The "Doorstop Pocket" In Thioredoxin Reductases─An Unexpected Druggable Regulator of the Catalytic Machinery.

Pyridine nucleotide-disulfide oxidoreductases are underexplored as drug targets, and thioredoxin reductases (TrxRs) stand out as compelling pharmacological targets. Selective TrxR inhibition is challenging primarily due to the reliance on covalent inhibition strategies. Recent studies identified a regulatory and druggable pocket in Schistosoma mansoni thioredoxin glutathione reductase (TGR), a TrxR-like enzyme, and an established drug target for schistosomiasis. This site is termed the "doorstop pocket" because compounds that bind there impede the movement of an aromatic side-chain necessary for the entry and exit of NADPH and NADP+ during enzymatic turnover. This discovery spearheaded the development of new TGR inhibitors with efficacies surpassing those of current schistosomiasis treatment. Targeting the "doorstop pocket" is a promising strategy, as the pocket is present in all members of the pyridine nucleotide-disulfide oxidoreductase family, opening new avenues for exploring therapeutic approaches in diseases where the importance of these enzymes is established, including cancer and inflammatory and infectious diseases.

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.

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.

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.

Synthesis, Optimization, and Structure-Activity Relationships of Nicotinamide Phosphoribosyltransferase (NAMPT) Positive Allosteric Modulators (N-PAMs).

Depletion of nicotinamide adenine dinucleotide (NAD+) is associated with aging and disease, spurring the study of dietary supplements to replenish NAD+. The catabolism of NAD+ to nicotinamide (NAM) requires the salvage of NAM to replenish cellular NAD+, which relies on the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT). Pharmacological activation of NAMPT provides an alternative to dietary supplements. Screening for activators of NAMPT identified small molecule NAMPT positive allosteric modulators (N-PAMs). N-PAMs bind to the rear channel of NAMPT increasing enzyme activity and alleviating feedback inhibition by NAM and NAD+. Synthesis of over 70 N-PAMs provided an excellent correlation between rear channel binding affinity and potency for enzyme activation, confirming the mechanism of allosteric activation via binding to the rear channel. The mechanism accounts for higher binding affinity leading to loss of efficacy. Enzyme activation translated directly to elevation of NAD+ measured in cells. Optimization led to an orally bioavailable N-PAM.

Non-covalent inhibitors of thioredoxin glutathione reductase with schistosomicidal activity in vivo.

Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target. TGR inhibitors identified to date are irreversible and/or covalent inhibitors with unacceptable off-target effects. In this work, we identify noncovalent TGR inhibitors with efficacy against schistosome infections in mice, meeting the criteria for lead progression indicated by WHO. Comparisons with previous in vivo studies with praziquantel suggests that these inhibitors outperform the drug of choice for schistosomiasis against juvenile worms.

TrkA expression directs the anti-neoplastic activity of MLK3 inhibitors in triple-negative breast cancer.

Mixed Lineage Kinase 3 (MLK3) is a viable target for neoplastic diseases; however, it is unclear whether its activators or inhibitors can act as anti-neoplastic agents. We reported that the MLK3 kinase activity was higher in triple-negative (TNBC) than in hormone receptor-positive human breast tumors, where estrogen inhibited MLK3 kinase activity and provided a survival advantage to ER+ breast cancer cells. Herein, we show that in TNBC, the higher MLK3 kinase activity paradoxically promotes cancer cell survival. Knockdown of MLK3 or MLK3 inhibitors, CEP-1347 and URMC-099, attenuated tumorigenesis of TNBC cell line and Patient-Derived (PDX) xenografts. The MLK3 kinase inhibitors decreased both the expression and activation of MLK3, PAK1, and NF-kB protein and caused cell death in TNBC breast xenografts. RNA-seq analysis identified several genes downregulated by MLK3 inhibition, and the NGF/TrkA MAPK pathway was significantly enriched in tumors sensitive to growth inhibition by MLK3 inhibitors. The TNBC cell line unresponsive to kinase inhibitor had substantially lower TrkA, and overexpression of TrkA restored the sensitivity to MLK3 inhibition. These results suggest that the functions of MLK3 in breast cancer cells depend on downstream targets in TNBC tumors expressing TrkA, and MLK3 kinase inhibition may provide a novel targeted therapy.

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.

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.

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.

Design of SARS-CoV-2 PLpro Inhibitors for COVID-19 Antiviral Therapy Leveraging Binding Cooperativity.

Antiviral agents that complement vaccination are urgently needed to end the COVID-19 pandemic. The SARS-CoV-2 papain-like protease (PLpro), one of only two essential cysteine proteases that regulate viral replication, also dysregulates host immune sensing by binding and deubiquitination of host protein substrates. PLpro is a promising therapeutic target, albeit challenging owing to featureless P1 and P2 sites recognizing glycine. To overcome this challenge, we leveraged the cooperativity of multiple shallow binding sites on the PLpro surface, yielding novel 2-phenylthiophenes with nanomolar inhibitory potency. New cocrystal structures confirmed that ligand binding induces new interactions with PLpro: by closing of the BL2 loop of PLpro forming a novel "BL2 groove" and by mimicking the binding interaction of ubiquitin with Glu167 of PLpro. Together, this binding cooperativity translates to the most potent PLpro inhibitors reported to date, with slow off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells.

MAP4K4 promotes pancreatic tumorigenesis via phosphorylation and activation of mixed lineage kinase 3.

MAP4K4 is a Ste20 member and reported to play important roles in various pathologies, including in cancer. However, the mechanism by which MAP4K4 promotes pancreatic cancer is not fully understood. It is suggested that MAP4K4 might function as a cancer promoter via specific downstream target(s) in an organ-specific manner. Here we identified MLK3 as a direct downstream target of MAP4K4. The MAP4K4 and MLK3 associates with each other, and MAP4K4 phosphorylates MLK3 on Thr738 and increases MLK3 kinase activity and downstream signaling. The phosphorylation of MLK3 by MAP4K4 promotes pancreatic cancer cell proliferation, migration, and colony formation. Moreover, MAP4K4 is overexpressed in human pancreatic tumors and directly correlates with the disease progression. The MAP4K4-specific pharmacological inhibitor, GNE-495, impedes pancreatic cancer cell growth, migration, induces cell death, and arrests cell cycle progression. Additionally, the GNE-495 reduced the tumor burden and extended survival of the KPC mice with pancreatic cancer. The MAP4K4 inhibitor also reduced MAP4K4 protein expression, tumor stroma, and induced cell death in murine pancreatic tumors. These findings collectively suggest that MLK3 phosphorylation by MAP4K4 promotes pancreatic cancer, and therefore therapies targeting MAP4K4 might alleviate the pancreatic cancer tumor burden in patients.

Identification of a potent Nrf2 displacement activator among aspirin-containing prodrugs.

Aspirin is a desired leaving group in prodrugs aimed at treatment of neurodegeneration and other conditions. A library of aspirin derivatives of various scaffolds potentially activating Nrf2 has been tested in Neh2-luc reporter assay which screens for direct Nrf2 protein stabilizers working via disruption of Nrf2-Keap1 interaction. Most aspirin prodrugs had a pro-alkylating or pro-oxidant motif in the structure and, therefore, were toxic at high concentrations. However, among the active compounds, we identified a molecule resembling a well-known Nrf2 displacement activator, bis-1,4-(4-methoxybenzenesulfonamidyl) naphthalene (NMBSA). The direct comparison of the newly identified compound with NMBSA and its improved analog in the reporter assay showed no quenching with N-acetyl cysteine, thus pointing to Nrf2 stabilization mechanism without cysteine alkylation. The potency of the newly identified compound in the reporter assay was much stronger than NMBSA, despite its inhibitory action in the commercial fluorescence polarization assay was observed only in the millimolar range. Molecular docking predicted that mono-deacetylation of the novel prodrug should generate a potent displacement activator. The time-course of reporter activation with the novel prodrug had a pronounced lag-period pointing to a plausible intracellular transformation leading to an active product. Treatment of the novel prodrug with blood plasma or cell lysate demonstrated stepwise deacetylation as judge by liquid chromatography-mass spectrometry (LC-MS). Hence, the esterase-catalyzed hydrolysis of the prodrug liberates only acetyl groups from aspirin moiety and generates a potent Nrf2 activator. The discovered mechanism of prodrug activation makes the newly identified compound a promising lead for future optimization studies.

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.

Discovery of Nonlipogenic ABCA1 Inducing Compounds with Potential in Alzheimer's Disease and Type 2 Diabetes.

Selective liver X receptor (LXR) agonists have been extensively pursued as therapeutics for Alzheimer's disease and related dementia (ADRD) and, for comorbidities such as type 2 diabetes (T2D) and cerebrovascular disease (CVD), disorders with underlying impaired insulin signaling, glucose metabolism, and cholesterol mobilization. The failure of the LXR-focused approach led us to pursue a novel strategy to discover nonlipogenic ATP-binding cassette transporter A1 (ABCA1) inducers (NLAIs): screening for ABCA1-luciferase activation in astrocytoma cells and counterscreening against lipogenic gene upregulation in hepatocarcinoma cells. Beneficial effects of LXRß agonists mediated by ABCA1 include the following: control of cholesterol and phospholipid efflux to lipid-poor apolipoproteins forming beneficial peripheral HDL and HDL-like particles in the brain and attenuation of inflammation. While rare, ABCA1 variants reduce plasma HDL and correlate with an increased risk of ADRD and CVD. In secondary assays, NLAI hits enhanced cholesterol mobilization and positively impacted in vitro biomarkers associated with insulin signaling, inflammatory response, and biogenic properties. In vivo target engagement was demonstrated after oral administration of NLAIs in (i) mice fed a high-fat diet, a model for obesity-linked T2D, (ii) mice administered LPS, and (iii) mice with accelerated oxidative stress. The lack of adverse effects on lipogenesis and positive effects on multiple biomarkers associated with T2D and ADRD supports this novel phenotypic approach to NLAIs as a platform for T2D and ADRD drug discovery.

Rapid Induction of the Unfolded Protein Response and Apoptosis by Estrogen Mimic TTC-352 for the Treatment of Endocrine-Resistant Breast Cancer.

Patients with long-term estrogen-deprived breast cancer, after resistance to tamoxifen or aromatase inhibitors develops, can experience tumor regression when treated with estrogens. Estrogen's antitumor effect is attributed to apoptosis via the estrogen receptor (ER). Estrogen treatment can have unpleasant gynecologic and nongynecologic adverse events; thus, the development of safer estrogenic agents remains a clinical priority. Here, we study synthetic selective estrogen mimics (SEM) BMI-135 and TTC-352, and the naturally occurring estrogen estetrol (E4), which are proposed as safer estrogenic agents compared with 17ß-estradiol (E2), for the treatment of endocrine-resistant breast cancer. TTC-352 and E4 are being evaluated in breast cancer clinical trials. Cell viability assays, real-time PCR, immunoblotting, ERE DNA pulldowns, mass spectrometry, X-ray crystallography, docking and molecular dynamic simulations, live cell imaging, and Annexin V staining were conducted in 11 biologically different breast cancer models. Results were compared with the potent full agonist E2, less potent full agonist E4, the benchmark partial agonist triphenylethylene bisphenol (BPTPE), and antagonists 4-hydroxytamoxifen and endoxifen. We report ERα's regulation and coregulators' binding profiles with SEMs and E4 We describe TTC-352's pharmacology as a weak full agonist and antitumor molecular mechanisms. This study highlights TTC-352's benzothiophene scaffold that yields an H-bond with Glu353, which allows Asp351-to-helix 12 (H12) interaction, sealing ERα's ligand-binding domain, recruiting E2-enriched coactivators, and triggering rapid ERα-induced unfolded protein response (UPR) and apoptosis, as the basis of its anticancer properties. BPTPE's phenolic OH yields an H-Bond with Thr347, which disrupts Asp351-to-H12 interaction, delaying UPR and apoptosis and increasing clonal evolution risk.

Screening and Reverse-Engineering of Estrogen Receptor Ligands as Potent Pan-Filovirus Inhibitors.

Filoviridae, including Ebola (EBOV) and Marburg (MARV) viruses, are emerging pathogens that pose a serious threat to public health. No agents have been approved to treat filovirus infections, representing a major unmet medical need. The selective estrogen receptor modulator (SERM) toremifene was previously identified from a screen of FDA-approved drugs as a potent EBOV viral entry inhibitor, via binding to EBOV glycoprotein (GP). A focused screen of ER ligands identified ridaifen-B as a potent dual inhibitor of EBOV and MARV. Optimization and reverse-engineering to remove ER activity led to a novel compound 30 (XL-147) showing potent inhibition against infectious EBOV Zaire (0.09 µM) and MARV (0.64 µM). Mutagenesis studies confirmed that inhibition of EBOV viral entry is mediated by the direct interaction with GP. Importantly, compound 30 displayed a broad-spectrum antifilovirus activity against Bundibugyo, Tai Forest, Reston, and Menglà viruses and is the first submicromolar antiviral agent reported for some of these strains, therefore warranting further development as a pan-filovirus inhibitor.

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.

Synthesis of α-Ketoamide-Based Stereoselective Calpain-1 Inhibitors as Neuroprotective Agents.

Calpain inhibitors have been proposed as drug candidates for neurodegenerative disorders, with ABT-957 entering clinical trials for Alzheimer's disease and mild cognitive impairment. The structure of ABT-957 was very recently disclosed, and trials were terminated owing to inadequate CNS concentrations to obtain a pharmacodynamic effect. The multistep synthesis of an α-ketoamide peptidomimetic inhibitor series potentially including ABT-957 was optimized to yield diastereomerically pure compounds that are potent and selective for calpain-1 over papain and cathepsins B and K. As the final oxidation step, with its optimized synthesis protocol, does not alter the configuration of the substrate, the synthesis of the diastereomeric pair (R)-1-benzyl-N-((S)-4-((4-fluorobenzyl)amino)-3,4-dioxo-1-phenylbutan-2-yl)-5-oxopyrrolidine-2-carboxamide (1 c) and (R)-1-benzyl-N-((R)-4-((4-fluorobenzyl)amino)-3,4-dioxo-1-phenylbutan-2-yl)-5-oxopyrrolidine-2-carboxamide (1 g) was feasible. This allowed the exploration of stereoselective inhibition of calpain-1, with 1 c (IC50 =78 nM) being significantly more potent than 1 g. Moreover, inhibitor 1 c restored cognitive function in amnestic mice.

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).