Structure-Activity Relationship of a Pyrrole Based Series of PfPKG Inhibitors as Anti-Malarials.

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.

4'-Ethynyl-2'-Deoxycytidine (EdC) Preferentially Targets Lymphoma and Leukemia Subtypes by Inducing Replicative Stress.

Anticancer nucleosides are effective against solid tumors and hematological 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 induced replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å co-crystal 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 to FDA approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a pre-clinical nucleoside prodrug candidate for DLBCL and ALL.

Liver Metabolomics and Inflammatory Profiles in Mouse Model of Fentanyl Overdose Treated with Beta-Lactams.

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.

A novel monobactam lacking antimicrobial activity, MC-100093, reduces sex-specific ethanol preference and depressive-like behaviors in mice.

Several ß-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 ß-lactams may cause adverse side effects for treating mood disorders and AUD. Studies suggest that MC-100093, a novel ß-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.

SAR Probing of KX2-391 Provided Analogues With Juxtaposed Activity Profile Against Major Oncogenic Kinases.

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 IC50 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.

Discovery of Imidazole-Based Inhibitors of Plasmodium falciparum cGMP-Dependent Protein Kinase.

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.

"I'll Be Back": The Resurrection of Dezocine.

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.

Discovery of novel class of histone deacetylase inhibitors as potential anticancer agents.

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.

Discovery and SAR of Novel Disubstituted Quinazolines as Dual PI3Kalpha/mTOR Inhibitors Targeting Breast Cancer.

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 IC50 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.

Novel allosteric PARP1 inhibitors for the treatment of BRCA-deficient leukemia.

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.

The Resurrection of Phenotypic Drug Discovery.

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.

Non-NAD-like PARP-1 inhibitors in prostate cancer treatment.

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.

Design and synthesis of functionalized piperazin-1yl-(E)-stilbenes as inhibitors of 17α-hydroxylase-C17,20-lyase (Cyp17).

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.

Novel inhibitors of Staphylococcus aureus RnpA that synergize with mupirocin.

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.

A Mitochondrial-targeted purine-based HSP90 antagonist for leukemia therapy.

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.

Design, synthesis, and evaluation of (2S,4R)-Ketoconazole sulfonamide analogs as potential treatments for Metabolic Syndrome.

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, 11ß-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.

Resistance to BET Bromodomain Inhibitors Is Mediated by Kinome Reprogramming in Ovarian Cancer.

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.

Ribosome-Templated Azide-Alkyne Cycloadditions: Synthesis of Potent Macrolide Antibiotics by In Situ Click Chemistry.

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).

Targeting Calcium Signaling Induces Epigenetic Reactivation of Tumor Suppressor Genes in Cancer.

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.

Estrogen receptor antagonists are anti-cryptococcal agents that directly bind EF hand proteins and synergize with fluconazole in vivo.

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.