotide binding in the D2 domain increases the catalytic efficiency (kcat/Km) of D1 ATP hydrolysis 280-fold, by increasing kcat 7-fold and decreasing Km about 40-fold. We further show that an ND1 construct lacking D2 but including the linker between D1 and D2 is catalytically active, resolving a conflict in the literature. Applying enzymatic observations to small-molecule inhibitors, we show that four p97 inhibitors (DBeQ, ML240, ML241, and NMS-873) have differential responses to Walker A and B mutations, to disease-causing IBMPFD mutations, and to the presence of the N domain binding cofactor protein p47. These differential effects provide the first evidence that p97 cofactors and disease mutations can alter p97 inhibitor potency and suggest the possibility of developing context-dependent inhibitors of p97.

Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins.

Antiapoptotic Bcl-2 family proteins are validated cancer targets composed of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). Although several isoform-selective inhibitors have been developed using structure-based design or high-throughput screening (HTS) of synthetic chemical libraries, no large-scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six antiapoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally relevant PPIs. The screens were conducted in 1536-well format and displayed satisfactory overall HTS statistics, with Z'-factor values ranging from 0.72 to 0.83 and a hit confirmation rate between 16% and 64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied, and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source, and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra-high-throughput screening using natural product sources and highlight some of the challenges associated with this approach.

From HTS to Phase I: the Institute for Therapeutics Discovery and Development at the University of Minnesota.

The high-throughput screening core at the University of Minnesota is part of the Institute for Therapeutics Discovery and Development (ITDD), a comprehensive drug discovery and development center. The Institute provides scientific services to both academic and business communities and supports translational medicine via collaborations and contractual work. The ITDD is well-known for its broad range of screening capabilities and offers extensive medicinal chemistry expertise along with GMP scale-up and pre-clinical pharmacology support.

Synthesis of novel analogs of cabergoline: improving cardiovascular safety by removing 5-HT2B receptor agonism.

The dopamine agonist cabergoline has been used to treat prolactinomas, Parkinson's disease, Cushing's disease and sexual dysfunction. However, its clinical use was severely curtailed when it was found that patients taking cabergoline had an increased risk of developing cardiac-valve regurgitation. This potentially life-threatening condition has been associated with drugs, such as cabergoline, that are 5-HT2B receptor agonists. We prepared analogs of cabergoline and have identified several that have limited or no agonism at the 5-HT2B receptor.

Hematologic and biochemical changes associated with human T lymphotropic virus type 1 infection in Jamaica: a report from the population-based blood donors study.

OBJECTIVE: We investigated changes in hematologic and biochemical parameters associated with human T lymphotropic virus type 1 (HTLV-1) infection, antibody titer, and provirus load. Additionally, on a subset of participants, we assessed the epidemiologic relationship of HTLV-1 with Strongyloides stercoralis. METHODS: Among volunteer blood donors in Jamaica, HTLV-1 carriers (n=482) were frequency matched with HTLV-1 negative subjects (n=355) by age (+/-5 years), sex, and date of blood donation (+/-3 months). HTLV-1 antibody titer, provirus load, S. stercoralis IgG antibodies, complete blood cell count, blood chemistry, and urinalysis parameters were measured. RESULTS: HTLV-1 carriers, compared with HTLV-1-negative individuals, had elevated levels of cleaved lymphocytes (24.5% vs. 16.4%), any lymphocyte abnormalities (atypical, cleaved, and reactive lymphocytes combined, 45.7% vs. 35.4%), and gamma-glutamyl transferase levels (21.2 vs. 19.6 IU/L), as well as lower eosinophil count (2.6% vs. 3.1%). Among carriers, HTLV-1 antibody titer (n=482) was inversely correlated with mean corpuscular volume (r=-0.10) and positively correlated with levels of total protein (r=0.16), phosphorus (r=0.12), and lactate dehydrogenase (r=0.24). HTLV-1-provirus load (n=326) was higher among carriers with cleaved lymphocytes and any lymphocyte abnormalities. Provirus load was inversely correlated with hemoglobin (r=-0.11), mean corpuscular volume (r=-0.15), neutrophil (r=-0.12), and eosinophil (r=-0.19) levels and was positively correlated with lactate dehydrogenase levels (r=0.12). Provirus load was significantly higher among male than female subjects. S. stercoralis antibodies were detected in 35 (12.1%) of 288 participants but were not associated with HTLV-1 status, antibody titer, or provirus load. CONCLUSIONS: Markers of HTLV-1 infection (infection status, antibody titer, and provirus load) are associated with hematologic and biochemical alterations, such as lymphocyte abnormalities, anemia, decreased eosinophils, and elevated lactate dehydrogenase levels.