In order to improve the discovery and development of new drugs, a broad effort is being made to assess the ‘drug-like’ properties of molecules in early stages of the discovery-research process. ...Although there are numerous approaches to this problem, perhaps the simplest and most widespread one is that developed by Chris Lipinski and his co-workers at Pfizer, which is generally referred either as the Lipinski Rules or the Rule of Five (ROF). The ROF is based on four properties of molecules, namely, molecular weight (MW), logP, number of hydrogen bond donors (HBD), and the number of hydrogen bond acceptors (HBA). A ‘flag’ is set if the value of a given property exceeds the chosen threshold value for that property—MW 500Da, logP 5, the number of HBDs 5, and the number of HBAs 10. Each flag corresponds to an ROF violation. The total number of violations is the ROF-Score, which lies between ‘0’ and ‘4’. Molecules with ROF-Scores greater than one are considered to be marginal for further development. The difficulty with this approach is that two molecules with nearly identical property values can, nonetheless, possess ROF-Scores that can differ by two or more. Thus, one molecule could be considered for further studies while the other, nearly identical molecule (in terms of its four ROF properties), would most likely not be. This problem arises because of the sharp thresholds imposed by the present formulation of the ROF, which is based upon classical sets. In the current work an alternative approach based on the use of utility functions, within the framework of the analytic hierarchy process (AHP), are employed to ‘soften’ the sharp boundaries inherent in classical sets. This provides a more realistic assessment of compounds in terms of their potential suitability in drug-discovery research programs.
The cellular cytotoxicity of APY0201, a PIKfyve inhibitor, against multiple myeloma was initially identified in an unbiased
chemical library screen. The activity of APY0201 was confirmed in all 25 ...cell lines tested and in 40% of 100
patient-derived primary samples, with increased activity in primary samples harboring trisomies and lacking t(11;14). The broad anti-multiple myeloma activity of PIKfyve inhibitors was further demonstrated in confirmatory screens and showed the superior potency of APY0201 when compared to the PIKfyve inhibitors YM201636 and apilimod, with a mid-point half maximal effective concentration (EC
) at nanomolar concentrations in, respectively, 65%, 40%, and 5% of the tested cell lines. Upregulation of genes in the lysosomal pathway and increased cellular vacuolization were observed
following APY0201 treatment, although these cellular effects did not correlate well with responsiveness. We confirm that PIKfyve inhibition is associated with activation of the transcription factor EB, a master regulator of lysosomal biogenesis and autophagy. Furthermore, we established an assay measuring autophagy as a predictive marker of APY0201 sensitivity. Overall, these findings indicate promising activity of PIKfyve inhibitors secondary to disruption of autophagy in multiple myeloma and suggest a strategy to enrich for likely responders.
Aquaporin (AQP) water channels, essential for fluid homeostasis, are expressed in perivascular brain end-feet regions of astroglia
(AQP4) and in choroid plexus (AQP1). At a high concentration, the ...loop diuretic bumetanide has been shown to reduce rat brain
edema after ischemic stroke by blocking Na + -K + -2Cl - cotransport. We hypothesized that an additional inhibition of AQP contributes to the protection. We show that osmotic water
flux in AQP4-expressing Xenopus laevis oocytes is reduced by extracellular bumetanide (â¥100 μM). The efficacy of block by bumetanide is increased by injection intracellularly.
Forty-five synthesized bumetanide derivatives were tested on oocytes expressing human AQP1 and rat AQP4. Of these, one of
the most effective was the 4-aminopyridine carboxamide analog, AqB013, which inhibits AQP1 and AQP4 (IC 50 â¼20 μM, applied extracellularly). The efficacy of block was enhanced by mutagenesis of intracellular AQP4 valine-189 to alanine
(V189A, IC 50 â¼8 μM), confirming the aquaporin as the molecular target of block. In silico docking of AqB013 supported an intracellular
candidate binding site in rat AQP4 and suggested that the block involves occlusion of the AQP water pore at the cytoplasmic
side. AqB013 at 2 μM had no effect, and 20 μM caused 20% block of human Na + -K + -2Cl - cotransporter activity, in contrast to >90% block of the transporter by bumetanide. AqB013 did not affect X. laevis oocyte Cl - currents and did not alter rhythmic electrical conduction in an ex vivo gastric muscle preparation. The identification of
AQP-selective pharmacological agents opens opportunities for breakthrough strategies in the treatment of edema and other fluid
imbalance disorders.
Multiple myeloma (MM) is an incurable plasma cell malignancy with dose-limiting toxicities and inter-individual variation in response/resistance to the standard-of-care/primary drugs, proteasome ...inhibitors (PIs), and immunomodulatory derivatives (IMiDs). Although newer therapeutic options are potentially highly efficacious, their costs outweigh the effectiveness. Previously, we have established that clofazimine (CLF) activates peroxisome proliferator-activated receptor-γ, synergizes with primary therapies, and targets cancer stem-like cells (CSCs) in drug-resistant chronic myeloid leukemia (CML) patients. In this study, we used a panel of human myeloma cell lines as
model systems representing drug-sensitive, innate/refractory, and clonally-derived acquired/relapsed PI- and cereblon (CRBN)-negative IMiD-resistant myeloma and bone marrow-derived CD138+ primary myeloma cells obtained from patients as
models to demonstrate that CLF shows significant cytotoxicity against drug-resistant myeloma as single-agent and in combination with PIs and IMiDs. Next, using genome-wide transcriptome analysis (RNA-sequencing), single-cell proteomics (CyTOF; Cytometry by time-of-flight), and ingenuity pathway analysis (IPA), we identified novel pathways associated with CLF efficacy, including induction of ER stress, autophagy, mitochondrial dysfunction, oxidative phosphorylation, enhancement of downstream cascade of p65-NFkB-IRF4-Myc downregulation, and ROS-dependent apoptotic cell death in myeloma. Further, we also showed that CLF is effective in killing rare refractory subclones like side populations that have been referred to as myeloma stem-like cells. Since CLF is an FDA-approved drug and also on WHO's list of safe and effective essential medicines, it has strong potential to be rapidly re-purposed as a safe and cost-effective anti-myeloma drug.
Bromodomains (BD) are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation of several genes including protooncogene cellular myelocytomatosis ...(c-Myc). c-Myc is difficult to target directly by agents due to its disordered alpha helical protein structure and predominant nuclear localization. The epigenetic targeting of c-Myc by BD inhibitors is an attractive therapeutic strategy for prostate cancer (PC) associated with increased c-Myc upregulation with advancing disease. MT-1 is a bivalent BD inhibitor that is 100-fold more potent than the first-in-class BD inhibitor JQ1. MT-1 decreased cell viability and causes cell cycle arrest in G0/G1 phase in castration-sensitive and resistant PC cell lines in a dose-dependent fashion. The inhibition of c-Myc function by MT-1 was molecularly corroborated by the de-repression of Protein Kinase D1 (PrKD) and increased phosphorylation of PrKD substrate proteins: threonine 120, serine 11, and serine 216 amino acid residues in β-Catenin, snail, and cell division cycle 25c (CDC25c) proteins, respectively. The treatment of 3D cell cultures derived from three unique clinically annotated heavily pretreated patient-derived PC xenografts (PDX) mice models with increasing doses of MT-1 demonstrated the lowest IC
in tumors with c-Myc amplification and clinically resistant to Docetaxel, Cabazitaxel, Abiraterone, and Enzalutamide. An intraperitoneal injection of either MT-1 or in combination with 3jc48-3, an inhibitor of obligate heterodimerization with MYC-associated protein X (MAX), in mice implanted with orthotopic PC PDX, decreased tumor growth. This is the first pre-clinical study demonstrating potential utility of MT-1 in the treatment of PC with c-Myc dysregulation.
There is substantial evidence that in addition to nicotine, other compounds found in tobacco smoke significantly influence smoking behavior. Further, recent years have seen an explosion in the ...availability of non-combusted products that deliver nicotine, such as e-cigarettes and “home-brew” vaping devices that are essentially unregulated. There are many thousands of compounds in tobacco smoke alone, and new products are constantly introducing new compounds. Uncovering which of these compounds are active, across multiple smoking-relevant subtypes of the nicotinic acetylcholine receptor (nAChR) that influence tobacco/nicotine addiction, requires a high-throughput screening (HTS) approach. Accordingly, we developed a panel of HTS-friendly cell-based assays, all performed in the same cellular background and using the same membrane potential dye readout, to measure the function of the α3β4-, α4β2-, and α6β2-nAChR subtypes. These subtypes have each been prominently and consistently associated with human smoking behavior. We validated our assays by performing pilot screening of an expanded set of the Prestwick FDA-approved drug library. The screens displayed excellent performance parameters, and moderate hit rates (mean of 1.2% across all three assays) were achieved when identifying antagonists (chosen since effects of endogenous antagonists on consumption of nicotine/tobacco products are under-studied). Validation rates using an orthogonal assay (86Rb+ efflux) averaged 73% across the three assays. The resulting panel of assays represents a valuable new platform with which to screen and identify nAChR subtype-selective compounds. This provides a resource for identifying smoking-related compounds in both combusted and non-combusted tobacco products, with potential relevance in the search for additional smoking-cessation therapies.
Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of ...tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a "proof-of-principle" that enzymatic inhibition of QSOX1 may have clinical relevancy.
Seventy-six FDA-approved oncology drugs and emerging therapeutics were evaluated in 25 multiple myeloma (MM) and 15 non-Hodgkin's lymphoma cell lines and in 113 primary MM samples. Ex vivo drug ...sensitivities were mined for associations with clinical phenotype, cytogenetic, genetic mutation, and transcriptional profiles. In primary MM samples, proteasome inhibitors, dinaciclib, selinexor, venetoclax, auranofin, and histone deacetylating agents had the broadest cytotoxicity. Of interest, newly diagnosed patient samples were globally less sensitive especially to bromodomain inhibitors, inhibitors of receptor tyrosine kinases or non-receptor kinases, and DNA synthesis inhibitors. Clustering demonstrated six broad groupings of drug sensitivity linked with genomic biomarkers and clinical outcomes. For example, our findings mimic clinical observations of increased venetoclax responsiveness in t(11;14) patients but also identify an increased sensitivity profile in untreated patients, standard genetic risk, low plasma cell S-Phase, and in the absence of Gain(1q) and t(4;14). In contrast, increased ex vivo responsiveness to selinexor was associated with biomarkers of poor prognosis and later relapse patients. This "direct to drug" screening resource, paired with functional genomics, has the potential to successfully direct appropriate individualized therapeutic approaches in MM and to enrich clinical trials for likely responders.
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In the search for new potential hypolipidemic agents, the present study focused on the synthesis of 2-acyl phenols (6a–c and 7a–c) and their saturated side-chain alkyl phenols (4a–c ...and 5a–c), and on the evaluation of their hypolipidemic activity using a murine Tyloxapol-induced hyperlipidemic protocol. The whole series of compounds 4–7 greatly and significantly reduced elevated serum levels of total cholesterol, LDL-cholesterol, and triglycerides, with series 6 and 7 showing the greatest potency ever found in our laboratory. At the minimum dose (25mg/kg/day), the latter compounds lowered cholesterol by 68–81%, LDL by 72–86%, and triglycerides by 59–80%. This represents a comparable performance than that shown by simvastatin. Experimental evidence and docking studies suggest that the activity of these derivatives is associated with the inhibition of HMG-CoA reductase.
Abnormally low plasma concentrations of thyroid hormones during sepsis often occur in the absence of thyroidal illness; however, the mechanisms involved in the "euthyroid sick syndrome" remain poorly ...understood. Here, we describe a previously unrecognized interaction between the thyroid hormone thyroxine (Tâ) and the proinflammatory cytokine macrophage migration inhibitory factor (MIF), together with its clinical relevance in sepsis. We found that in both patients with severe sepsis, and our rodent model, low plasma Tâ concentrations were inversely correlated with plasma MIF concentrations. The MIF molecule contains a hydrophobic pocket that is important for many of its proinflammatory activities. Binding of L-Tâ (or its hormonally inert isomer D-Tâ) significantly, and dose-dependently, inhibited the catalytic activity of this pocket. Moreover, administration of exogenous D-Tâ significantly improved survival in mice with severe sepsis. To examine the specificity of the MIF:Tâ interaction, wild-type and MIF knockout mice were subjected to the carrageenan-air pouch model of inflammation and then treated with D-Tâ or vehicle. D-Tâ significantly inhibited leukocyte infiltration in wild-type mice but not in MIF knockout mice, providing evidence that in vivo Tâ may influence MIF-mediated inflammatory responses via inhibition of its hydrophobic proinflammatory pocket. These findings demonstrate a new physiological role for Tâ as a natural inhibitor of MIF proinflammatory activity. The data may also, in part, explain the low plasma Tâ concentrations in critically ill, euthyroid patients and suggest that targeting the imbalance between MIF and Tâ may be beneficial in improving outcome from sepsis.
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