XP-524 is a dual-BET/EP300 inhibitor that represses oncogenic KRAS and potentiates immune checkpoint inhibition in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is associated with extensive dysregulation of the epigenome and epigenetic regulators, such as bromodomain and extraterminal motif (BET) proteins, have been suggested as potential targets for therapy. However, single-agent BET inhibition has shown poor efficacy in clinical trials, and no epigenetic approaches are currently used in PDAC. To circumvent the limitations of the current generation of BET inhibitors, we developed the compound XP-524 as an inhibitor of the BET protein BRD4 and the histone acetyltransferase EP300/CBP, both of which are ubiquitously expressed in PDAC tissues and cooperate to enhance tumorigenesis. XP-524 showed increased potency and superior tumoricidal activity than the benchmark BET inhibitor JQ-1 in vitro, with comparable efficacy to higher-dose JQ-1 combined with the EP300/CBP inhibitor SGC-CBP30. We determined that this is in part due to the epigenetic silencing of KRAS in vitro, with similar results observed using ex vivo slice cultures of human PDAC tumors. Accordingly, XP-524 prevented KRAS-induced, neoplastic transformation in vivo and extended survival in two transgenic mouse models of aggressive PDAC. In addition to the inhibition of KRAS/MAPK signaling, XP-524 also enhanced the presentation of self-peptide and tumor recruitment of cytotoxic T lymphocytes, though these lymphocytes remained refractory from full activation. We, therefore, combined XP-524 with an anti-PD-1 antibody in vivo, which reactivated the cytotoxic immune program and extended survival well beyond XP-524 in monotherapy. Pending a comprehensive safety evaluation, these results suggest that XP-524 may benefit PDAC patients and warrant further exploration, particularly in combination with immune checkpoint inhibition.

Mechanism of Allosteric Modulation of Nicotinamide Phosphoribosyltransferase to Elevate Cellular NAD.

In aging and disease, cellular nicotinamide adenine dinucleotide (NAD+) is depleted by catabolism to nicotinamide (NAM). NAD+ supplementation is being pursued to enhance human healthspan and lifespan. Activation of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD+ biosynthesis, has the potential to increase the salvage of NAM. Novel NAMPT-positive allosteric modulators (N-PAMs) were discovered in addition to the demonstration of NAMPT activation by biogenic phenols. The mechanism of activation was revealed through the synthesis of novel chemical probes, new NAMPT co-crystal structures, and enzyme kinetics. Binding to a rear channel in NAMPT regulates NAM binding and turnover, with biochemical observations being replicated by NAD+ measurements in human cells. The mechanism of action of N-PAMs identifies, for the first time, the role of the rear channel in the regulation of NAMPT turnover coupled to productive and nonproductive NAM binding. The tight regulation of cellular NAMPT via feedback inhibition by NAM, NAD+, and adenosine 5'-triphosphate (ATP) is differentially regulated by N-PAMs and other activators, indicating that different classes of pharmacological activators may be engineered to restore or enhance NAD+ levels in affected tissues.

Evidence for distinct mechanisms of small molecule inhibitors of filovirus entry.

Many small molecules have been identified as entry inhibitors of filoviruses. However, a lack of understanding of the mechanism of action for these molecules limits further their development as anti-filoviral agents. Here we provide evidence that toremifene and other small molecule entry inhibitors have at least three distinctive mechanisms of action and lay the groundwork for future development of anti-filoviral agents. The three mechanisms identified here include: (1) direct binding to the internal fusion loop region of Ebola virus glycoprotein (GP); (2) the HR2 domain is likely the main binding site for Marburg virus GP inhibitors and a secondary binding site for some EBOV GP inhibitors; (3) lysosome trapping of GP inhibitors increases drug exposure in the lysosome and further improves the viral inhibition. Importantly, small molecules targeting different domains on GP are synergistic in inhibiting EBOV entry suggesting these two mechanisms of action are distinct. Our findings provide important mechanistic insights into filovirus entry and rational drug design for future antiviral development.

Mixed lineage kinase 3 inhibition induces T cell activation and cytotoxicity.

Mixed lineage kinase 3 (MLK3), also known as MAP3K11, was initially identified in a megakaryocytic cell line and is an emerging therapeutic target in cancer, yet its role in immune cells is not known. Here, we report that loss or pharmacological inhibition of MLK3 promotes activation and cytotoxicity of T cells. MLK3 is abundantly expressed in T cells, and its loss alters serum chemokines, cytokines, and CD28 protein expression on T cells and its subsets. MLK3 loss or pharmacological inhibition induces activation of T cells in in vitro, ex vivo, and in vivo conditions, irrespective of T cell activating agents. Conversely, overexpression of MLK3 decreases T cell activation. Mechanistically, loss or inhibition of MLK3 down-regulates expression of a prolyl-isomerase, Ppia, which is directly phosphorylated by MLK3 to increase its isomerase activity. Moreover, MLK3 also phosphorylates nuclear factor of activated T cells 1 (NFATc1) and regulates its nuclear translocation via interaction with Ppia, and this regulates T cell effector function. In an immune-competent mouse model of breast cancer, MLK3 inhibitor increases Granzyme B-positive CD8+ T cells and decreases MLK3 and Ppia gene expression in tumor-infiltrating T cells. Likewise, the MLK3 inhibitor in pan T cells, isolated from breast cancer patients, also increases cytotoxic CD8+ T cells. These results collectively demonstrate that MLK3 plays an important role in T cell biology, and targeting MLK3 could serve as a potential therapeutic intervention via increasing T cell cytotoxicity in cancer.

Effect of a brain-penetrant selective estrogen receptor degrader (SERD) on binge drinking in female mice.

BACKGROUND: Greater circulating levels of the steroid hormone 17ß-estradiol (E2) are associated with higher levels of binge drinking in women. In female mice, estrogen receptors in the ventral tegmental area, a dopaminergic region of the brain involved in the motivation to consume ethanol, regulate binge-like ethanol intake. We recently developed a brain-penetrant selective estrogen receptor degrader (SERD), YL3-122, that could be used to test the behavioral role of brain estrogen receptors. We hypothesized that treating female mice with this compound would reduce binge-like ethanol drinking. METHODS: Female C57BL/6J mice were treated systemically with YL3-122 and a related SERD with low brain penetrance, XR5-27, and tested for binge-like ethanol consumption in the drinking in the dark (DID) test. Mice were also tested for sucrose and water consumption and blood ethanol clearance after treatment with the SERDs. Finally, the effect of ethanol exposure on Esr1 gene expression was measured in the ventral tegmental area (VTA), prefrontal cortex (PFC), and ventral hippocampus (vHPC) of male and female mice by quantitative real-time PCR after 4 DID sessions. RESULTS: YL3-122 reduced ethanol consumption when mice were in diestrus but not estrus. YL3-122 also decreased sucrose consumption but did not alter water intake or blood ethanol clearance. XR5-27 did not affect any of these measures. Binge-like ethanol drinking resulted in increased Esr1 transcript in the VTA of both sexes, male vHPC, and female PFC. CONCLUSIONS: These results indicate that SERD treatment can decrease binge-like ethanol drinking in female mice. Thus, it could be a novel strategy to reduce binge drinking in women, with the caveat that effectiveness may depend on menstrual cycle phase. In addition, Esr1 transcript is increased by binge ethanol exposure in both sexes but in a brain region-specific manner.

Pharmacology and Molecular Mechanisms of Clinically Relevant Estrogen Estetrol and Estrogen Mimic BMI-135 for the Treatment of Endocrine-Resistant Breast Cancer.

Long-term estrogen deprivation (LTED) with tamoxifen (TAM) or aromatase inhibitors leads to endocrine-resistance, whereby physiologic levels of estrogen kill breast cancer (BC). Estrogen therapy is effective in treating patients with advanced BC after resistance to TAM and aromatase inhibitors develops. This therapeutic effect is attributed to estrogen-induced apoptosis via the estrogen receptor (ER). Estrogen therapy can have unpleasant gynecologic and nongynecologic adverse events. Here, we study estetrol (E4) and a model Selective Human ER Partial Agonist (ShERPA) BMI-135. Estetrol and ShERPA TTC-352 are being evaluated in clinical trials. These agents are proposed as safer estrogenic candidates compared with 17ß-estradiol (E2) for the treatment of endocrine-resistant BC. Cell viability assays, real-time polymerase chain reaction, luciferase reporter assays, chromatin immunoprecipitation, docking and molecular dynamics simulations, human unfolded protein response (UPR) RT2 PCR profiler arrays, live cell microscopic imaging and analysis, and annexin V staining assays were conducted. Our work was done in eight biologically different human BC cell lines and one human endometrial cancer cell line, and results were compared with full agonists estrone, E2, and estriol, a benchmark partial agonist triphenylethylene bisphenol (BPTPE), and antagonists 4-hydroxytamoxifen and endoxifen. Our study shows the pharmacology of E4 and BMI-135 as less-potent full-estrogen agonists as well as their molecular mechanisms of tumor regression in LTED BC through triggering a rapid UPR and apoptosis. Our work concludes that the use of a full agonist to treat BC is potentially superior to a partial agonist given BPTPE's delayed induction of UPR and apoptosis, with a higher probability of tumor clonal evolution and resistance. SIGNIFICANCE STATEMENT: Given the unpleasant gynecologic and nongynecologic adverse effects of estrogen treatment, the development of safer estrogens for endocrine-resistant breast cancer (BC) treatment and hormone replacement therapy remains a priority. The naturally occurring estrogen estetrol and Selective Human Estrogen-Receptor Partial Agonists are being evaluated in endocrine-resistant BC clinical trials. This work provides a comprehensive evaluation of their pharmacology in numerous endocrine-resistant BC models and an endometrial cancer model and their molecular mechanisms of tumor regression through the unfolded protein response and apoptosis.

Phase 1 study of TTC-352 in patients with metastatic breast cancer progressing on endocrine and CDK4/6 inhibitor therapy.

PURPOSE: TTC-352 is a selective human estrogen receptor (ER) partial agonist developed for treatment of hormone-refractory ER + breast cancer. METHODS: This was an accelerated dose escalation study with the primary endpoint of maximum tolerated dose that evaluated five dose levels of TTC-352 in breast cancer progressing after at least two lines of hormonal therapy including one in combination with a CDK4/6 inhibitor. The secondary objectives were to determine treatment tolerability, pharmacokinetics of TTC-352, best response, progression-free survival (PFS), and PKCα expression in tumors. RESULTS: The study enrolled 15 patients. No dose-limiting toxicity was observed. Patients experienced the following grade 3 toxicities: asymptomatic pulmonary embolism, diarrhea, aspartate transaminase elevation, and myalgia, and one grade 4 toxicity of gamma glutamyltransferase elevation. Pharmacokinetic half-life was 7.6-14.3 h. The intra- and inter-individual variability for AUC0-∞ hampered assessment of the relationship between dose and AUC0-∞. Median PFS was 58 days (95% CI = 28,112). Higher PKCα expression in tumor stroma was associated with a trend toward longer PFS. CONCLUSIONS: TTC-352 demonstrates safety and early clinical evidence of antitumor activity against heavily pretreated hormone-refractory breast cancer. Based upon TTC-352 plasma concentrations and tolerability, the 180 mg twice a day is recommended for further testing. (ClinicalTrials.gov Identifier: NCT03201913).

G1T48, an oral selective estrogen receptor degrader, and the CDK4/6 inhibitor lerociclib inhibit tumor growth in animal models of endocrine-resistant breast cancer.

PURPOSE: The combination of targeting the CDK4/6 and estrogen receptor (ER) signaling pathways with palbociclib and fulvestrant is a proven therapeutic strategy for the treatment of ER-positive breast cancer. However, the poor physicochemical properties of fulvestrant require monthly intramuscular injections to patients, which limit the pharmacokinetic and pharmacodynamic activity of the compound. Therefore, an orally available compound that more rapidly reaches steady state may lead to a better clinical response in patients. Here, we report the identification of G1T48, a novel orally bioavailable, non-steroidal small molecule antagonist of ER. METHODS: The pharmacological effects and the antineoplastic mechanism of action of G1T48 on tumors was evaluated using human breast cancer cells (in vitro) and xenograft efficacy models (in vivo). RESULTS: G1T48 is a potent and efficacious inhibitor of estrogen-mediated transcription and proliferation in ER-positive breast cancer cells, similar to the pure antiestrogen fulvestrant. In addition, G1T48 can effectively suppress ER activity in multiple models of endocrine therapy resistance including those harboring ER mutations and growth factor activation. In vivo, G1T48 has robust antitumor activity in a model of estrogen-dependent breast cancer (MCF7) and significantly inhibited the growth of tamoxifen-resistant (TamR), long-term estrogen-deprived (LTED) and patient-derived xenograft tumors with an increased response being observed with the combination of G1T48 and the CDK4/6 inhibitor lerociclib. CONCLUSIONS: These data show that G1T48 has the potential to be an efficacious oral antineoplastic agent in ER-positive breast cancer.

Pharmacological manipulation of cGMP and NO/cGMP in CNS drug discovery.

The development of small molecule modulators of NO/cGMP signaling for use in the CNS has lagged far behind the use of such clinical agents in the periphery, despite the central role played by NO/cGMP in learning and memory, and the substantial evidence that this signaling pathway is perturbed in neurodegenerative disorders, including Alzheimer's disease. The NO-chimeras, NMZ and Nitrosynapsin, have yielded beneficial and disease-modifying responses in multiple preclinical animal models, acting on GABAA and NMDA receptors, respectively, providing additional mechanisms of action relevant to synaptic and neuronal dysfunction. Several inhibitors of cGMP-specific phosphodiesterases (PDE) have replicated some of the actions of these NO-chimeras in the CNS. There is no evidence that nitrate tolerance is a phenomenon relevant to the CNS actions of NO-chimeras, and studies on nitroglycerin in the periphery continue to challenge the dogma of nitrate tolerance mechanisms. Hybrid nitrates have shown much promise in the periphery and CNS, but to date only one treatment has received FDA approval, for glaucoma. The potential for allosteric modulation of soluble guanylate cyclase (sGC) in brain disorders has not yet been fully explored nor exploited; whereas multiple applications of PDE inhibitors have been explored and many have stalled in clinical trials.

Time-Gated Luminescence Detection of Enzymatically Produced Hydrogen Sulfide: Design, Synthesis, and Application of a Lanthanide-Based Probe.

Hydrogen sulfide (H2S) is now recognized as an important gaseous transmitter that is involved in a variety of biological processes. Here, we report the design and synthesis of a luminescent lanthanide biosensor for H2S, LP2-Cu(II)-Ln(III), a heterobinuclear metal complex that uses Cu(II) decomplexation to control millisecond-scale-lifetime-Tb(III)- or Eu(III)-emission intensity. LP2-Cu(II)-Ln(III) responded rapidly, selectively, and with high sensitivity to aqueous H2S. The probe's potential for biological applications was verified by measuring the H2S generated by the slow-releasing chemical-sulfide-donor GYY4147, by cystathionine γ-lyase (CSE), and by Na2S-stimulated HeLa cells.

Dimethyl Fumarate Inhibits the Nuclear Factor κB Pathway in Breast Cancer Cells by Covalent Modification of p65 Protein.

In breast tumors, activation of the nuclear factor κB (NFκB) pathway promotes survival, migration, invasion, angiogenesis, stem cell-like properties, and resistance to therapy--all phenotypes of aggressive disease where therapy options remain limited. Adding an anti-inflammatory/anti-NFκB agent to breast cancer treatment would be beneficial, but no such drug is approved as either a monotherapy or adjuvant therapy. To address this need, we examined whether dimethyl fumarate (DMF), an anti-inflammatory drug already in clinical use for multiple sclerosis, can inhibit the NFκB pathway. We found that DMF effectively blocks NFκB activity in multiple breast cancer cell lines and abrogates NFκB-dependent mammosphere formation, indicating that DMF has anti-cancer stem cell properties. In addition, DMF inhibits cell proliferation and significantly impairs xenograft tumor growth. Mechanistically, DMF prevents p65 nuclear translocation and attenuates its DNA binding activity but has no effect on upstream proteins in the NFκB pathway. Dimethyl succinate, the inactive analog of DMF that lacks the electrophilic double bond of fumarate, is unable to inhibit NFκB activity. Also, the cell-permeable thiol N-acetyl l-cysteine, reverses DMF inhibition of the NFκB pathway, supporting the notion that the electrophile, DMF, acts via covalent modification. To determine whether DMF interacts directly with p65, we synthesized and used a novel chemical probe of DMF by incorporating an alkyne functionality and found that DMF covalently modifies p65, with cysteine 38 being essential for the activity of DMF. These results establish DMF as an NFκB inhibitor with anti-tumor activity that may add therapeutic value in the treatment of aggressive breast cancers.

Time-Gated Detection of Cystathionine γ-Lyase Activity and Inhibition with a Selective, Luminogenic Hydrogen Sulfide Sensor.

Herein, we report the design, synthesis, and characterization of a lanthanideIII complex-based probe for the time-gated luminescence detection of hydrogen sulfide (H2 S) in aqueous media. The probe's unique sensing mechanism relies on the selective reduction of azide to amine by sulfide, followed by intramolecular cyclization to form a quinolinone. The quinolinone is a sensitizer that absorbs near-UV light and transfers excitation energy to coordinated TbIII or EuIII ions to trigger a strong "turn-on" luminescence response with ms-scale lifetimes characteristic of lanthanide complexes. Using this probe, we developed a robust, high throughput screening (HTS) assay for detecting H2 S generated by cystathionine γ-lyase (CSE), one of the main producers of H2 S in mammalian cells. In a 240-compound screen to identify potential CSE inhibitors, the EuIII analogue of the sensor showed a low false-positive rate and high Z'-factor (>0.7).

Prodrugs Bioactivated to Quinones Target NF-κB and Multiple Protein Networks: Identification of the Quinonome.

Electrophilic reactive intermediates resulting from drug metabolism have been associated with toxicity and off-target effects and in some drug discovery programs trigger NO-GO decisions. Many botanicals and dietary supplements are replete with such reactive electrophiles, notably Michael acceptors, which have been demonstrated to elicit chemopreventive mechanisms; and Michael acceptors are gaining regulatory approval as contemporary cancer therapeutics. Identifying protein targets of these electrophiles is central to understanding potential therapeutic benefit and toxicity risk. NO-donating NSAID prodrugs (NO-NSAIDs) have been the focus of extensive clinical and preclinical studies in inflammation and cancer chemoprevention and therapy: a subset exemplified by pNO-ASA, induces chemopreventive mechanisms following bioactivation to an electrophilic quinone methide (QM) Michael acceptor. Having previously shown that these NO-independent, QM-donors activated Nrf2 via covalent modification of Keap-1, we demonstrate that components of canonical NF-κB signaling are also targets, leading to the inhibition of NF-κB signaling. Combining bio-orthogonal probes of QM-donor ASA prodrugs with mass spectrometric proteomics and pathway analysis, we proceeded to characterize the quinonome: the protein cellular targets of QM-modification by pNO-ASA and its ASA pro-drug congeners. Further comparison was made using a biorthogonal probe of the "bare-bones", Michael acceptor, and clinical anti-inflammatory agent, dimethyl fumarate, which we have shown to inhibit NF-κB signaling. Identified quinonome pathways include post-translational protein folding, cell-death regulation, protein transport, and glycolysis; and identified proteins included multiple heat shock elements, the latter functionally confirmed by demonstrating activation of heat shock response.

Amyloid-ß pathology and APOE genotype modulate retinoid X receptor agonist activity in vivo.

Previous data demonstrate that bexarotene (Bex), retinoid X receptor (RXR) agonist, reduces soluble and insoluble amyloid-ß (Aß) in Alzheimer disease (AD)-transgenic mice either by increasing the levels of mouse apolipoprotein E (apoE) or increasing ABCA1/ABCG1-induced apoE lipoprotein association/lipidation. However, although the mechanism of action of RXR agonists remains unclear, a major concern for their use is human (h)-APOE4, the greatest AD genetic risk factor. If APOE4 imparts a toxic gain-of-function, then increasing apoE4 may increase soluble Aß, likely the proximal AD neurotoxin. If the APOE4 loss-of-function is lipidation of apoE4, then induction of ABCA1/ABCG1 may be beneficial. In novel EFAD-Tg mice (overexpressing h-Aß42 with h-APOE), levels of soluble Aß (Aß42 and oligomeric Aß) are highest in E4FAD hippocampus (HP) > E3FAD-HP > E4FAD cortex (CX) > E3FAD-CX, whereas levels of lipoprotein-associated/lipidated apoE have the opposite pattern (6 months). In E4FAD-HP, short-term RXR agonist treatment (Bex or LG100268; 5.75-6 months) increased ABCA1, apoE4 lipoprotein-association/lipidation, and apoE4/Aß complex, decreased soluble Aß, and increased PSD95. In addition, hydrogel delivery, which mimics low sustained release, was equally effective as gavage for Bex and LG100268. RXR agonists induced no beneficial effects in the E4FAD-HP in a prevention protocol (5-6 months) and actually increased soluble Aß levels in E3FAD-CX and E4FAD-CX with the short-term protocol, possibly the result of systemic hepatomegaly. Thus, RXR agonists address the loss-of-function associated with APOE4 and exacerbated by Aß pathology, i.e. low levels of apoE4 lipoprotein association/lipidation. Further studies are vital to address whether RXR agonists are an APOE4-specific AD therapeutic and the systemic side effects that limit translational application.

Re-engineering a neuroprotective, clinical drug as a procognitive agent with high in vivo potency and with GABAA potentiating activity for use in dementia.

BACKGROUND: Synaptic dysfunction is a key event in pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) where synapse loss pathologically correlates with cognitive decline and dementia. Although evidence suggests that aberrant protein production and aggregation are the causative factors in familial subsets of such diseases, drugs singularly targeting these hallmark proteins, such as amyloid-ß, have failed in late stage clinical trials. Therefore, to provide a successful disease-modifying compound and address synaptic dysfunction and memory loss in AD and mixed pathology dementia, we repurposed a clinically proven drug, CMZ, with neuroprotective and anti-inflammatory properties via addition of nitric oxide (NO) and cGMP signaling property. RESULTS: The novel compound, NMZ, was shown to retain the GABAA potentiating actions of CMZ in vitro and sedative activity in vivo. Importantly, NMZ restored LTP in hippocampal slices from AD transgenic mice, whereas CMZ was without effect. NMZ reversed amnestic blockade of acetylcholine receptors by scopolamine as well as NMDA receptor blockade by a benzodiazepine and a NO synthase inhibitor in the step-through passive avoidance (STPA) test of learning and working memory. A PK/PD relationship was developed based on STPA analysis coupled with pharmacokinetic measures of drug levels in the brain: at 1 nM concentration in brain and plasma, NMZ was able to restore memory consolidation in mice. CONCLUSION: Our findings show that NMZ embodies a promising pharmacological approach targeting synaptic dysfunction and opens new avenues for neuroprotective intervention strategies in mixed pathology AD, neurodegeneration, and dementia.

A novel aspirin prodrug inhibits NFκB activity and breast cancer stem cell properties.

INTRODUCTION: Activation of cyclooxygenase (COX)/prostaglandin and nuclear factor κB (NFκB) pathways can promote breast tumor initiation, growth, and progression to drug resistance and metastasis. Thus, anti-inflammatory drugs have been widely explored as chemopreventive and antineoplastic agents. Aspirin (ASA), in particular, is associated with reduced breast cancer incidence but gastrointestinal toxicity has limited its usefulness. To improve potency and minimize toxicity, ASA ester prodrugs have been developed, in which the carboxylic acid of ASA is masked and ancillary pharmacophores can be incorporated. To date, the effects of ASA and ASA prodrugs have been largely attributed to COX inhibition and reduced prostaglandin production. However, ASA has also been reported to inhibit the NFκB pathway at very high doses. Whether ASA prodrugs can inhibit NFκB signaling remains relatively unexplored. METHODS: A library of ASA prodrugs was synthesized and screened for inhibition of NFκB activity and cancer stem-like cell (CSC) properties, an important PGE2-and NFκB-dependent phenotype of aggressive breast cancers. Inhibition of NFκB activity was determined by dual luciferase assay, RT-QPCR, p65 DNA binding activity and Western blots. Inhibition of CSC properties was determined by mammosphere growth, CD44(+)CD24(-)immunophenotype and tumorigenicity at limiting dilution. RESULTS: While we identified multiple ASA prodrugs that are capable of inhibiting the NFκB pathway, several were associated with cytotoxicity. Of particular interest was GTCpFE, an ASA prodrug with fumarate as the ancillary pharmacophore. This prodrug potently inhibits NFκB activity without innate cytotoxicity. In addition, GTCpFE exhibited selective anti-CSC activity by reducing mammosphere growth and the CD44(+)CD24(-)immunophenotype. Moreover, GTCpFE pre-treated cells were less tumorigenic and, when tumors did form, latency was increased and growth rate was reduced. Structure-activity relationships for GTCpFE indicate that fumarate, within the context of an ASA prodrug, is essential for anti-NFκB activity, whereas both the ASA and fumarate moieties contributed to attenuated mammosphere growth. CONCLUSIONS: These results establish GTCpFE as a prototype for novel ASA-and fumarate-based anti-inflammatory drugs that: (i) are capable of targeting CSCs, and (ii) may be developed as chemopreventive or therapeutic agents in breast cancer.

Structure-Guided Design and Synthesis of Pyridinone-Based Selective Bromodomain and Extra-Terminal Domain (BET)-First Bromodomain (BD1) Inhibitors.

The bromodomain and extra-terminal domain (BET) proteins are epigenetic readers, regulating transcription via two highly homologous tandem bromodomains, BD1 and BD2. Clinical development of nonselective pan-BD BET inhibitors has been challenging, partly due to dose-limiting side effects such as thrombocytopenia. This has prompted the push for domain-selective BET inhibitors to achieve a more favorable therapeutic window. We report a structure-guided drug design campaign that led to the development of a potent BD1-selective BET inhibitor, 33 (XL-126), with a Kd of 8.9 nM and 185-fold BD1/BD2 selectivity. The high selectivity was first assayed by SPR, validated by a secondary time-resolved fluorescence energy transfer assay, and further corroborated by BROMOscan (∼57-373 fold selectivity). The cocrystal of 33 with BRD4 BD1 and BD2 demonstrates the source of selectivity: repulsion with His437 and lost binding with the leucine clamp. Notably, the BD1 selectivity of BET inhibitor 33 leads to both the preservation of platelets and potent anti-inflammatory efficacy.

Channeling Nicotinamide Phosphoribosyltransferase (NAMPT) to Address Life and Death.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in NAD+ biosynthesis via salvage of NAM formed from catabolism of NAD+ by proteins with NADase activity (e.g., PARPs, SIRTs, CD38). Depletion of NAD+ in aging, neurodegeneration, and metabolic disorders is addressed by NAD+ supplementation. Conversely, NAMPT inhibitors have been developed for cancer therapy: many discovered by phenotypic screening for cancer cell death have low nanomolar potency in cellular models. No NAMPT inhibitor is yet FDA-approved. The ability of inhibitors to act as NAMPT substrates may be associated with efficacy and toxicity. Some 3-pyridyl inhibitors become 4-pyridyl activators or "NAD+ boosters". NAMPT positive allosteric modulators (N-PAMs) and boosters may increase enzyme activity by relieving substrate/product inhibition. Binding to a "rear channel" extending from the NAMPT active site is key for inhibitors, boosters, and N-PAMs. A deeper understanding may fulfill the potential of NAMPT ligands to regulate cellular life and death.

Estradiol improves behavior in FAD transgenic mice that express APOE3 but not APOE4 after ovariectomy.

Increasing evidence suggests that female individuals have a higher Alzheimer's disease (AD) risk associated with post-menopausal loss of circulating estradiol (E2). However, clinical data are conflicting on whether E2 lowers AD risk. One potential contributing factor is APOE. The greatest genetic risk factor for AD is APOE4, a factor that is pronounced in female individuals post-menopause. Clinical data suggests that APOE impacts the response of AD patients to E2 replacement therapy. However, whether APOE4 prevents, is neutral, or promotes any positive effects of E2 is unclear. Therefore, our goal was to determine whether APOE modulates the impact of E2 on behavior and AD pathology in vivo. To that end, mice that express human APOE3 (E3FAD) or APOE4 (E4FAD) and overproduce Aß42 were ovariectomized at either 4 months (early) or 8 months (late) and treated with vehicle or E2 for 4 months. In E3FAD mice, we found that E2 mitigated the detrimental effect of ovariectomy on memory, with no effect on Aß in the early paradigm and only improved learning in the late paradigm. Although E2 lowered Aß in E4FAD mice in the early paradigm, there was no impact on learning or memory, possibly due to higher Aß pathology compared to E3FAD mice. In the late paradigm, there was no effect on learning/memory and Aß pathology in E4FAD mice. Collectively, these data support the idea that, in the presence of Aß pathology, APOE impacts the response to E2 supplementation post-menopause.

Nicotinamide Phosphoribosyltransferase Positive Allosteric Modulators Attenuate Neuronal Oxidative Stress.

Evidence supports boosting nicotinamide adenine dinucleotide (NAD+) to counteract oxidative stress in aging and neurodegenerative disease. One approach is to enhance the activity of nicotinamide phosphoribosyltransferase (NAMPT). Novel NAMPT positive allosteric modulators (N-PAMs) were identified. A cocrystal structure confirmed N-PAM binding to the NAMPT rear channel. Early hit-to-lead efforts led to a 1.88-fold maximum increase in the level of NAD+ in human THP-1 cells. Select N-PAMs were assessed for mitigation of reactive oxygen species (ROS) in HT-22 neuronal cells subject to inflammatory stress using tumor necrosis factor alpha (TNFα). N-PAMs that increased NAD+ more effectively in THP-1 cells attenuated TNFα-induced ROS more effectively in HT-22 cells. The most efficacious N-PAM completely attenuated ROS elevation in glutamate-stressed HT-22 cells, a model of neuronal excitotoxicity. This work demonstrates for the first time that N-PAMs are capable of mitigating elevated ROS in neurons stressed with TNFα and glutamate and provides support for further N-PAM optimization for treatment of neurodegenerative diseases.