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Cryptococcosis is an infectious disease of global significance for which new therapies are needed. Repurposing previously developed drugs for new indications can expedite the translation of new therapies from bench to beside. Here, we characterized the ant

Drug addiction is considered a worldwide concern and one of the most prevailing causes of death globally. Opioids are highly addictive drugs, and one of the most common opioids that is frequently used clinically is fentanyl. The potential harmful effects of chronic exposure to opioids on the heart are still to be elucidated. Although β-lactam antibiotics are well recognized for their ability to fight bacteria, its protective effect in the brain and liver has been reported. In this study, we hypothesize that β-lactam antibiotic, ceftriaxone, and the novel synthetic non-antibiotic β-lactam, MC-100093, are cardioprotective against fentanyl induced-cardiac injury by upregulating xCT expression. Mice were exposed to repeated low dose (0.05 mg/kg, i.p.) of fentanyl for one week and then challenged on day 9 with higher dose of fentanyl (1 mg/kg, i.p.). This study investigated cardiac histopathology and target genes and proteins in serum and cardiac tissues in mice exposed to fentanyl overdose and β-lactams. We revealed that fentanyl treatment induced cardiac damage as evidenced by elevated cardiac enzymes (troponin I). Furthermore, fentanyl treatment caused large aggregations of inflammatory cells and elevation in the areas and volumes of myocardial fibers, indicating hypertrophy and severe cardiac damage. Ceftriaxone and MC-100093 treatment, However, induced cardioprotective effects as evidenced by marked reduction in cardiac enzymes (troponin I) and changes in histopathology. Furthermore, ceftriaxone and MC-100093 treatment decreased the levels of hypertrophic genes (α-MHC & β-MHC), apoptotic (caspase-3), and inflammatory markers (IL-6 & NF-κB). This study reports for the first time the cardioprotective effect of β-lactams against fentanyl-induced cardiac injury. Further studies are greatly encouraged to completely identify the cardioprotective properties of ceftriaxone and MC-100093., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Opioid-related deaths are attributed to overdoses, and fentanyl overdose has been on the rise in many parts of the world, including the USA. Glutamate transporter 1 (GLT-1) has been identified as a therapeutic target in several preclinical models of substance use disorders, and β-lactams effectively enhance its expression and function. In the current study, we characterized the metabolomic profile of the nucleus accumbens (NAc) in fentanyl-overdose mouse models, and we evaluated the protective effects of the functional enhancement of GLT-1 using β-lactams, ceftriaxone, and MC-100093. BALB/c mice were divided into four groups: control, fentanyl, fentanyl/ceftriaxone, and fentanyl/MC-100093. While the control group was intraperitoneally (i.p.) injected with normal saline simultaneously with other groups, all fentanyl groups were i.p. injected with 1 mg/kg of fentanyl as an overdose after habituation with four repetitive non-consecutive moderate doses (0.05 mg/kg) of fentanyl for a period of seven days. MC-100093 (50 mg/kg) and ceftriaxone (200 mg/kg) were i.p. injected from days 5 to 9. Gas chromatography-mass spectrometry (GC-MS) was used for metabolomics, and Western blotting was performed to determine the expression of target proteins. Y-maze spontaneous alternation performance and the open field activity monitoring system were used to measure behavioral manifestations. Fentanyl overdose altered the abundance of about 30 metabolites, reduced the expression of GLT-1, and induced the expression of inflammatory mediators IL-6 and TLR-4 in the NAc. MC-100093 and ceftriaxone attenuated the effects of fentanyl-induced downregulation of GLT-1 and upregulation of IL-6; however, only ceftriaxone attenuated fentanyl-induced upregulation of TRL4 expression. Both of the β-lactams attenuated the effects of fentanyl overdose on locomotor activities but did not induce significant changes in the overall metabolomic profile. Our findings revealed that the exposure to a high dose of fentanyl causes alterations in key metabolic pathways in the NAc. Pretreatment with ceftriaxone and MC-100093 normalized fentanyl-induced downregulation of GLT-1 expression with subsequent attenuation of neuroinflammation as well as the hyperactivity, indicating that β-lactams may be promising drugs for treating fentanyl use disorder.

Chronic exposure to opioids can lead to downregulation of astrocytic glutamate transporter 1 (GLT-1), which regulates the majority of glutamate uptake. Studies from our lab revealed that beta-lactam antibiotic, ceftriaxone, attenuated hydrocodone-induced downregulation of GLT-1 as well as cystine/glutamate antiporter (xCT) expression in central reward brain regions. In this study, we investigated the effects of escalating doses of morphine and tested the efficacy of novel synthetic non-antibiotic drug, MC-100093, and ceftriaxone in attenuating the effects of morphine exposure in the expression of GLT-1, xCT, and neuroinflammatory factors (IL-6 and TGF-β) in the nucleus accumbens (NAc). This study also investigated the effects of morphine and beta-lactams in locomotor activity, spontaneous alternation percentage (SAP) and number of entries in Y maze since opioids have effects in locomotor sensitization. Mice were exposed to moderate dose of morphine (20 mg/kg, i.p.) on days 1, 3, 5, 7, and a higher dose of morphine (150 mg/kg, i.p.) on day 9, and these mice were then behaviorally tested and euthanized on Day 10. Western blot analysis showed that exposure to morphine downregulated GLT-1 and xCT expression in the NAc, and both MC-100093 and ceftriaxone attenuated these effects. In addition, morphine exposure increased IL-6 mRNA and TGF-β mRNA expression, and MC-100093 and ceftriaxone attenuated only the effect on IL-6 mRNA expression in the NAc. Furthermore, morphine exposure induced an increase in distance travelled, and MC-100093 and ceftriaxone attenuated this effect. In addition, morphine exposure decreased the SAP and increased the number of arm entries in Y maze, however, neither MC-100093 nor ceftriaxone showed any attenuating effect. Our findings demonstrated for the first time that MC-100093 and ceftriaxone attenuated morphine-induced downregulation of GLT-1 and xCT expression, and morphine-induced increase in neuroinflammatory factor, IL-6, as well as hyperactivity. These findings revealed the beneficial therapeutic effects of MC-100093 and ceftriaxone against the effects of exposure to escalated doses of morphine., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Anticancer nucleosides are effective against solid tumors and hematologic malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induces replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å cocrystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared with FDA-approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a preclinical nucleoside prodrug candidate for DLBCL and ALL., (©2023 American Association for Cancer Research.)

Controlling malaria requires new drugs against Plasmodium falciparum . The P. falciparum cGMP-dependent protein kinase (PfPKG) is a validated target whose inhibitors could block multiple steps of the parasite's life cycle. We defined the structure-activity relationship (SAR) of a pyrrole series for PfPKG inhibition. Key pharmacophores were modified to enable full exploration of chemical diversity and to gain knowledge about an ideal core scaffold. In vitro potency against recombinant PfPKG and human PKG were used to determine compound selectivity for the parasite enzyme. P. berghei sporozoites and P. falciparum asexual blood stages were used to assay multistage antiparasitic activity. Cellular specificity of compounds was evaluated using transgenic parasites expressing PfPKG carrying a substituted "gatekeeper" residue. The structure of PfPKG bound to an inhibitor was solved, and modeling using this structure together with computational tools was utilized to understand SAR and establish a rational strategy for subsequent lead optimization.

Fentanyl is a highly potent opioid analgesic that is approved medically to treat acute and chronic pain. There is a high potential for overdose-induced organ toxicities, including liver toxicity, and this might be due to the increase of recreational use of opioids. Several preclinical studies have demonstrated the efficacy of beta-lactams in modulating the expression of glutamate transporter-1 (GLT-1) in different body organs, including the liver. The upregulation of GLT-1 by beta-lactams is associated with the attenuation of hyperglutamatergic state, which is a characteristic feature of opioid use disorders. A novel experimental beta-lactam compound with no antimicrobial properties, MC-100093, has been developed to attenuate dysregulation of glutamate transport, in part by normalizing GLT-1 expression. A previous study showed that MC-100093 modulated hepatic GLT-1 expression with subsequent attenuation of alcohol-increased fat droplet content in the liver. In this study, we investigated the effects of fentanyl overdose on liver metabolites, and determined the effects of MC-100093 and ceftriaxone in the liver of a fentanyl overdose mouse model. Liver samples from control, fentanyl overdose, and fentanyl overdose ceftriaxone- or MC-100093-treated mice were analyzed for metabolomics using gas chromatography-mass spectrometry. Heatmap analysis revealed that both MC-100093 and ceftriaxone attenuated the effects of fentanyl overdose on several metabolites, and MC-100093 showed superior effects. Statistical analysis showed that MC-100093 reversed the effects of fentanyl overdose in some metabolites. Moreover, enrichment analysis revealed that the altered metabolites were strongly linked to the glucose-alanine cycle, the Warburg effect, gluconeogenesis, glutamate metabolism, lactose degradation, and ketone body metabolism. The changes in liver metabolites induced by fentanyl overdose were associated with liver inflammation, an effect attenuated with ceftriaxone pre-treatments. Ceftriaxone normalized fentanyl-overdose-induced changes in liver interleukin-6 and cytochrome CYP3A11 (mouse homolog of human CYP3A4) expression. Our data indicate that fentanyl overdose impaired liver metabolites, and MC-100093 restored certain metabolites.

Several β-lactam derivatives upregulate astrocytic glutamate transporter type 1expression and are known to improve measures in models of mood and alcohol use disorders (AUD) through normalizing glutamatergic states. However, long-term, and high doses of β-lactams may cause adverse side effects for treating mood disorders and AUD. Studies suggest that MC-100093, a novel β-lactam lacking antimicrobial activity, rescues GLT1 expression. Thus, we sought to investigate whether MC-100093 improves affective behaviors and reduces voluntary ethanol drinking. We intraperitoneally administered MC-100093 (50 mg/kg) or vehicle once per day to C57BL/6J male and female mice (8-10 weeks old) over 6 days. We employed the open field test and the elevated plus maze to examine the effect of MC-100093 on anxiety-like behaviors. We assayed MC-100093's effects on depressive-like behaviors using the tail suspension and forced swim tests. Next, utilizing a separate cohort of male and female C57BL6 mice, we assessed the effects MC100093 treatment on voluntary ethanol drinking utilizing the 2-bottle choice continuous access drinking paradigm. After screening and selecting high-drinking mice, we systematically administered MC-100093 (50 mg/kg) or vehicle to the high-drinking mice over 6 days. Overall, we found that MC-100093 treatment resulted in sex-specific pharmacological effects with female mice displaying reduced innate depressive-like behaviors during the tail suspension and force swim testing juxtaposed with male treated mice who displayed no changes in tail suspension and a paradoxical increased depressive-like behavior during the forced swim testing. Additionally, we found that MC100093 treatment reduced female preference for 10% EtOH during the 2-bottle choice continuous access drinking with no effects of MC100093 treatment detected in male mice. Overall, this data suggests sex-specific regulation of innate depressive-like behavior and voluntary EtOH drinking by MC100093 treatment. Western blot analysis of the medial prefrontal cortex and hippocampus revealed no changes in male or female GLT1 protein abundance relative to GAPDH., Competing Interests: Declaration of competing interest Dr. DS Choi is a scientific advisory board member to Peptron Inc. Peptron had no role in the preparation, review, or approval of the manuscript; nor the decision to submit the manuscript for publication. All the other authors declare no biomedical financial interests or potential conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Tirbanibulin (KX2-391, KX-01), a dual non-ATP (substrate site) Src kinase and tubulin-polymerization inhibitor, demonstrated a universal anti-cancer activity for variety of cancer types. The notion that KX2-391 is a highly selective Src kinase inhibitor have been challenged by recent reports on the activities of this drug against FLT3-ITD mutations in some leukemic cell lines. Therefore, we hypothesized that analogues of KX2-391 may inhibit oncogenic kinases other than Src. A set of 4-aroylaminophenyl- N -benzylacetamides were synthesized and found to be more active against leukemia cell lines compared to solid tumor cell lines. N -(4-(2-(benzylamino)-2-oxoethyl)phenyl)-4-chlorobenzamide ( 4e ) exhibited activities at IC 50 0.96 µM, 1.62 µM, 1.90 µM and 4.23 µM against NB4, HL60, MV4-11 and K562 leukemia cell lines, respectively. We found that underlying mechanisms of 4e did not include tubulin polymerization or Src inhibition. Such results interestingly suggested that scaffold-hopping of KX2-391 may change the two main underlying cytotoxic mechanisms (Src and tubulin). Kinase profiling using two methods revealed that 4e significantly reduces the activities of some other potent oncogenic kinases like the MAPK member ERK1/2 (>99%) and it also greatly upregulates the pro-apoptotic c-Jun kinase (84%). This research also underscores the importance of thorough investigation of total kinase activities as part of the structure-activity relationship studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Omar, Khayat, Ahmed, Muhammad, Malebari, Ibrahim, Khan, Shah, Childers and El-Araby.)

The discovery of new targets for the treatment of malaria, in particular those aimed at the pre-erythrocytic stage in the life cycle, advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, this paper presents initial structure-activity relationships in an imidazole scaffold at four positions, representative in vitro ADME, hERG characterization, and cell-based antiparasitic activity. This series of PfPKG inhibitors has good in vitro PfPKG potency, low hERG activity, and cell-based antiparasitic activity against multiple Plasmodium species that appears to be correlated with the in vitro potency., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

Selective inhibition of histone deacetylases (HDACs) is an important strategy in the field of anticancer drug discovery. However, lack of inhibitors that possess high selectivity toward certain HDACs isozymes is associated with adverse side effects that limits their clinical applications. We have initiated a collaborative initiatives between multi-institutions aimed at the discovery of novel and selective HDACs inhibitors. To this end, a phenotypic screening of an in-house pilot library of about 70 small molecules against various HDAC isozymes led to the discovery of five compounds that displayed varying degrees of HDAC isozyme selectivity. The anticancer activities of these molecules were validated using various biological assays including transcriptomic studies. Compounds 15, 14, and 19 possessed selective inhibitory activity against HDAC5, while 28 displayed selective inhibition of HDAC1 and HDAC2. Compound 22 was found to be a selective inhibitor for HDAC3 and HDAC9. Importantly, we discovered a none-hydroxamate based HDAC inhibitor, compound 28, representing a distinct chemical probe of HDAC inhibitors. It contains a trifluoromethyloxadiazolyl moiety (TFMO) as a non-chelating metal-binding group. The new compounds showed potent anti-proliferative activity when tested against MCF7 breast cancer cell line, as well as increased acetylation of histones and induce cells apoptosis. The new compounds apoptotic effects were validated through the upregulation of proapoptotic proteins caspases3 and 7 and downregulation of the antiapoptotic biomarkers C-MYC, BCL2, BCL3 and NFĸB genes. Furthermore, the new compounds arrested cell cycle at different phases, which was confirmed through downregulation of the CDK1, 2, 4, 6, E2F1 and RB1 proteins. Taken together, our findings provide the foundation for the development of new chemical probes as potential lead drug candidates for the treatment of cancer., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Beginning with opium itself, natural and synthetic opioids have been used as analgesics for over 8000 years and were likely abused as drugs of recreation for that long as well. However, the "opioid crisis" resulted in attempts to avoid or limit opioid analgesics in favor of other therapies and methods. Mu opioid agonists can be effective analgesics but suffer from addiction, tolerance, and dangerous, sometimes fatal, side effects. One exception to this generalization is dezocine (Dalgan), a mixed mu/kappa opioid partial agonist. Dezocine is at least as effective as morphine in reducing acute pain in animal models and clinical applications such as postoperative pain. And while dezocine was discontinued in western markets in 2011, it has become the favored opioid analgesic in China, capturing over 40% of the market. Additionally, dezocine possesses norepinephrine uptake inhibitory activity, which may synergize with mu agonism in the case of acute pain treatment and possibly endow the drug with antinociceptive activity in neuropathic pain conditions. This Innovations article summarizes the history and properties of dezocine and presents evidence and rationale for why dezocine has undergone a resurrection., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

The dual PI3Kα/ m TOR inhibitors represent a promising molecularly targeted therapy for cancer. Here, we documented the discovery of new 2,4-disubstituted quinazoline analogs as potent dual PI3Kα/sm TOR inhibitors. Our structure based chemistry endeavor yielded six excellent compounds 9e, 9f, 9g, 9k, 9m , and 9o with single/double digit nanomolar IC 50 values against both enzymes and acceptable aqueous solubility and stability to oxidative metabolism. One of those analogs, 9m , possessed a sulfonamide substituent, which has not been described for this chemical scaffold before. The short direct synthetic routes, structure-activity relationship, in vitro 2D cell culture viability assays against normal fibroblasts and 3 breast cancer cell lines, and in vitro 3D culture viability assay against MCF7 cells for this series are described., Competing Interests: The authors declare no competing financial interest., (© 2020 American Chemical Society.)

The successful use of PARP1 inhibitors like olaparib (Loparza ® ) in the treatment of BRCA1/2- deficient breast cancer has provided clinical proof of concept for applying personalized medicine based on synthetic lethality to the treatment of cancer. Unfortunately, all marketed PARP1 inhibitors act by competing with the cofactor NAD + and resistance is already developing to this anti-cancer mechanism. Allosteric PARP1 inhibitors could provide a means of overcoming this resistance. A high throughput screen performed by Tulin et al. identified 5F02 as an allosteric PARP inhibitor that acts by preventing the enzymatic activation of PARP1 by histone H4. 5F02 demonstrated anti-cancer activity in several cancer cell lines and was more potent than olaparib and synergistic with olaparib in these assays. In the present study we explored the structure-activity relationship of 5F02 by preparing analogs that possessed structural variation in four regions of the chemical scaffold. Our efforts led to lead molecule 7 , which demonstrated potent anti-clonogenic activity against BRCA-deficient NALM6 leukemia cells in culture and a therapeutic index for the BRCA-deficient cells over their BRCA-proficient isogenic counterparts., Competing Interests: Conflict of Interest The authors declare that they have no conflict of interest.

Prior to genetic mapping, the majority of drug discovery efforts involved phenotypic screening, wherein compounds were screened in either in vitro or in vivo models thought to mimic the disease state of interest. While never completely abandoning phenotypic approaches, the labor intensive nature of such tests encouraged the pharmaceutical industry to move away from them in favor of target-based drug discovery, which facilitated throughput and allowed for the efficient screening of large numbers of compounds. However, a consequence of reliance on target-based screening was an increased number of failures in clinical trials due to poor correlation between novel mechanistic targets and the actual disease state. As a result, the field has seen a recent resurrection in phenotypic drug discovery approaches. In this work, we highlight some recent phenotypic projects from our industrial past and in our current academic drug discovery environment that have provided encouraging results., Competing Interests: The authors declare no competing financial interest.

In our previous studies of the molecular mechanisms of poly(ADP-ribose) polymerase 1 (PARP-1)-mediated transcriptional regulation we identified a novel class of PARP-1 inhibitors targeting the histone-dependent route of PARP-1 activation. Because histone-dependent activation is unique to PARP-1, non-NAD-like PARP-1 inhibitors have the potential to bypass the off-target effects of classical NAD-dependent PARP-1 inhibitors, such as olaparib, veliparib, and rucaparib. Furthermore, our recently published studies demonstrate that, compared to NAD-like PARP-1 inhibitors that are used clinically, the non-NAD-like PARP-1 inhibitor 5F02 exhibited superior antitumor activity in cell and animal models of human prostate cancer (PC). In this study, we further evaluated the antitumor activity of 5F02 and several of its novel analogues against PC cells. In contrast to NAD-like PARP-1 inhibitors, non-NAD-like PARP-1 inhibitors demonstrated efficacy against androgen-dependent and -independent routes of androgen receptor signaling activation. Our experiments reveal that methylation of the quaternary ammonium salt and the presence of esters were critical for the antitumor activity of 5F02 against PC cells. In addition, we examined the role of a related regulatory protein of PARP-1, called Poly(ADP-ribose) glycohydrolase (PARG), in prostate carcinogenesis. Our study reveals that PARG expression is severely disrupted in PC cells, which is associated with decreased integrity and localization of Cajal bodies (CB). Overall, the results of our study strengthen the justification for using non-NAD-like PARP-1 inhibitors as a novel therapeutic strategy for the treatment of advanced prostate cancer., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The synthesis of steroid hormones is critical to human physiology and improper regulation of either the synthesis of these key molecules or activation of the associated receptors can lead to disease states. This has led to intense interest in developing compounds capable of modulating the synthesis of steroid hormones. Compounds capable of inhibiting Cyp19 (Aromatase), a key enzyme in the synthesis of estrogens, have been successfully employed as breast cancer therapies, while inhibitors of Cyp17 (17α-hydroxylase-17,20-lyase), a key enzyme in the synthesis of glucocorticoids, mineralocorticoids and steroidal sex hormones, are a key component of prostate cancer therapy. Inhibition of CYP17 has also been suggested as a possible target for the treatment of Cushing Syndrome and Metabolic Syndrome. We have identified two novel series of stilbene based CYP17 inhibitors and demonstrated that exemplary compounds in these series have pharmacokinetic properties consistent with orally delivered drugs. These findings suggest that compounds in these classes may be useful for the treatment of diseases and conditions associated with improper regulation of glucocorticoids synthesis and glucocorticoids receptor activation., (Copyright © 2018. Published by Elsevier Ltd.)

We recently discovered RnpA as a promising new drug discovery target for methicillin-resistant S. aureus (MRSA). RnpA is an essential protein that is thought to perform two required cellular processes. As part of the RNA degrasome Rnpa mediates RNA degradation. In combination with rnpB it forms RNase P haloenzymes which are required for tRNA maturation. A high throughput screen identified RNPA2000 as an inhibitor of both RnpA-associated activities that displayed antibacterial activity against clinically relevant strains of S. aureus, including MRSA. Structure-activity studies aimed at improving potency and replacing the potentially metabotoxic furan moiety led to the identification of a number of more potent analogs. Many of these new analogs possessed overt cellular toxicity that precluded their use as antibiotics but two derivatives, including compound 5o, displayed an impressive synergy with mupirocin, an antibiotic used for decolonizing MSRA whose effectiveness has recently been jeopardized by bacterial resistance. Based on our results, compounds like 5o may ultimately find use in resensitizing mupirocin-resistant bacteria to mupirocin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Reprogramming of mitochondrial functions sustains tumor growth and may provide therapeutic opportunities. Here, we targeted the protein folding environment in mitochondria by coupling a purine-based inhibitor of the molecular chaperone Heat Shock Protein-90 (Hsp90), PU-H71 to the mitochondrial-targeting moiety, triphenylphosphonium (TPP). Binding of PU-H71-TPP to ADP-Hsp90, Hsp90 co-chaperone complex or mitochondrial Hsp90 homolog, TRAP1 involved hydrogen bonds, π-π stacking, cation-π contacts and hydrophobic interactions with the surrounding amino acids in the active site. PU-H71-TPP selectively accumulated in mitochondria of tumor cells (17-fold increase in mitochondria/cytosol ratio), whereas unmodified PU-H71 showed minimal mitochondrial localization. Treatment of tumor cells with PU-H71-TPP dissipated mitochondrial membrane potential, inhibited oxidative phosphorylation in sensitive cell types, and reduced ATP production, resulting in apoptosis and tumor cell killing. Unmodified PU-H71 had no effect. Bioinformatics analysis identified a "mitochondrial Hsp90" signature in Acute Myeloid Leukemia (AML), which correlates with worse disease outcome. Accordingly, inhibition of mitochondrial Hsp90s killed primary and cultured AML cells, with minimal effects on normal peripheral blood mononuclear cells. These data demonstrate that directing Hsp90 inhibitors with different chemical scaffolds to mitochondria is feasible and confers improved anticancer activity. A potential "addiction" to mitochondrial Hsp90s may provide a new therapeutic target in AML., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article.

Metabolic Syndrome, also referred to as 'Syndrome X' or 'Insulin Resistance Syndrome,' remains a major, unmet medical need despite over 30years of intense effort. Recent research suggests that there may be a causal link between this condition and abnormal glucocorticoid processing. Specifically, dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) axis leads to increased systemic cortisol concentrations. Cushing' syndrome, a disorder that is also typified by a marked elevation in levels of cortisol, produces clinical symptomology that is similar to those observed in MetS, and they can be alleviated by decreasing circulating cortisol concentrations. As a result, it has been suggested that decreasing systemic cortisol concentration might have a positive impact on the progression of MetS. This could be accomplished through inhibition of enzymes in the cortisol synthetic pathway, 11β-hydroxylase (Cyp11B1), 17α-hydroxylase-C17,20-lyase (Cyp17), and 21-hydroxylase (Cyp21). We have identified a series of novel sulfonamide analogs of (2S,4R)-Ketoconazole that are potent inhibitors of these enzymes. In addition, selected members of this class of compounds have pharmacokinetic properties consistent with orally delivered drugs, making them well suited to further investigation as potential therapies for MetS., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Small-molecule BET bromodomain inhibitors (BETis) are actively being pursued in clinical trials for the treatment of a variety of cancers, but the mechanisms of resistance to BETis remain poorly understood. Using a mass spectrometry approach that globally measures kinase signaling at the proteomic level, we evaluated the response of the kinome to targeted BETi treatment in a panel of BRD4-dependent ovarian carcinoma (OC) cell lines. Despite initial inhibitory effects of BETi, OC cells acquired resistance following sustained treatment with the BETi JQ1. Through application of multiplexed inhibitor beads (MIBs) and mass spectrometry, we demonstrate that BETi resistance is mediated by adaptive kinome reprogramming, where activation of compensatory pro-survival kinase networks overcomes BET protein inhibition. Furthermore, drug combinations blocking these kinases may prevent or delay the development of drug resistance and enhance the efficacy of BETi therapy., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Targeting epigenetic pathways is a promising approach for cancer therapy. Here, we report on the unexpected finding that targeting calcium signaling can reverse epigenetic silencing of tumor suppressor genes (TSG). In a screen for drugs that reactivate silenced gene expression in colon cancer cells, we found three classical epigenetic targeted drugs (DNA methylation and histone deacetylase inhibitors) and 11 other drugs that induced methylated and silenced CpG island promoters driving a reporter gene (GFP) as well as endogenous TSGs in multiple cancer cell lines. These newly identified drugs, most prominently cardiac glycosides, did not change DNA methylation locally or histone modifications globally. Instead, all 11 drugs altered calcium signaling and triggered calcium-calmodulin kinase (CamK) activity, leading to MeCP2 nuclear exclusion. Blocking CamK activity abolished gene reactivation and cancer cell killing by these drugs, showing that triggering calcium fluxes is an essential component of their epigenetic mechanism of action. Our data identify calcium signaling as a new pathway that can be targeted to reactivate TSGs in cancer., (©2015 American Association for Cancer Research.)

Over half of all antibiotics target the bacterial ribosome-nature's complex, 2.5 MDa nanomachine responsible for decoding mRNA and synthesizing proteins. Macrolide antibiotics, exemplified by erythromycin, bind the 50S subunit with nM affinity and inhibit protein synthesis by blocking the passage of nascent oligopeptides. Solithromycin (1), a third-generation semisynthetic macrolide discovered by combinatorial copper-catalyzed click chemistry, was synthesized in situ by incubating either E. coli 70S ribosomes or 50S subunits with macrolide-functionalized azide 2 and 3-ethynylaniline (3) precursors. The ribosome-templated in situ click method was expanded from a binary reaction (i.e., one azide and one alkyne) to a six-component reaction (i.e., azide 2 and five alkynes) and ultimately to a 16-component reaction (i.e., azide 2 and 15 alkynes). The extent of triazole formation correlated with ribosome affinity for the anti (1,4)-regioisomers as revealed by measured Kd values. Computational analysis using the site-identification by ligand competitive saturation (SILCS) approach indicated that the relative affinity of the ligands was associated with the alteration of macrolactone+desosamine-ribosome interactions caused by the different alkynes. Protein synthesis inhibition experiments confirmed the mechanism of action. Evaluation of the minimal inhibitory concentrations (MIC) quantified the potency of the in situ click products and demonstrated the efficacy of this method in the triaging and prioritization of potent antibiotics that target the bacterial ribosome. Cell viability assays in human fibroblasts confirmed 2 and four analogues with therapeutic indices for bactericidal activity over in vitro mammalian cytotoxicity as essentially identical to solithromycin (1).

The pharmaceutical industry is facing enormous challenges, including reduced efficiency, stagnant success rate, patent expirations for key drugs, fierce price competition from generics, high regulatory hurdles, and the industry's perceived tarnished image. Pharma has responded by embarking on a range of initiatives. Other sectors, including NIH, have also responded. Academic drug discovery groups have appeared to support the transition of innovative academic discoveries and ideas into attractive drug discovery opportunities. Part 1 of this two-part series discussed the criticisms that have been leveled at the pharmaceutical industry over the past 3 decades and summarized the supporting data for and against these criticisms. This second installment will focus on the current challenges facing the pharmaceutical industry and Pharma's responses, focusing on the industry's changing perspective and new business models for coping with the loss of talent and declining clinical pipelines as well as presenting some examples of recent drug discovery successes.

Unlabelled: Cryptococcosis is an infectious disease of global significance for which new therapies are needed. Repurposing previously developed drugs for new indications can expedite the translation of new therapies from bench to beside. Here, we characterized the anti-cryptococcal activity and antifungal mechanism of estrogen receptor antagonists related to the breast cancer drugs tamoxifen and toremifene. Tamoxifen and toremifene are fungicidal and synergize with fluconazole and amphotericin B in vitro. In a mouse model of disseminated cryptococcosis, tamoxifen at concentrations achievable in humans combines with fluconazole to decrease brain burden by ~1 log10. In addition, these drugs inhibit the growth of Cryptococcus neoformans within macrophages, a niche not accessible by current antifungal drugs. Toremifene and tamoxifen directly bind to the essential EF hand protein calmodulin, as determined by thermal shift assays with purified C. neoformans calmodulin (Cam1), prevent Cam1 from binding to its well-characterized substrate calcineurin (Cna1), and block Cna1 activation. In whole cells, toremifene and tamoxifen block the calcineurin-dependent nuclear localization of the transcription factor Crz1. A large-scale chemical genetic screen with a library of C. neoformans deletion mutants identified a second EF hand-containing protein, which we have named calmodulin-like protein 1 (CNAG_05655), as a potential target, and further analysis showed that toremifene directly binds Cml1 and modulates its ability to bind and activate Cna1. Importantly, tamoxifen analogs (idoxifene and methylene-idoxifene) with increased calmodulin antagonism display improved anti-cryptococcal activity, indicating that calmodulin inhibition can be used to guide a systematic optimization of the anti-cryptococcal activity of the triphenylethylene scaffold., Importance: Worldwide, cryptococcosis affects approximately 1 million people annually and kills more HIV/AIDS patients per year than tuberculosis. The gold standard therapy for cryptococcosis is amphotericin B plus 5-flucytosine, but this regimen is not readily available in regions where resources are limited and where the burden of disease is highest. Herein, we show that molecules related to the breast cancer drug tamoxifen are fungicidal for Cryptococcus and display a number of pharmacological properties desirable for an anti-cryptococcal drug, including synergistic fungicidal activity with fluconazole in vitro and in vivo, oral bioavailability, and activity within macrophages. We have also demonstrated that this class of molecules targets calmodulin as part of their mechanism of action and that tamoxifen analogs with increased calmodulin antagonism have improved anti-cryptococcal activity. Taken together, these results indicate that tamoxifen is a pharmacologically attractive scaffold for the development of new anti-cryptococcal drugs and provide a mechanistic basis for its further optimization.

Spermidine/spermine-N1-acetyltransferase (SSAT) is a mitochondrial-localized enzyme that is highly inducible and tightly controlled and is the rate-limiting enzyme in polyamine catabolism. It is known that SSAT is induced when polyamine level increases. Although multiple mechanisms have been implicated, translational control is thought to be paramount. Previous studies with transgenic and knockout mice suggested that for certain human conditions, the modulation of SSAT levels could offer therapeutic benefits. Besides polyamines and their analogs, certain stimuli can increase SSAT levels, suggesting that the development of reporters for high throughput screening can lead to the identification of novel pharmacophores that can modulate SSAT translation. Here we report the development and validation of a luciferase-based biosensor system for the identification of compounds that are able to either promote or prevent the translation of SSAT. The system uses HEK293T cells transfected with a construct composed of SSAT mRNA modified to lack upstream open reading frame (uORF) function, is mutated to reduce translational repression and is linked with luciferase. As a proof of principle of the utility of the SSAT translation sensor, we screened the Prestwick drug library (1,200 FDA Approved compounds). The library contained 15 compounds that activated SSAT translation by at least 40% more than the basal expression, but none exceeded the positive control N1, N11-diethylnorspermine. On the other hand, 38 compounds were found to strongly inhibit SSAT translation. We conclude that this biosensor can lead to the identification of novel pharmacophores that are able to modulate the translation of SSAT.

The pharmaceutical industry is facing enormous challenges, including reduced efficiency, declining innovation, key patent expirations, fierce price competition from generics, high regulatory hurdles, and a tarnished image. There is a clear need for change in the paradigms designed to address these challenges. Pharma has responded by embarking on a range of initiatives. However, along the way the industry has accrued critics whose accusations have tainted its reputation. The first part of this two-part series will discuss the criticisms that have been leveled at the pharmaceutical industry and summarize the supporting data for and against these criticisms. The second installment will focus on the current challenges facing the pharmaceutical industry and Pharma's responses to address these challenges. It will describe the industry's changing perspective and new business models for coping with the recent loss of talent and declining clinical pipelines as well as present some examples of recent drug discovery successes.

As part of an effort to identify 5-HT(1A) antagonists that did not possess typical arylalkylamine or keto/amido-alkyl aryl piperazine scaffolds, prototype compound 10a was identified from earlier work in a combined 5-HT(1A) antagonist/SSRI program. This quinolyl-piperazinyl piperidine analogue displayed potent, selective 5-HT(1A) antagonism but suffered from poor oxidative metabolic stability, resulting in low exposure following oral administration. SAR studies, driven primarily by in vitro liver microsomal stability assessment, identified compound 10b, which displayed improved oral bioavailability and lower intrinsic clearance. Further changes to the scaffold (e.g., 10r) resulted in a loss in potency. Compound 10b displayed cognitive enhancing effects in a number of animal models of learning and memory, enhanced the antidepressant-like effects of the SSRI fluoxetine, and reversed the sexual dysfunction induced by chronic fluoxetine treatment.

Cysteine-dependant aspartyl protease (caspase) activation has been implicated as a part of the signal transduction pathway leading to apoptosis. It has been postulated that caspase-3 inhibition could attenuate cell damage after an ischemic event and thereby providing for a novel neuroprotective treatment for stroke. As part of a program to develop a small molecule inhibitor of caspase-3, a novel series of 3,4-dihydropyrimido(1,2-a)indol-10(2H)-ones (pyrimidoindolones) was identified. The synthesis, biological evaluation and structure-activity relationships of the pyrimidoindolones are described.

Previous studies with perzinfotel (1), a potent, selective, competitive NMDA receptor antagonist, showed it to be efficacious in inflammatory and neuropathic pain models. To increase the low oral bioavailability of 1 (3-5%), prodrug derivatives (3a-h) were synthesized and evaluated. The oxymethylene-spaced diphenyl analogue 3a demonstrated good stability at acidic and neutral pH, as well as in simulated gastric fluid. In rat plasma, 3a was rapidly converted to 1 via 2a. Pharmacokinetic studies indicated that the amount of systemic exposure of 1 produced by a 10 mg/kg oral dose of 3a was 2.5-fold greater than that produced by a 30 mg/kg oral dose of 1. Consistent with these results, 3a was significantly more potent and had a longer duration of activity than 1 following oral administration in a rodent model of inflammatory pain. Taken together, these results demonstrate that an oxymethylene-spaced prodrug approach increased the bioavailability of 1.

5-Hydroxytryptamine (5-HT)(1A) receptors play an important role in multiple cognitive processes, and compelling evidence suggests that 5-HT(1A) antagonists can reverse cognitive impairment. We have examined the therapeutic potential of a potent (K(i) = 1.1 nM), selective (>100-fold), orally bioavailable, silent 5-HT(1A) receptor antagonist (K(B) = 1.3 nM) (R)-N-(2-methyl-(4-indolyl-1-piperazinyl)-ethyl)-N-(2-pyridinyl)-cyclohexane carboxamide (WAY-101405). Oral administration of WAY-101405 was shown to be effective in multiple rodent models of learning and memory. In a novel object recognition paradigm, 1 mg/kg enhanced retention (memory) for previously learned information, and it was able to reverse the memory deficits induced by scopolamine. WAY-101405 (1 mg/kg) was also able to reverse scopolamine-induced deficits in a rat contextual fear conditioning model. In the Morris water maze, WAY-101405 (3 mg/kg) significantly improved learning in a paradigm of increasing task difficulty. In vivo microdialysis studies in the dorsal hippocampus of freely moving adult rats demonstrated that acute administration of WAY-101405 (10 mg/kg) increased extracellular acetylcholine levels. The selective radioligand [(3)H]WAY-100635, administered i.v., was used for in vivo receptor occupancy studies, where WAY-101405 occupied 5-HT(1A) receptors in the rat cortex, with an ED(50) value of 0.1 mg/kg p.o. Taken together, these studies demonstrate that WAY-101405 is a potent and selective, brain penetrant, orally bioavailable 5-HT(1A) receptor "silent" antagonist that is effective in preclinical memory paradigms at doses where approximately 90% of the postsynaptic 5-HT(1A) receptors are occupied. These results further support the rationale for use of this compound class in the treatment of cognitive dysfunction associated with psychiatric and neurological conditions.

A series of benzodioxanylpiperazine derivatives possessing a 4-aryl amide substituent was prepared and evaluated for 5-HT(1A) affinity and functional antagonist activity in vitro and in vivo. All of the compounds in this series possessed high affinity for the human 5-HT(1A) receptor and many displayed potent antagonist activity in vitro and varying degrees of intrinsic activity in vivo. Compound 11c (Lecozotan) was selected for further development and is currently in clinical trials.

In some A-type voltage-gated K channels, rapid inactivation is achieved through the binding of an N-terminal domain of the pore-forming alpha-subunit or an associated beta-subunit to a cytoplasmic acceptor located at or near the channel pore using the ball-and-chain machinery (1-5). This inactivation involving the N terminus is known as N-type inactivation. Here, we describe an erbstatin (Erb) analogue as a small molecule inhibitor of the N-type inactivation in channels of Kv1.4 and Kv1.1+Kvbeta1. We show that this inhibition of inactivation (designated as "disinactivation") is potent and selective for N-type inactivation in heterologous cells (Chinese hamster ovary and Xenopus oocytes) expressing these A-type channels. In Chinese hamster ovary cells, Erb increased the inactivation time constant of Kv1.4 from 86.5 +/- 9.5 to 150 +/- 10 ms (n = 6, p < 0.0 1). Similarly, Erb increased the inactivation time constant of Kv1.1+Kvbeta1 from 10 +/- 0.9 to 49.3 +/- 7 ms (n = 7, p < 0.01). The EC(50) for disinactivating Kv1.1+Kvbeta1 was 10.4 +/- 0.9 microm (n = 2-9). Erb had no effect upon another A-channel, Kv4.3, which does not utilize the ball-and-chain mechanism. The mechanism of Erb-induced disinactivation was also investigated. Neither cysteine oxidation nor tyrosine kinase inhibition was involved. The results demonstrate that Erb can be used as a base structure to identify potent, selective small molecule inhibitors of intracellular protein-protein interactions, and that these disinactivators may offer another therapeutic approach to the treatment of seizure disorders.

Several novel functionalized adamantyl aryl- and heteroarylpiperazine derivatives were prepared and examined in various receptor binding and behavioral tests to determine their serotonin receptor activities. Many compounds demonstrated modest to high affinity for 5-HT(1A) receptors, with compounds 9, 13, 23, 33, 34, and 43 being the most potent at this site. Compound 1, 2-[4-(2-pyrimidinyl)-1-piperazinyl]ethyl adamantyl-1-carboxylate, demonstrated relatively high affinity for 5-HT(1A) receptors (K(i) = 8 nM) and acceptable selectivity versus D(2) receptors (K(i) = 708 mM); however, it lacked in vivo activity in serotonergic behavioral models. In contrast, compounds 9 (WY-50,324, SEB-324, adatanserin), adamantyl-1-carboxylic acid 2-[4-(2-pyrimidinyl)-1-piperazinyl]ethylamide, and 13, adamantyl-1-carboxylic acid 2-[4-(2-methoxyphenyl)-1-piperazinyl]ethylamide, demonstrated high affinity for 5-HT(1A) binding sites (K(i) = 1 nM for both) and moderate affinity for 5-HT(2) receptors (K(i) = 73 and 75 nM, respectively). Both compounds also demonstrated partial 5-HT(1A) agonist activity in vivo in rat serotonin syndrome and 5-HT(2) antagonist activity in quipazine- and DOI-induced head shake paradigms. The selective 5-HT(1A) partial agonist and 5-HT(2) antagonist activity of 9 was accompanied by significant anxiolytic activity in an animal conflict model. On the basis of this profile, compound 9 entered development as a combined anxiolytic and antidepressant agent.

Novel homologated 19-oxiranyl- and 9-thiiranylandrost-4-ene-3,17-diones (8a,b and 9a,b, respectively) have been synthesized. The configuration and conformation of compound 8a have been established by X-ray crystallographic analysis. All four compounds have been shown to be competitive inhibitors of human placental aromatase. The thiiranes were more potent inhibitors than the corresponding oxiranes, and the 2'S isomers (8b and 9b) were better inhibitors than the 2'R (8a and 9a) diastereomers in each series. Spectroscopic studies with purified human placental aromatase suggest that the oxiranyl oxygen and thiiranyl sulfur of 2'S compounds 8b and 9b coordinate to the enzyme's heme iron.

We have synthesized the (19R)- and (19S)-isomers (2 and 3 respectively) of 10 beta-oxiranylestr-4-ene-3,17-dione. The configurations and conformations of these compounds were established by X-ray crystallographic analysis. Each of these compounds is a powerful competitive inhibitor of human placental microsomal aromatase, and stereoselectivity of inhibition was observed (Ki values for 2 and 3 were 7 and 75 nanomolar, respectively). Spectroscopic studies with purified aromatase indicate that the inhibition process involves reversible binding of oxirane oxygen to the heme iron of the enzyme. The (19R)- and (19S)-10 beta-thiiranes (6 and 7) corresponding to 2 and 3 have been synthesized from the oxiranes by a stereospecific process. The thiiranes are very effective competitive inhibitors of placental aromatase, and show even greater stereoselectivity in binding than the oxiranes (Ki values for 6 and 7 were 1 and 75 nanomolar, respectively). Spectroscopic studies with purified aromatase indicate that the inhibition process involves reversible binding of thiirane sulfur to heme iron.

A 22-year-old man underwent palliative surgery for superior vena cava syndrome due to a benign obstructive process compatible with fibrosing mediastinitis. By using a spiral vein constructed from autologous saphenous vein, an innominate vein to the right atrial appendage shunt was created. This technique, which was first described experimentally in 1974 and performed clinically in 1976, may warrant more liberal application in patients with superior vena cava syndrome caused by malignant and benign diseases.

The mechanism of inhibition of estrogen synthetase (P-450arom) by 19R- and 19S-isomers of 10-oxiranyl-and 10-thiiranyl-4-estrene-3,17-dione was investigated using human placental microsomes and purified enzyme preparations. The 19R-isomers were potent inhibitors and exhibited affinities 36-fold (10-oxirane) and 80-fold (10-thiirane) greater than the respective 19S-isomers. Kinetic experiments showed that inhibition by the 19R-isomers is competitive with respect to substrate; inhibition constants for the (19R)-10-oxirane (Ki = 10 nM) and the 19R-10-thiirane (Ki = 2 nM) indicate that each binds with greater affinity than the androgen substrates androstenedione and testosterone. Inhibition time courses and kinetic data were consistent with high affinity, reversible binding. Spectral titrations of microsomal preparations and purified P-450arom showed that binding of the 19R-isomers shifts the Soret maximum of the ferric enzyme to 411 nm (10-oxirane) or 425 nm (10-thiirane); addition of excess androstenedione reversed the spectral changes, producing the high spin form of the enzyme with a Soret peak at 393 nm. These spectral shifts suggest that the oxygen atom of the 10-oxirane and the sulfur atom of the 10-thiirane are bound to the heme iron in the inhibitor complexes. These results suggest that the high affinities of the inhibitors arise from their dual interaction with the androgen binding site and with the heme. Coordination of the C19 heteroatom to the heme indicates that C19 of androgen substrates may be positioned sufficiently close to the heme to allow direct attack by an iron-bound oxidant. Stereoselective binding of the 19R-isomers by P-450arom further suggests that the heme is likely to be positioned above C1 and C2 of the A ring.