Covalent Modification of the JH2 Domain of Janus Kinase 2.

Probe molecules that covalently modify the JAK2 pseudokinase domain (JH2) are reported. Selective targeting of JH2 domains over the kinase (JH1) domains is a necessary feature for ligands intended to evaluate JH2 domains as therapeutic targets. The JH2 domains of three Janus kinases (JAK1, JAK2, and TYK2) possess a cysteine residue in the catalytic loop that does not occur in their JH1 domains. Starting from a non-selective kinase binding molecule, computer-aided design directed attachment of substituents terminating in acrylamide warheads to modify Cys675 of JAK2 JH2. Successful covalent attachment was demonstrated first through observation of enhanced binding with increasing incubation time in fluorescence polarization experiments. Covalent binding also increased selectivity to as much as ca. 30-fold for binding the JAK2 JH2 domain over the JH1 domain after a 20-h incubation. Covalency was confirmed through HPLC electrospray quadrupole time-of-flight HRMS experiments, which revealed the expected mass shifts.

Insights on JAK2 Modulation by Potent, Selective, and Cell-Permeable Pseudokinase-Domain Ligands.

JAK2 is a non-receptor tyrosine kinase that regulates hematopoiesis through the JAK-STAT pathway. The pseudokinase domain (JH2) is an important regulator of the activity of the kinase domain (JH1). V617F mutation in JH2 has been associated with the pathogenesis of various myeloproliferative neoplasms, but JAK2 JH2 has been poorly explored as a pharmacological target. In light of this, we aimed to develop JAK2 JH2 binders that could selectively target JH2 over JH1 and test their capacity to modulate JAK2 activity in cells. Toward this goal, we optimized a diaminotriazole lead compound into potent, selective, and cell-permeable JH2 binders leveraging computational design, synthesis, binding affinity measurements for the JH1, JH2 WT, and JH2 V617F domains, permeability measurements, crystallography, and cell assays. Optimized diaminotriazoles are capable of inhibiting STAT5 phosphorylation in both WT and V617F JAK2 in cells.

Conversion of a False Virtual Screen Hit into Selective JAK2 JH2 Domain Binders Using Convergent Design Strategies.

The Janus kinase 2 (JAK2) pseudokinase domain (JH2) is an ATP-binding domain that regulates the activity of the catalytic tyrosine kinase domain (JH1). Dysregulation of JAK2 JH1 signaling caused by the V617F mutation in JH2 is implicated in various myeloproliferative neoplasms. To explore if JAK2 activity can be modulated by a small molecule binding to the ATP site in JH2, we have developed several ligand series aimed at selectively targeting the JAK2 JH2 domain. We report here the evolution of a false virtual screen hit into a new JAK2 JH2 series. Optimization guided by computational modeling has yielded analogues with nanomolar affinity for the JAK2 JH2 domain and >100-fold selectivity for the JH2 domain over the JH1 domain. A crystal structure for one of the potent compounds bound to JAK2 JH2 clarifies the origins of the strong binding and selectivity. The compounds expand the platform for seeking molecules to regulate JAK2 signaling, including V617F JAK2 hyperactivation.

Indoloxytriazines as binding molecules for the JAK2 JH2 pseudokinase domain and its V617F variant.

Small molecules that selectively bind to the pseudokinase JH2 domain over the JH1 kinase domain of JAK2 kinase are sought. Virtual screening led to the purchase of 17 compounds among which 9 were found to bind to V617F JAK2 JH2 with affinities of 40 - 300 µM in a fluorogenic assay. Ten analogues were then purchased yielding 9 additional active compounds. Aminoanilinyltriazine 22 was particularly notable as it shows no detectable binding to JAK2 JH1, and it has a 65-µM dissociation constant K d with V617F JAK2 JH2. A crystal structure for 22 in complex with wild-type JAK2 JH2 was obtained to elucidate the binding mode. Additional de novo design led to the synthesis of 19 analogues of 22 with the most potent being 33n with K d values of 2-3 µM for WT and V617F JAK2 JH2, and with 16-fold selectivity relative to binding with WT JAK2 JH1.

Metadynamics as a Postprocessing Method for Virtual Screening with Application to the Pseudokinase Domain of JAK2.

With standard scoring methods, top-ranked compounds from virtual screening by docking often turn out to be inactive. For this reason, metadynamics, a method used to sample rare events, was studied to further evaluate docking poses with the aim of reducing false positives. Specifically, virtual screening was performed with Glide SP to seek potential molecules to bind to the ATP site in the pseudokinase domain of JAK2 kinase, and promising compounds were selected from the top-ranked 1000 based on visualization. Rescoring with Glide XP, GOLD, and MM/GBSA was unable to differentiate well between active and inactive compounds. Metadynamics was then used to gauge the relative binding affinity from the required time or the potential of mean force needed to dissociate the ligand from the bound complex. With consideration of previously known binders of varying affinities, metadynamics was able to differentiate between the most active compounds and inactive or weakly active ones, and it could identify correctly most of the selected virtual screening compounds as false positives. Thus, metadynamics has the potential to be a viable postprocessing method for virtual screening, minimizing the expense of buying or synthesizing inactive compounds.

Structure-Guided Identification of DNMT3B Inhibitors.

Methyltransferase 3 beta (DNMT3B) inhibitors that interfere with cancer growth are emerging possibilities for treatment of melanoma. Herein we identify small molecule inhibitors of DNMT3B starting from a homology model based on a DNMT3A crystal structure. Virtual screening by docking led to purchase of 15 compounds, among which 5 were found to inhibit the activity of DNMT3B with IC50 values of 13-72 µM in a fluorogenic assay. Eight analogues of 7, 10, and 12 were purchased to provide 2 more active compounds. Compound 11 is particularly notable as it shows good selectivity with no inhibition of DNMT1 and 22 µM potency toward DNMT3B. Following additional de novo design, exploratory synthesis of 17 analogues of 11 delivered 5 additional inhibitors of DNMT3B with the most potent being 33h with an IC50 of 8.0 µM. This result was well confirmed in an ultrahigh-performance liquid chromatography (UHPLC)-based analytical assay, which yielded an IC50 of 4.8 µM. Structure-activity data are rationalized based on computed structures for DNMT3B complexes.

Selective Janus Kinase 2 (JAK2) Pseudokinase Ligands with a Diaminotriazole Core.

Janus kinases (JAKs) are non-receptor tyrosine kinases that are essential components of the JAK-STAT signaling pathway. Associated aberrant signaling is responsible for many forms of cancer and disorders of the immune system. The present focus is on the discovery of molecules that may regulate the activity of JAK2 by selective binding to the JAK2 pseudokinase domain, JH2. Specifically, the Val617Phe mutation in JH2 stimulates the activity of the adjacent kinase domain (JH1) resulting in myeloproliferative disorders. Starting from a non-selective screening hit, we have achieved the goal of discovering molecules that preferentially bind to the ATP binding site in JH2 instead of JH1. We report the design and synthesis of the compounds and binding results for the JH1, JH2, and JH2 V617F domains, as well as five crystal structures for JH2 complexes. Testing with a selective and non-selective JH2 binder on the autophosphorylation of wild-type and V617F JAK2 is also contrasted.

Predictive models for identifying risk of readmission after index hospitalization for heart failure: A systematic review.

AIMS: Readmission rates for patients with heart failure have consistently remained high over the past two decades. As more electronic data, computing power, and newer statistical techniques become available, data-driven care could be achieved by creating predictive models for adverse outcomes such as readmissions. We therefore aimed to review models for predicting risk of readmission for patients admitted for heart failure. We also aimed to analyze and possibly group the predictors used across the models. METHODS: Major electronic databases were searched to identify studies that examined correlation between readmission for heart failure and risk factors using multivariate models. We rigorously followed the review process using PRISMA methodology and other established criteria for quality assessment of the studies. RESULTS: We did a detailed review of 334 papers and found 25 multivariate predictive models built using data from either health system or trials. A majority of models was built using multiple logistic regression followed by Cox proportional hazards regression. Some newer studies ventured into non-parametric and machine learning methods. Overall predictive accuracy with C-statistics ranged from 0.59 to 0.84. We examined significant predictors across the studies using clinical, administrative, and psychosocial groups. CONCLUSIONS: Complex disease management and correspondingly increasing costs for heart failure are driving innovations in building risk prediction models for readmission. Large volumes of diverse electronic data and new statistical methods have improved the predictive power of the models over the past two decades. More work is needed for calibration, external validation, and deployment of such models for clinical use.

Adding a Hydrogen Bond May Not Help: Naphthyridinone vs Quinoline Inhibitors of Macrophage Migration Inhibitory Factor.

Coordination of the ammonium group of Lys32 in the active site of human macrophage migration inhibitory factor (MIF) using a 1,7-naphthyridin-8-one instead of a quinoline is investigated. Both gas- and aqueous-phase DFT calculations for model systems indicate potential benefits for the added hydrogen bond with the lactam carbonyl group, while FEP results are neutral. Three crystal structures are reported for complexes of MIF with 3a, 4a, and 4b, which show that the desired hydrogen bond is formed with O-N distances of 2.8-3.0 Å. Compound 4b is the most potent new MIF inhibitor with Ki and Kd values of 90 and 94 nM; it also has excellent aqueous solubility, 288 µg/mL. Consistent with the FEP results, the naphthyridinones are found to have similar potency as related quinolines in spite of the additional protein-ligand hydrogen bond.

JAK2 JH2 Fluorescence Polarization Assay and Crystal Structures for Complexes with Three Small Molecules.

A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe molecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent 5 and 6 used in the assay are reported as well as Kd results for 10 compounds, including JNJ7706621, NVP-BSK805, and filgotinib (GLPG0634). X-ray crystal structures of JAK2 JH2 in complex with NVP-BSK805, filgotinib, and diaminopyrimidine 8 elucidate the binding poses.

Identification and Characterization of JAK2 Pseudokinase Domain Small Molecule Binders.

Janus kinases (JAKs) regulate hematopoiesis via the cytokine-mediated JAK-STAT signaling pathway. JAKs contain tandem C-terminal pseudokinase (JH2) and tyrosine kinase (JH1) domains. The JAK2 pseudokinase domain adopts a protein kinase fold and, despite its pseudokinase designation, binds ATP with micromolar affinity. Recent evidence shows that displacing ATP from the JAK2 JH2 domain alters the hyperactivation state of the oncogenic JAK2 V617F protein while sparing the wild type JAK2 protein. In this study, small molecule binders of JAK2 JH2 were identified via an in vitro screen. Top hits were characterized using biophysical and structural approaches. Development of pseudokinase-selective compounds may offer novel pharmacological opportunities for treating cancers driven by JAK2 V617F and other oncogenic JAK mutants.

A Validated Risk Model for 30-Day Readmission for Heart Failure.

One of the goals of the Precision Medicine Initiative launched in the United States in 2016 is to use innovative tools and sources in data science. We realized this goal by implementing a use case that identified patients with heart failure at Veterans Health Administration using data from the Electronic Health Records from multiple health domains between 2005 and 2013. We applied a regularized logistic regression model and predicted 30-day readmission risk for 1210 unique patients. Our validation cohort resulted in a C-statistic of 0.84. Our top predictors of readmission were prior diagnosis of heart failure, vascular and renal diseases, and malnutrition as comorbidities, compliance with outpatient follow-up, and low socioeconomic status. This validated risk prediction scheme delivered better performance than the published models so far (C-Statistics: 0.69). It can be used to stratify patients for readmission and to aid clinicians in delivering precise health interventions.

Probing the aurone scaffold against Plasmodium falciparum: design, synthesis and antimalarial activity.

A library comprising 44 diversely substituted aurones derivatives was synthesized by straightforward aldol condensation reactions of benzofuranones and the appropriately substituted benzaldehydes. Microwave enhanced synthesis using palladium catalyzed protocols was introduced as a powerful strategy for extending the chemical space around the aurone scaffold. Additionally, Mannich-base derivatives, containing a 7-aminomethyl-6-hydroxy substitution pattern at ring A, were also prepared. Screening against the chloroquine resistant Plasmodium falciparum W2 strain identified novel aurones with IC50 values in the low micromolar range. The most potent compounds contained a basic moiety, with the ability to accumulate in acidic digestive vacuole of the malaria parasite. However, none of those aurones revealed significant activity against hemozoin formation and falcipain-2, two validated targets expressed during the blood stage of P. falciparum infection and functional in digestive vacuole of the parasite. Overall, this study highlight (i) the usefulness of aurones as platforms for synthetic procedures using palladium catalyzed protocols to rapidly deliver lead compounds for further optimization and (ii) the potential of novel aurone derivatives as promising antimalarial compounds.

Novel anti-plasmodial hits identified by virtual screening of the ZINC database.

Increased resistance of Plasmodium falciparum to most available drugs challenges the control of malaria. Studies with protease inhibitors have suggested important roles for the falcipain family of cysteine proteases. These enzymes act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. In order to find potential new antimalarial drugs, we screened in silico the ZINC database using two different protocols involving structure- and ligand-based methodologies. Our search identified 19 novel low micromolar inhibitors of cultured chloroquine resistant P. falciparum. The most active compound presented an IC50 value of 0.5 µM against cultured parasites and it also inhibited the cysteine protease falcipain-2 (IC50 = 25.5 µM). These results identify novel classes of antimalarials that are structurally different from those currently in use and which can be further derivatized to deliver leads suitable for optimisation.

An endoperoxide-based hybrid approach to deliver falcipain inhibitors inside malaria parasites.

The emergence of artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia has reinforced the urgent need to discover novel chemotherapeutic strategies to treat and control malaria. To address this problem, we prepared a set of dual-acting tetraoxane-based hybrid molecules designed to deliver a falcipain-2 (FP-2) inhibitor upon activation by iron(II) in the parasite digestive vacuole. These hybrids are active in the low nanomolar range against chloroquine-sensitive and chloroquine-resistant P. falciparum strains. We also demonstrate that in the presence of FeBr2 or within infected red blood cells, these molecules fragment to release falcipain inhibitors with nanomolar protease inhibitory activity. Molecular docking studies were performed to better understand the molecular interactions established between the tetraoxane-based hybrids and the cysteine protease binding pocket residues. Our results further indicate that the intrinsic activity of the tetraoxane partner compound can be masked, suggesting that a tetraoxane-based delivery system offers the potential to attenuate the off-target effects of known drugs.

Aspartic vinyl sulfones: inhibitors of a caspase-3-dependent pathway.

In this article we describe an expanded structure-activity relationship study for vinyl sulfones as caspase-3 inhibitors, a topic virtually unexplored in the existing literature. Most remarkably, and to our surprise, tripeptidyl vinyl sulfones were not active for caspase-3, opposite to other examples described in literature for peptidyl vinyl sulfones as potent cysteine protease inhibitors of clan CA. Moreover, the caspase-3 inhibitory activity of vinyl sulfones using an in vitro assay was then confirmed using a yeast cell-based assay. The results show that Fmoc-protected vinyl sulfones containing only the Asp moiety are inhibitors of a caspase-3-dependent pathway and the IC50 values obtained in the yeast assay are in the same order of magnitude of that obtained with the caspase-3 inhibitor tetrapeptidyl chloromethyl ketone, Ac-DEVD-CMK. This observation is consistent with appropriate cell permeability properties displayed by the vinyl sulfone inhibitors, as reflected by logP values ranging from 1.1 to 3.4. Overall, these results suggest that vinyl sulfones containing Asp at P1 should be considered for further optimization as caspase inhibitors and modulators of caspase-3-dependent pathways.

Synthesis and evaluation of vinyl sulfones as caspase-3 inhibitors. A structure-activity study.

The first structure-activity relationship study of vinyl sulfones as caspase-3 inhibitors is reported. A series of 12 vinyl sulfones was synthesized and evaluated for two downstream caspases (caspases-3 and -7). Dipeptidyl derivatives were significantly superior to their counterparts containing only Asp at P(1), as caspase-3 inhibitors. Fmoc-Val-Asp-trans-CH=CH-SO(2)Me was the most potent inhibitor of caspase-3 in the series, with a IC(50) of 29 microM and a second-order rate constant of inactivation, k(inact)/K(i), of 1.5 M(-1) s(-1). Computational studies suggest that the second amino acid occupies position S(3) of the enzyme. In addition, Fmoc-Val-Asp-trans-CH=CH-SO(2)Ph was inactive for caspase-7 for the tested concentrations.

Alkyl deoxy-arabino-hexopyranosides: synthesis, surface properties, and biological activities.

Octyl and dodecyl glycosides possessing 2-deoxy-arabino-hexopyranoside moieties belonging to the D- and L-series in their alpha- and beta-forms were synthesized by reaction of an acetyl protected glycal with octanol or dodecanol, catalyzed by triphenylphosphine hydrobromide, followed by deprotection. Their surface properties were studied and discussed in terms of the adsorption and aggregation parameters, pC(20), CMC, and gamma(CMC). The antimicrobial activities were assessed using the paper disk diffusion and broth dilution methods. Both the octyl and dodecyl 2-deoxy beta-D-glycosides inhibited significantly Enterococcus faecalis, a microbe also highly susceptible to dodecyl 2,6-dideoxy-alpha-L-arabino-hexopyranoside. This compound was particularly active against Bacillus cereus and Bacillus subtilis, presenting for both Bacillus species a minimal inhibitory concentration of the same order of magnitude and a minimal lethal concentration even smaller than that obtained for chloramphenicol, a bioactivity which remained unaltered after 1 year solution storage at 4 degrees C. In addition, activity over Listeria monocytogenes was also observed. Direct cytotoxicity and genotoxicity of the glycosides were determined by proliferative index (mitotic index) evaluation in peripheral human lymphocytes of healthy donors. All compounds induced acute toxicity effects, and the response was dose dependent for the alpha-anomer of both the alkyl 2-deoxy-arabino-hexopyranosides and for the corresponding dodecyl beta-anomer, what suggests that non-toxic but still bioactive concentrations may be found for these compounds.

Effect of triclosan (TRN) on energy-linked functions of rat liver mitochondria.

Bisphenols are a class of compounds that exhibit a broad spectrum of antimicrobial activity. One of the most widely used member of this group is triclosan (TRN). TRN is a synthetic, non-ionic, broad-spectrum antimicrobial agent, which is incorporated into several products, including hand soaps and detergents and those of skin care and oral hygiene. The effects of TRN on mitochondrial respiratory parameters and the inner mitochondrial membrane potential (DeltaPsi) are described. That of TRN (up to 60 nmol mg(-1) protein) on isolated liver mitochondria decreased oxygen consumption of state 3 respiration, as well as DeltaPsi, but increased oxygen consumption of state 4 respiration, characteristic of an uncoupler effect. Analysis of segments of the respiratory chain suggested that the TRN inhibition site is located between complexes II and III. Mitochondrial swelling, energized or driven by the K+ diffusion potential using valinomycin, was also inhibited by TRN, the former being completely inhibited at concentrations greater than 10 nmol TRN mg(-1) protein, suggesting that it is also able to interfere with fluidity of the inner mitochondrial membrane. These results suggest that, besides its antibacterial effect, TRN can also impair the mitochondrial function of animal cells.

New data on biological effects of chlorhexidine: Fe2+ induced lipid peroxidation and mitochondrial permeability transition.

Chlorhexidine (CHX) is a bis-bis-guanide with anphipatic and antiseptic properties and is largely used in dentistry, mainly for management of periodontal problems and in oral pre-operatory procedures. The present study concerns the effect of CHX on lipid peroxidation, mitochondrial permeability transition (MPT), and the interaction of CHX with ferritin (HoSF). CHX (100 microM) increased iron release from HoSF by approximately 13-fold when compared to control values. CHX also increased iron-dependent lipid peroxidation. MPT induced by CHX was protected by ethylene glycol-bis(beta-aminoethyl-ether)-N,N,N',N'-tetraacetic acid (EGTA), dithiothreitol (DTT), and cyclosporin A (CsA), showing a Ca2+-dependent effect, in which oxidation of thiol groups is involved, as well as the involvement of the transmembrane proteinaceous pore. BHT, catalase or o-phenanthroline did not protect MPT induced by CHX. This suggests that a ROS-independent mechanism is involved in the induction of MPT.