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The Chromodomain helicase DNA-binding protein 1-like (CHD1L) is a nucleosome remodeling enzyme, which plays a key role in chromatin relaxation during the DNA damage response. Genome editing has shown that deletion of CHD1L sensitizes cells to PARPi, but the effect of its pharmacological inhibition has not been defined. Triple-negative breast cancer SUM149PT, HCC1937, and MDA-MB-231 cells were used to assess the mechanism of action of the CHD1Li OTI-611. Cytotoxicity as a single agent or in combination with standard-of-care treatments was assessed in tumor organoids. Immunofluorescence was used to assess the translocation of PAR and AIF to the cytoplasm or the nucleus and to study markers of DNA damage or apoptosis. Trapping of PARP1/2 or CHD1L onto chromatin was also assessed by in situ subcellular fractionation and immunofluorescence and validated by Western blot. We show that the inhibition of CHD1L's ATPase activity by OTI-611 is cytotoxic to triple-negative breast cancer tumor organoids and synergizes with PARPi and chemotherapy independently of the BRCA mutation status. The inhibition of the remodeling function blocks the phosphorylation of H2AX, traps CHD1L on chromatin, and leaves PAR chains on PARP1/2 open for hydrolysis. PAR hydrolysis traps PARP1/2 at DNA damage sites and mediates PAR translocation to the cytoplasm, release of AIF from the mitochondria, and induction of PARthanatos. The targeted inhibition of CHD1L's oncogenic function by OTI-611 signifies an innovative therapeutic strategy for breast cancer and other cancers. This approach capitalizes on CHD1L-mediated DNA repair and cell survival vulnerabilities, thereby creating synergy with standard-of-care therapies.

Chromodomain helicase DNA-binding protein 1 like (CHD1L) is an oncogene that promotes tumor progression, metastasis, and multidrug resistance. CHD1L expression is indicative of poor outcomes and low survival in cancer patients with various cancer types. Herein, we report a set of CHD1L inhibitors (CHD1Li) discovered from high-throughput screening and evaluated using enzyme inhibition, 3D tumor organoid cytotoxicity and mechanistic assays. The structurally distinct compounds 8-11 emerged as hits with promising bioactivity by targeting CHD1L. CHD1Li were further examined for their stability in human and mouse liver microsomes, which showed compounds 9 and 11 to be the most metabolically stable. Additionally, molecular modeling studies of CHD1Li with the target protein shed light on key pharmacophore features driving CHD1L binding. Taken together, these results expand the chemical space of CHD1Li as a potential targeted therapy for colorectal cancer and other cancers., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: D.V.L. is the founder of Onconaut Therapeutics Incorporated, which is developing CHD1Li as a lead drug therapies for the treatment of cancer. D.V.L, H.E., P.A., Q.Z. are inventors on patents pertaining to this research., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

PARP1/2 inhibitors (PARPi) are effective clinically used drugs for the treatment of cancers with BRCA deficiencies. PARPi have had limited success and applicability beyond BRCA deficient cancers, and their effect is diminished by resistance mechanisms. The recent discovery of Histone PARylation Factor (HPF1) and the role it plays in the PARylation reaction by forming a shared active site with PARP1 raises the possibility that novel inhibitors that target the PARP1-HPF1 complex can be identified. Herein we describe a simple and cost-effective high-throughput screening (HTS) method aimed at discovering inhibitors of the PARP1-HPF1 complex. Upon HTS validation, we first applied this method to screen a small PARP-focused library of compounds and then scale up our approach using robotic automation to conduct a pilot screen of 10,000 compounds and validating >100 hits. This work demonstrates for the first time the capacity to discover potent inhibitors of the PARP1-HPF1 complex, which may have utility as probes to better understand the DNA damage response and as therapeutics for cancer., Competing Interests: Declaration of Competing Interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mutations in the promoter of the human Telomerase Reverse Transcriptase (hTERT) gene are common and associated with its elevated expression in bladder cancer, melanoma, and glioblastoma. Though these mutations and TERT overexpression are associated with aggressive disease and poor outcome, an incomplete understanding of mutant TERT regulation limits treatment options directed at this gene. Herein, we unravel a signaling pathway that leads to upregulated hTERT expression resulting from the -124 bp promoter mutation, the most frequent variant across human cancer. We employed engineered bladder cancer cells that harbor a GFP insertion at the TSS region on -124 hTERT promoter for high-content screening drug discovery using a focused library of ~800 kinase inhibitors. Studies using in vitro and in vivo models prioritized AST-487, an inhibitor of the wild-type, and mutant RET (rearranged during transfection) proto-oncogene as a novel drug inhibitor of both wild-type and mutant promoter-driven hTERT expression. We also identified the RET kinase pathway, targeted by AST-487, as a novel regulator of mutant hTERT promoter-driven transcription in bladder cancer cells. Collectively, our work provides new potential precision medicine approaches for cancer patients with upregulated hTERT expression, perhaps, especially those harboring mutations in both the RET gene and the hTERT promoter, such as in thyroid cancer.

Molecular subtypes of muscle-invasive bladder cancer (MIBC) display differential survival and drug sensitivities in clinical trials. To date, they have not been used as a paradigm for phenotypic drug discovery. This study aimed to discover novel subtype-stratified therapy approaches based on high-content screening (HCS) drug discovery. Transcriptome expression data of CCLE and BLA-40 cell lines were used for molecular subtype assignment in basal, luminal, and mesenchymal-like cell lines. Two independent HCSs, using focused compound libraries, were conducted to identify subtype-specific drug leads. We correlated lead drug sensitivity data with functional genomics, regulon analysis, and in-vitro drug response-based enrichment analysis. The basal MIBC subtype displayed sensitivity to HDAC and CHK inhibitors, while the luminal subtype was sensitive to MDM2 inhibitors. The mesenchymal-like cell lines were exclusively sensitive to the ITGAV inhibitor SB273005. The role of integrins within this mesenchymal-like MIBC subtype was confirmed via its regulon activity and gene essentiality based on CRISPR-Cas9 knock-out data. Patients with high ITGAV expression showed a significant decrease in the median overall survival. Phenotypic high-content drug screens based on bladder cancer cell lines provide rationales for novel stratified therapeutic approaches as a framework for further prospective validation in clinical trials.

The ubiquity of cognitive deficits and early onset Alzheimer's disease in Down syndrome (DS) has focused much DS iPSC-based research on neuron degeneration and regeneration. Despite reports of elevated oxidative stress in DS brains, few studies assess the impact of this oxidative burden on iPSC differentiation. Here, we evaluate cellular specific redox differences in DS and euploid iPSCs and neural progenitor cells (NPCs) during critical intermediate stages of differentiation. Despite successful generation of NPCs, our results indicate accelerated neuroectodermal differentiation of DS iPSCs compared to isogenic, euploid controls. Specifically, DS embryoid bodies (EBs) and neural rosettes prematurely develop with distinct morphological differences from controls. Additionally, we observed developmental stage-specific alterations in mitochondrial superoxide production and SOD1/2 abundance, coupled with modulations in thioredoxin, thioredoxin reductase, and peroxiredoxin isoforms. Disruption of intracellular redox state and its associated signaling has the potential to disrupt cellular differentiation and development in DS lending to DS-specific phenotypes., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Chromodomain helicase DNA-binding protein 1 like (CHD1L) is an oncogene implicated in tumor progression, multidrug resistance, and metastasis in many types of cancer. In this article, we described the optimization of the first lead CHD1L inhibitors (CHD1Li) through drug design and medicinal chemistry. More than 30 CHD1Li were synthesized and evaluated using a variety of colorectal cancer (CRC) tumor organoid models and functional assays. The results led to the prioritization of six lead CHD1Li analogues with improved potency, antitumor activity, and drug-like properties including metabolic stability and in vivo pharmacokinetics. Furthermore, lead CHD1Li 6.11 proved to be an orally bioavailable antitumor agent, significantly reducing the tumor volume of CRC xenografts generated from isolated quasi mesenchymal cells (M-phenotype), which possess enhanced tumorigenic properties. In conclusion, we reported the optimization of first-in-class inhibitors of oncogenic CHD1L as a novel therapeutic strategy with potential for the treatment of cancer.

Bladder cancer (BC) has a 70% telomerase reverse transcriptase (TERT or hTERT in humans) promoter mutation prevalence, commonly at -124 base pairs, and this is associated with increased hTERT expression and poor patient prognosis. We inserted a green fluorescent protein (GFP) tag in the mutant hTERT promoter allele to create BC cells expressing an hTERT-GFP fusion protein. These cells were used in a fluorescence-activated cell sorting-based pooled CRISPR-Cas9 Kinome knockout genetic screen to identify tripartite motif containing 28 (TRIM28) and TRIM24 as regulators of hTERT expression. TRIM28 activates, while TRIM24 suppresses, hTERT transcription from the mutated promoter allele. TRIM28 is recruited to the mutant promoter where it interacts with TRIM24, which inhibits its activity. Phosphorylation of TRIM28 through the mTOR complex 1 (mTORC1) releases it from TRIM24 and induces hTERT transcription. TRIM28 expression promotes in vitro and in vivo BC cell growth and stratifies BC patient outcome. mTORC1 inhibition with rapamycin analog Ridaforolimus suppresses TRIM28 phosphorylation, hTERT expression, and cell viability. This study may lead to hTERT-directed cancer therapies with reduced effects on normal progenitor cells., Competing Interests: Competing interest statement: T.R.C. is on the board of directors of Merck, Inc. and a scientific advisor to Storm Therapeutics and Eikon Therapeutics.

Epithelial-mesenchymal transition (EMT) is a driving force in promoting malignant cancer, including initiation, growth, and metastasis. EMT is a dynamic process that can undergo a mesenchymal-epithelial transition (MET) and partial transitions between both phenotypes, termed epithelial-mesenchymal plasticity (EMP). In cancer, the acquisition of EMP results in a spectrum of phenotypes, promoting tumor cell heterogeneity and resistance to standard of care therapy. Here we describe a real-time fluorescent dual-reporter for vimentin and E-cadherin, biomarkers of the mesenchymal and epithelial cell phenotypes, respectively. Stable dual-reporter cell lines generated from colorectal (SW620), lung (A549), and breast (MDA-MB-231) cancer demonstrate a spectrum of EMT cell phenotypes. We used the dual-reporter to isolate the quasi epithelial, epithelial/mesenchymal, and mesenchymal phenotypes. Although EMT is a dynamic process, these isolated quasi-EMT-phenotypes remain stable to spontaneous EMP in the absence of stimuli and during prolonged cell culture. However, the quasi-EMT phenotypes can readily be induced to undergo EMT or MET with growth factors or small molecules. Moreover, isolated EMT phenotypes display different tumorigenic properties and are morphologically and metabolically distinct. 3D high-content screening of ~23,000 compounds using dual-reporter mesenchymal SW620 tumor organoids identified small molecule probes that modulate EMT, and a subset of probes that effectively induced MET. The tools, probes, and models described herein provide a coherent mechanistic understanding of mesenchymal cell plasticity. Future applications utilizing this technology and probes are expected to advance our understanding of EMT and studies aimed at therapeutic strategies targeting EMT.

Since its first report in 1956 by Puck and Marcus, the clonogenic assay has not been completely adapted into high-content-screening (HCS) workflows despite the numerous automated systems available. Initially, clonogenic assays were used to observe the effects of radiation on cell survival, particularly with cancer cells. The clonogenic assay has since been well characterized as a measure of cancer stem cell (CSC) stemness, demonstrating that a single CSC can generate clonogenic colonies. CSCs are highly tumorigenic with an unlimited proliferation potential and capacity to generate malignant tumors. Furthermore, CSCs are also known to resist conventional chemotherapy as well as more contemporary targeted therapies alike. Therefore, given the complexity of CSCs and their clinical relevance, new methods must follow to more effectively study and characterize CSC mechanisms that allow them to proliferate and persist, and to develop drugs and other therapies that can more effectively target these populations. Herein, we present a HCS method to quantify the number and size of colonies in 2D and 3D culture models and to distinguish colonies based on fluorescent markers using an Opera Phenix high-content-screening system. In addition, we present a method to scan at low magnification and rescan at a higher magnification to capture in greater detail colonies or even single cells of interest. These methods can be adapted to numerous applications or other imaging systems to study CSC biology using high-content analysis and for high-throughput drug discovery.

Objective: Our recent work demonstrates that PTEN (phosphatase and tensin homolog) is an important regulator of smooth muscle cell (SMC) phenotype. SMC-specific PTEN deletion promotes spontaneous vascular remodeling and PTEN loss correlates with increased atherosclerotic lesion severity in human coronary arteries. In mice, PTEN overexpression reduces plaque area and preserves SMC contractile protein expression in atherosclerosis and blunts Ang II (angiotensin II)-induced pathological vascular remodeling, suggesting that pharmacological PTEN upregulation could be a novel therapeutic approach to treat vascular disease. Approach and Results: To identify novel PTEN activators, we conducted a high-throughput screen using a fluorescence based PTEN promoter-reporter assay. After screening ≈3400 compounds, 11 hit compounds were chosen based on level of activity and mechanism of action. Following in vitro confirmation, we focused on 5-azacytidine, a DNMT1 (DNA methyltransferase-1) inhibitor, for further analysis. In addition to PTEN upregulation, 5-azacytidine treatment increased expression of genes associated with a differentiated SMC phenotype. 5-Azacytidine treatment also maintained contractile gene expression and reduced inflammatory cytokine expression after PDGF (platelet-derived growth factor) stimulation, suggesting 5-azacytidine blocks PDGF-induced SMC de-differentiation. However, these protective effects were lost in PTEN-deficient SMCs. These findings were confirmed in vivo using carotid ligation in SMC-specific PTEN knockout mice treated with 5-azacytidine. In wild type controls, 5-azacytidine reduced neointimal formation and inflammation while maintaining contractile protein expression. In contrast, 5-azacytidine was ineffective in PTEN knockout mice, indicating that the protective effects of 5-azacytidine are mediated through SMC PTEN upregulation., Conclusions: Our data indicates 5-azacytidine upregulates PTEN expression in SMCs, promoting maintenance of SMC differentiation and reducing pathological vascular remodeling in a PTEN-dependent manner.

Since the discovery of CHD1L in 2008, it has emerged as an oncogene implicated in the pathology and poor prognosis of a variety of cancers, including gastrointestinal cancers. However, a mechanistic understanding of CHD1L as a driver of colorectal cancer has been limited. Until now, there have been no reported inhibitors of CHD1L, also limiting its development as a molecular target. We sought to characterize the clinicopathologic link between CHD1L and colorectal cancer, determine the mechanism(s) by which CHD1L drives malignant colorectal cancer, and discover the first inhibitors with potential for novel treatments for colorectal cancer. The clinicopathologic characteristics associated with CHD1L expression were evaluated using microarray data from 585 patients with colorectal cancer. Further analysis of microarray data indicated that CHD1L may function through the Wnt/TCF pathway. Thus, we conducted knockdown and overexpression studies with CHD1L to determine its role in Wnt/TCF-driven epithelial-to-mesenchymal transition (EMT). We performed high-throughput screening (HTS) to identify the first CHD1L inhibitors. The mechanism of action, antitumor efficacy, and drug-like properties of lead CHD1L inhibitors were determined using biochemical assays, cell models, tumor organoids, patient-derived tumor organoids, and in vivo pharmacokinetics and pharmacodynamics. Lead CHD1L inhibitors display potent in vitro antitumor activity by reversing TCF-driven EMT. The best lead CHD1L inhibitor possesses drug-like properties in pharmacokinetic/pharmacodynamic mouse models. This work validates CHD1L as a druggable target and establishes a novel therapeutic strategy for the treatment of colorectal cancer., (©2020 American Association for Cancer Research.)

Metastasis is the cause of 90% of mortality in cancer patients. For metastatic colorectal cancer (mCRC), the standard-of-care drug therapies only palliate the symptoms but are ineffective, evidenced by a low survival rate of ∼11%. T-cell factor (TCF) transcription is a major driving force in CRC, and we have characterized it to be a master regulator of epithelial-mesenchymal transition (EMT). EMT transforms relatively benign epithelial tumor cells into quasi-mesenchymal or mesenchymal cells that possess cancer stem cell properties, promoting multidrug resistance and metastasis. We have identified topoisomerase IIα (TOP2A) as a DNA-binding factor required for TCF-transcription. Herein, we describe the design, synthesis, biological evaluation, and in vitro and in vivo pharmacokinetic analysis of TOP2A ATP-competitive inhibitors that prevent TCF-transcription and modulate or reverse EMT in mCRC. Unlike TOP2A poisons, ATP-competitive inhibitors do not damage DNA, potentially limiting adverse effects. This work demonstrates a new therapeutic strategy targeting TOP2A for the treatment of mCRC and potentially other types of cancers.

Cancer cell lines are critical models to study tumor progression and response to therapy. In 2008, we showed that approximately 50% of thyroid cancer cell lines were redundant or not of thyroid cancer origin. We therefore generated new authenticated thyroid cancer cell lines and patient-derived xenograft (PDX) models using in vitro and feeder cell approaches, and characterized these models in vitro and in vivo . We developed four thyroid cancer cell lines, two derived from 2 different patients with papillary thyroid cancer (PTC) pleural effusions, CUTC5, and CUTC48; one derived from a patient with anaplastic thyroid cancer (ATC), CUTC60; and one derived from a patient with follicular thyroid cancer (FTC), CUTC61. One PDX model (CUTC60-PDX) was also developed. Short tandem repeat (STR) genotyping showed that each cell line and PDX is unique and match the original patient tissue. The CUTC5 and CUTC60 cells harbor the BRAF (V600E) mutation, the CUTC48 cell line expresses the RET/PTC1 rearrangement, and the CUTC61 cells have the HRAS (Q61R) mutation. Moderate to high levels of PAX8 and variable levels of NKX2-1 were detected in each cell line and PDX. The CUTC5 and CUTC60 cell lines form tumors in orthotopic and flank xenograft mouse models. IMPLICATIONS: We have developed the second RET/PTC1-expressing PTC-derived cell line in existence, which is a major advance in studying RET signaling. We have further linked all cell lines to the originating patients, providing a set of novel, authenticated thyroid cancer cell lines and PDX models to study advanced thyroid cancer., (©2019 American Association for Cancer Research.)

HIV-1 protease autoprocessing liberates the free mature protease from its Gag-Pol polyprotein precursor through a series of highly regulated autoproteolysis reactions. Herein, we report the development and validation (Z' ≥ 0.50) of a cell-based functional assay for high-throughput screening (HTS) of autoprocessing inhibitors using fusion precursors in combination with AlphaLISA (amplified luminescent proximity homogeneous assay ELISA). Through pilot screening of a collection of 130 known protease inhibitors, the AlphaLISA assay confirmed all 11 HIV protease inhibitors in the library capable of suppressing precursor autoprocessing at low micromolar concentrations. Meanwhile, other protease inhibitors had no impact on precursor autoprocessing. We next conducted HTS of ~23,000 compounds but found no positive hits. Such high selectivity is advantageous for large-scale HTS campaigns and as anticipated based on assay design because a positive hit needs simultaneously to be nontoxic, cell permeable, and inhibiting precursor autoprocessing. Furthermore, AlphaLISA quantification of fusion precursors carrying mutations known to cause resistance to HIV protease inhibitors faithfully recapitulated the reported resistance, suggesting that precursor autoprocessing is a critical step contributing to drug resistance. Taken together, this reported AlphaLISA platform will provide a useful tool for drug discovery targeting HIV-1 protease autoprocessing and for quantification of PI resistance.

Purpose: To define the genetic landscape of advanced differentiated and anaplastic thyroid cancer (ATC) and identify genetic alterations of potential diagnostic, prognostic, and therapeutic significance. Experimental Design: The genetic profiles of 583 advanced differentiated and 196 ATCs generated with targeted next-generation sequencing cancer-associated gene panels MSK-IMPACT and FoundationOne were analyzed. Results: ATC had more genetic alterations per tumor, and pediatric papillary thyroid cancer had fewer genetic alterations per tumor when compared with other thyroid cancer types. DNA mismatch repair deficit and activity of APOBEC cytidine deaminases were identified as mechanisms associated with high mutational burden in a subset of differentiated thyroid cancers and ATCs. Copy number losses and mutations of CDKN2A and CDKN2B , amplification of CCNE1 , amplification of receptor tyrosine kinase genes KDR, KIT , and PDGFRA , amplification of immune evasion genes CD274, PDCD1LG2 , and JAK2 , and activating point mutations in small GTPase RAC1 were associated with ATC. An association of KDR, KIT , and PDGFRA amplification with the sensitivity of thyroid cancer cells to lenvatinib was shown in vitro Three genetically distinct types of ATCs are proposed. Conclusions: This large-scale analysis describes genetic alterations in a cohort of thyroid cancers enriched in advanced cases. Many novel genetic events previously not seen in thyroid cancer were found. Genetic alterations associated with anaplastic transformation were identified. An updated schematic of thyroid cancer genetic evolution is proposed. Clin Cancer Res; 24(13); 3059-68. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Chikungunya virus (CHIKV) is an arthropod-borne alphavirus. Alphaviruses are positive strand RNA viruses that require a 5' cap structure to direct translation of the viral polyprotein and prevent degradation of the viral RNA genome by host cell nucleases. Formation of the 5' RNA cap is orchestrated by the viral protein nsP1, which binds GTP and provides the N-7 methyltransferase and guanylyltransferase activities that are necessary for cap formation. Viruses with aberrant nsP1 activity are unable to replicate effectively suggesting that nsP1 is a promising target for antiviral drug discovery. Given the absence of commercially available antiviral therapies for CHIKV, it is imperative to identify compounds that could be developed as potential therapeutics. This study details a high-throughput screen of 3051 compounds from libraries containing FDA-approved drugs, natural products, and known bioactives against CHIKV nsP1 using a fluorescence polarization-based GTP competition assay. Several small molecule hits from this screen were able to compete with GTP for the CHIKV nsP1 GTP binding site at low molar concentrations. Compounds were also evaluated with an orthogonal assay that measured the ability of nsP1 to perform the guanylation step of the capping reaction in the presence of inhibitor. In addition, live virus assays with CHIKV and closely related alphavirus, Sindbis virus, were used in conjunction with cell toxicity assays to determine the antiviral activity of compounds in cell culture. The naturally derived compound lobaric acid was found to inhibit CHIKV nsP1 GTP binding and guanylation as well as attenuate viral growth in vitro at both 24 hpi and 48 hpi in hamster BHK21 and human Huh 7 cell lines. These data indicate that development of lobaric acid and further exploration of CHIKV nsP1 as a drug target may aid in the progress of anti-alphaviral drug development strategies., (Copyright © 2018. Published by Elsevier B.V.)

Diabetes mellitus is a chronic disease that is becoming a serious global health problem. Diabetes has been considered to be one of the major risks of cataract and retinopathy. Synthetic and natural product inhibitors of carbohydrate degrading enzymes are able to reduce type 2 diabetes and its complications. For a long time, potatoes have been portrayed as unhealthy for diabetic patients by some nutritionist due to their high starch content. However, purple and red potato cultivars have received considerable attention from consumers because they have high levels of polyphenolic compounds that have potent antioxidant activities. In this study, we screened the total phenolics (TP) and total anthocyanins (TA) and analyzed the phenolic and anthocyanin compounds in selected potato cultivars and advanced selections with distinct flesh colors (purple, red, yellow and white). Purple and red potato cultivars had higher levels of TP and TA than tubers with other flesh colors. Chlorogenic acid is the predominant phenolic acid, and major anthocyanin is composed of the derivatives of petunidin, peonidin, malvidin and pelargonidin. We tested the potential inhibitory effect of potato extracts on the activities of α-amylase and α-glucosidase, which were targeted to develop antidiabetic therapeutic agents. We also measured inhibitory effect of potato extracts on aldose reductase (AR) which is a key enzyme that has been a major drug target for the development of therapies to treat diabetic complications. Purple flesh tubers extract showed the most effective inhibition of α-amylase, α-glucosidase, and aldose reductase with IC50 values 25, 42, and 32 μg/ml, respectively. Kinetic studies showed that anthocyanins are noncompetitive inhibitors of these enzymes, whereas phenolic acids behaved as mixed inhibitors for α-amylase and α-glucosidase and noncompetitive inhibitors for AR. This study supports the development of a positive and healthful image of potatoes, which is an important issue for consumers.

Half of estrogen receptor-positive breast cancers contain a subpopulation of cytokeratin 5 (CK5)-expressing cells that are therapy resistant and exhibit increased cancer stem cell (CSC) properties. We and others have demonstrated that progesterone (P4) increases CK5+ breast cancer cells. We previously discovered that retinoids block P4 induction of CK5+ cells. Here we investigated the mechanisms by which progesterone receptors (PR) and retinoic acid receptors (RAR) regulate CK5 expression and breast CSC activity. After P4 treatment, sorted CK5+ compared to CK5- cells were more tumorigenic in vivo. In vitro, P4-treated breast cancer cells formed larger mammospheres and silencing of CK5 using small hairpin RNA abolished this P4-dependent increase in mammosphere size. Retinoic acid (RA) treatment blocked the P4 increase in CK5+ cells and prevented the P4 increase in mammosphere size. Dual small interfering RNA (siRNA) silencing of RARα and RARγ reversed RA blockade of P4-induced CK5. Using promoter deletion analysis, we identified a region 1.1 kb upstream of the CK5 transcriptional start site that is necessary for P4 activation and contains a putative progesterone response element (PRE). We confirmed by chromatin immunoprecipitation that P4 recruits PR to the CK5 promoter near the -1.1 kb essential PRE, and also to a proximal region near -130 bp that contains PRE half-sites and a RA response element (RARE). RA induced loss of PR binding only at the proximal site. Interestingly, RARα was recruited to the -1.1 kb PRE and the -130 bp PRE/RARE regions with P4, but not RA alone or RA plus P4. Treatment of breast cancer xenografts in vivo with the retinoid fenretinide reduced the accumulation of CK5+ cells during estrogen depletion. This reduction, together with the inhibition of CK5+ cell expansion through RAR/PR cross talk, may explain the efficacy of retinoids in prevention of some breast cancer recurrences.

Protozoan parasites infect and kill millions of people worldwide every year, particularly in developing countries where access to clean fresh water is limited. Among the most common are intestinal parasites, including Giardia lamblia and Entamoeba histolytica. These parasites wreak havoc on the epithelium lining the small intestines (G. lamblia) and colon (E. histolytica) causing giardiasis and amebiasis, respectively. In addition, there are less common but far more deadly pathogens such as Naegleria fowleri that thrive in warm waters and infect the central nervous systems of their victims via the nasal passages. Despite their prevalence and associated high mortality rates, there remains an unmet need to identify more effective therapeutics for people infected with these opportunistic parasites. To address this unmet need, we have surveyed plants and traditional herbal medicines known throughout the world to identify novel antiparasitic agents with activity against G. lamblia, E. histolytica, and N. fowleri. Herein, we report Larrea tridentata, known as creosote bush, as a novel source for secondary metabolites that display antiparasitic activity against all three pathogens. This report also characterizes the lignan compound classes, nordihydroguairetic acid and demethoxyisoguaiacin, as novel antiparasitic lead agents to further develop more effective drug therapy options for millions of people worldwide.

Aberrant T-cell factor (TCF) transcription is implicated in the majority of colorectal cancers (CRCs). TCF transcription induces epithelial-mesenchymal transition (EMT), promoting a tumor-initiating cell (TIC) phenotype characterized by increased proliferation, multidrug resistance (MDR), invasion and metastasis. The data presented herein characterize topoisomerase IIα (TopoIIα) as a required component of TCF transcription promoting EMT. Using chromatin immunoprecipitation (ChIP) and protein co-immunoprecipitation (co-IP) studies, we show that TopoIIα forms protein-protein interactions with β-catentin and TCF4 and interacts with Wnt response elements (WREs) and promoters of direct target genes of TCF transcription, including: MYC, vimentin, AXIN2 and LEF1. Moreover, both TopoIIα and TCF4 ChIP with the N-cadherin promoter, which is a new discovery indicating that TCF transcription may directly regulate N-cadherin expression. TopoIIα N-terminal ATP-competitive inhibitors, exemplified by the marine alkaloid neoamphimedine (neo), block TCF activity in vitro and in vivo. Neo effectively inhibits TopoIIα and TCF4 from binding WREs/promoter sites, whereas protein-protein interactions remain intact. Neo inhibition of TopoIIα-dependent TCF transcription also correlates with significant antitumor effects in vitro and in vivo, including the reversion of EMT, the loss of TIC-mediated clonogenic colony formation, and the loss of cell motility and invasion. Interestingly, non-ATP-competitive inhibitors of TopoIIα, etoposide and merbarone, were ineffective at preventing TopoIIα-dependent TCF transcription. Thus, we propose that TopoIIα participation in TCF transcription may convey a mechanism of MDR to conventional TopoIIα inhibitors. However, our results indicate that TopoIIα N-terminal ATP-binding sites remain conserved and available for drug targeting. This article defines a new strategy for targeted inhibition of TCF transcription that may lead to effective therapies for the treatment of CRC and potentially other Wnt-dependent cancers.

Aldose reductase (AR) in the lens plays an important role in the pathogenesis of diabetic cataract (DC) by contributing to osmotic and oxidative stress associated with accelerated glucose metabolism through the polyol pathway. Therefore, inhibition of AR in the lens may hold the key to prevent DC formation. Emodin, a bioactive compound isolated from plants, has been implicated as a therapy for diabetes. However, its inhibitory activity against AR remains unclear. Our results showed that emodin has good selectively inhibitory activity against AR (IC50 = 2.69 ± 0.90 μM) but not other aldo-keto reductases and is stable at 37 °C for at least 7 days. Enzyme kinetic studies demonstrated an uncompetitive inhibition against AR with a corresponding inhibition constant of 2.113 ± 0.095 μM. In in vivo studies, oral administration of emodin reduced the incidence and severity of morphological markers of cataract in lenses of AR transgenic mice. Computational modeling of the AR-NADP(+)-emodin ternary complex indicated that the 3-hydroxy group of emodin plays an essential role by interacting with Ser302 through hydrogen bonding in the specificity pocket of AR. All the findings above provide encouraging evidence for emodin as a potential therapeutic agent to prevent cataract in diabetic patients.

3D organotypic culture models such as organoids and multicellular tumor spheroids (MCTS) are becoming more widely used for drug discovery and toxicology screening. As a result, 3D culture technologies adapted for high-throughput screening formats are prevalent. While a multitude of assays have been reported and validated for high-throughput imaging (HTI) and high-content screening (HCS) for novel drug discovery and toxicology, limited HTI/HCS with large compound libraries have been reported. Nonetheless, 3D HTI instrumentation technology is advancing and this technology is now on the verge of allowing for 3D HCS of thousands of samples. This review focuses on the state-of-the-art high-throughput imaging systems, including hardware and software, and recent literature examples of 3D organotypic culture models employing this technology for drug discovery and toxicology screening., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Docking studies of tubulin-targeting C2-substituted 7-deazahypoxanthine analogues of marine alkaloid rigidins led to the design and synthesis of compounds containing linear C2-substituents. The C2-alkynyl analogue was found to have double- to single-digit nanomolar antiproliferative IC50 values and showed statistically significant tumor size reduction in a colon cancer mouse model at nontoxic concentrations. These results provide impetus and further guidance for the development of these rigidin analogues as anticancer agents.

Chronic hyperglycemia is an important risk factor involved in the onset and progression of diabetic retinopathy (DR). Among other effectors, aldose reductase (AR) has been linked to the pathogenesis of this degenerative disease. The purpose of this study was to investigate whether the novel AR inhibitor, beta-glucogallin (BGG), can offer protection against various hyperglycemia-induced abnormalities in human adult retinal pigment epithelial (ARPE-19) cells. AR is an enzyme that contributes to cellular stress by production of reactive oxygen species (ROS) under high glucose conditions. A marked decrease in cell viability (from 100% to 78%) following long-term exposure (4 days) of RPE cells to high glucose (HG) was largely prevented by siRNA-mediated knockdown of AR gene expression (from 79% to 97%) or inhibition using sorbinil (from 66% to 86%). In HG, BGG decreased sorbitol accumulation (44%), ROS production (27%) as well as ER stress (22%). Additionally, we demonstrated that BGG prevented loss of mitochondrial membrane potential (MMP) under HG exposure. We also showed that AR inhibitor pretreatment reduced retinal microglia-induced apoptosis in APRE-19 cells. These results suggest that BGG may be useful as a therapeutic agent against retinal degeneration in the diabetic eye by preventing RPE cell death., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Recently, we characterized neoamphimedine (neo) as an ATP-competitive inhibitor of the ATPase domain of human Topoisomerase IIα. Thus far, neo is the only pyridoacridine with this mechanism of action. One limiting factor in the development of neo as a therapeutic agent has been access to sufficient amounts of material for biological testing. Although there are two reported syntheses of neo, both require 12 steps with low overall yields (≤6%). In this article, we report an improved total synthesis of neo achieved in 10 steps with a 25% overall yield. In addition, we report an expanded cytotoxicity study using a panel of human cancer cell lines, including: breast, colorectal, lung, and leukemia. Neo displays potent cytotoxicity (nM IC50 values) in all, with significant potency against colorectal cancer (lowest IC50 = 6 nM). We show that neo is cytotoxic not cytostatic, and that neo exerts cytotoxicity by inducing G2-M cell cycle arrest and apoptosis.

Purpose: Retinal microglia become activated in diabetes and produce pro-inflammatory molecules associated with changes in retinal vasculature and increased apoptosis of retinal neurons and glial cells. We sought to determine if the action of aldose reductase (AR), an enzyme linked to the pathogenesis of diabetic retinopathy, contributes to activation of microglial cells., Methods: Involvement of AR in the activation process was studied using primary cultures of retinal microglia (RMG) isolated from wild-type and AR-null mice, or in mouse macrophage cultures treated with either AR inhibitors or small interfering RNA (siRNA) directed to AR. Inflammatory cytokines were measured by ELISA. Cell migration was measured using a transwell assay. Gelatin zymography was used to detect active matrix metalloproteinase (MMP)-9, while RMG-induced apoptosis of adult retinal pigment epithelium (ARPE-19) cells was studied in a cell coculture system., Results: Aldose reductase inhibition or genetic deficiency substantially reduced lipopolysacharide (LPS)-induced cytokine secretion from macrophages and RMG. Aldose reductase inhibition or deficiency also reduced the activation of MMP-9 and attenuated LPS-induced cell migration. Additionally, blockade of AR by sorbinil or through genetic means caused a reduction in the ability of activated RMG to induce apoptosis of ARPE-19 cells., Conclusions: These results demonstrate that the action of AR contributes to the activation of RMG. Inhibition of AR may be a therapeutic strategy to reduce inflammation associated with activation of RMG in disease.

The multi cellular tumor spheroid (MCTS) model has been used for decades with proven superiority over monolayer cell culture models at recapitulating in vivo tumor growth. Yet its use in high-throughput drug discovery has been limited, particularly with image based screening, due to practical and technical hurdles. Here we report a significant advance in utilizing live MCTS models for high-content image based drug discovery. Using a validated GFP reporter (CK5Pro-GFP) of luminal breast cancer stem cells (CSC), we developed an algorithm to quantify changes in CK5Pro-GFP expression levels for individual Z-stack planes (local) or as maximal projections of the summed Z-stacks (global) of MCTS. From these image sets, we can quantify the cross-sectional area of GFP positive cells, the fluorescence intensity of the GFP positive cells, and the percent of spheroid cross-sectional area that expresses CK5Pro-GFP.We demonstrate that acquiring data in this manner can be done in real time and is statistically robust (Z'=0.85) for use in primary high-content screening cancer drug discovery.

β-Glucogallin (BGG), a major component of the Emblica officinalis medicinal plant, is a potent and selective inhibitor of aldose reductase (AKR1B1). New linkages (ether/triazole/amide) were introduced via high yielding, efficient syntheses to replace the labile ester, and an original two-step (90%) preparation of BGG was developed. Inhibition of AKR1B1was assessed in vitro and using transgenic lens organ cultures, which identified the amide linked glucoside (BGA) as a stable, potent, and selective therapeutic lead toward the treatment of diabetic eye disease.

We report herein a convenient synthetic methodology for the conversion of meta-dinitro heterocyclic rings to iminoquinones with vinylogous amidine functionality. These structures are found in nature, particularly in marine organisms, and may be important for the pigments and biological activity observed with such marine secondary metabolites. Using benzimidazole and indole ring systems we show the versatility of these vinylogous amidines for organic synthesis, including the following: transamination substitution reactions with virtually any primary amine, regional control of the substitution with substituents between the vinylogous amidine, and hydrolytic properties that can be controlled or optimized based on the properties of the chosen ring system. Taken together, this versatile chemistry and functionalization of organic molecules may be useful in the preparation of a variety of chemical products such as drug pharmacophores or assembling macromolecular structures.

Aldose reductase (AR) catalyzes the reduction of toxic lipid aldehydes to their alcohol products and mediates inflammatory signals triggered by lipopolysaccharide (LPS). Beta-glucogallin (BGG), a recently described AR inhibitor, was purified from extracts of the Indian gooseberry (Emblica officinalis). In this study, we found that BGG showed low cytotoxicity in Raw264.7 murine macrophages and effectively inhibited AR activity as measured by a decrease in sorbitol accumulation. In addition, BGG-mediated inhibition of AR prevented LPS-induced activation of JNK and p38 and lowered ROS levels, which could inhibit LPS-induced apoptosis. Uveitis is a disease of the eye associated with chronic inflammation. In this study, we also demonstrated that treatment with BGG decreased the number of inflammatory cells that infiltrate the ocular media of mice with experimental uveitis. Accordingly, these results suggest BGG is a potential therapy for inflammatory diseases., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Progestins play a deleterious role in the onset of breast cancer, yet their influence on existing breast cancer and tumor progression is not well understood. In luminal estrogen receptor (ER)- and progesterone receptor (PR)-positive breast cancer, progestins induce a fraction of cells to express cytokeratin 5 (CK5), a marker of basal epithelial and progenitor cells in the normal breast. CK5(+) cells lose expression of ER and PR and are relatively quiescent, increasing their resistance to endocrine and chemotherapy compared to intratumoral CK5(-)ER(+)PR(+) cells. Characterization of live CK5(+) cells has been hampered by a lack of means for their direct isolation. Here, we describe optical (GFP) and bioluminescent (luciferase) reporter models to quantitate and isolate CK5(+) cells in luminal breast cancer cell lines utilizing the human KRT5 gene promoter and a viral vector approach. Using this system, we confirmed that the induction of GFP(+)/CK5(+) cells is specific to progestins, is dependent on PR, can be blocked by antiprogestins, and does not occur with other steroid hormones. Progestin-induced, fluorescence-activated cell sorting-isolated CK5(+) cells had lower ER and PR mRNA, were slower cycling, and were relatively more invasive and sphere forming than their CK5(-) counterparts in vitro. Repeated progestin treatment and selection of GFP(+) cells enriched for a persistent population of CK5(+) cells, suggesting that this transition can be semi-permanent. These data support that in PR(+) breast cancers, progestins induce a subpopulation of CK5(+)ER(-)PR(-) cells with enhanced progenitor properties and have implications for treatment resistance and recurrence in luminal breast cancer.

Breast cancers expressing hormone receptors for estrogen (ER) and progesterone (PR) represent ~70% of all cases and are treated with both ER-targeted and chemotherapies, with near 40% becoming resistant. We have previously described that in some ER(+) tumors, the resistant cells express cytokeratin 5 (CK5), a putative marker of breast stem and progenitor cells. CK5(+) cells have lost expression of ER and PR, express the tumor-initiating cell surface marker CD44, and are relatively quiescent. In addition, progestins, which increase breast cancer incidence, expand the CK5(+) subpopulation in ER(+)PR(+) breast cancer cell lines. We have developed models to induce and quantitate CK5(+)ER(-)PR(-) cells, using CK5 promoter-driven luciferase (Fluc) or green fluorescent protein (GFP) reporters stably transduced into T47D breast cancer cells (CK5Pro-GFP or CK5Pro-Luc). We validated the CK5Pro-GFP-T47D model for high-content screening in 96-well microplates and performed a pilot screen using a focused library of 280 compounds from the National Institutes of Health clinical collection. Four hits were obtained that significantly abrogated the progestin-induced CK5(+) cell population, three of which were members of the retinoid family. Hence, this approach will be useful in discovering small molecules that could potentially be developed as combination therapies, preventing the acquisition of a drug-resistant subpopulation.

Introduction: For the past 30 years 2D-cell-based assay models have dominated preclinical cancer drug discovery efforts. 2D-cell-based models fail to predict in vivo efficacy, contributing to a lower success rate and higher cost required to translate an investigational new drug to clinical approval. Technological advances in 3D-cell culture models bridge the gap between 2D and in vivo models to improve upon the current success rates of cancer drug discovery., Areas Covered: This review focuses on the multicellular tumor spheroid (MCTS), particularly how this model can be utilized for HTS drug discovery. We discuss the current technologies for uniform culture of MCTS suitable for HTS and detection methods utilized for assay development and drug screening., Expert Opinion: Substantial hurdles remain before we reach the ultimate goal of robust HTS of large compound libraries with MCTS models. Specifically, we can group these challenges into three categories: MCTS growth, data collection, and data analysis. The MCTS model should be utilized with fluorescent readouts and high-content imaging with a systems biology approach to model human tumors in vitro. Such models will be more predictive of in vivo efficacy, improving on the current success rates of cancer drug discovery from bench to bedside.

Diabetes mellitus is recognized as a leading cause of new cases of blindness. The prevalence of diabetic eye disease is expected to continue to increase worldwide as a result of the dramatic increase in the number of people with diabetes. At present, there is no medical treatment to delay or prevent the onset and progression of cataract or retinopathy, the most common causes of vision loss in diabetics. The plant Emblica officinalis (gooseberry) has been used for thousands of years as a traditional Indian Ayurvedic preparation for the treatment of diabetes in humans. Extracts from this plant have been shown to be efficacious against the progression of cataract in a diabetic rat model. Aldose reductase (ALR2) is implicated in the development of secondary complications of diabetes including cataract and, therefore, has been a major drug target for the development of therapies to treat diabetic disease. Herein, we present the bioassay-guided isolation and structure elucidation of 1-O-galloyl-β-D-glucose (β-glucogallin), a major component from the fruit of the gooseberry that displays selective as well as relatively potent inhibition (IC(50) = 17 µM) of AKR1B1 in vitro. Molecular modeling demonstrates that this inhibitor is able to favorably bind in the active site. Further, we show that β-glucogallin effectively inhibits sorbitol accumulation by 73% at 30 µM under hyperglycemic conditions in an ex-vivo organ culture model of lenses excised from transgenic mice overexpressing human ALR2 in the lens. This study supports the continued development of natural products such as β-glucogallin as therapeutic leads in the development of novel therapies to treat diabetic complications such as cataract.

Despite advancements in therapies developed for the treatment of cancer, patient prognosis and mortality rates have improved minimally, and metastasis remains the primary cause of cancer mortality worldwide. An underlying mechanism promoting metastasis in many types of cancer is epithelial-mesenchymal transition (EMT). Here the authors report a novel 3D model of EMT and metastatic breast cancer suitable for high-throughput screening (HTS) drug discovery. The primary assay incorporates the expression of the prognostic biomarker vimentin, as a luciferase reporter of EMT, in basil-like/triple-negative MDA-MB-231 breast carcinoma spheroids. Using this model, the authors developed a number of known antitumor agents as control modulators of EMT. U0126, PKC412, PF2341066, dasatinib, and axitinib downregulated vimentin expression by 70% to 90% as compared to untreated spheroids. Counterassays were developed to measure spheroid viability and the invasive potential of MDA-MB-231 spheroids after small-molecule treatment and used to confirm hits from primary screening. Finally, the authors conducted a pilot screen to validate this model for HTS using a purified library of marine secondary metabolites. From 230 compounds screened, they obtained a Z' score of 0.64, indicative of an excellent assay, and confirmed 4 hits, including isonaamidine B, papuamine, mycalolide E, and jaspamide. This HTS model demonstrates the potential to identify small-molecule modulators of EMT that could be used to discover novel antimetastatic agents for the treatment of cancer.

Type IIα DNA topoisomerase (TopoIIα) is among the most important clinical drug targets for the treatment of cancer. Recently, the DNA repair protein Metnase was shown to enhance TopoIIα activity and increase resistance to TopoIIα poisons. Using in vitro DNA decatenation assays we show that neoamphimedine potently inhibits TopoIIα-dependent DNA decatenation in the presence of Metnase. Cell proliferation assays demonstrate that neoamphimedine can inhibit Metnase-enhanced cell growth with an IC(50) of 0.5 μM. Additionally, we find that the apparent K(m) of TopoIIα for ATP increases linearly with higher concentrations of neoamphimedine, indicating ATP-competitive inhibition, which is substantiated by molecular modeling. These findings support the continued development of neoamphimedine as an anticancer agent, particularly in solid tumors that over-express Metnase.

Apoptosis is important for normal development and removal of damaged cells. Evasion of apoptosis by cancer cells is one of the key characteristics of many tumor types. Thus, discovering agents that promote apoptosis in tumor cells could have great therapeutic value. Marine natural products have demonstrated great potential as anticancer agents, and the proapoptotic activity of some of these products is emerging as a potentially useful property for cancer treatments. Using a tumor xenograft assay in rodents, we previously found that the marine alkaloid naamidine A is a potent antitumor agent. In this study, we further characterize the mechanism of action of naamidine A. In cultured tumor cells, we find that naamidine A induces cell death, which is accompanied with annexin V staining, disruption of the mitochondrial membrane potential, and cleavage and activation of caspases 3, 8, and 9, all of which are hallmarks of apoptosis. Furthermore, naamidine A-induced cell death is caspase dependent. We also find that under conditions where naamidine A inhibits tumor xenograft growth, it induces activation of caspase 3, suggesting that apoptosis is part of its antitumorigenic activity in vivo. Apoptosis is not dependent on extracellular signal-regulated kinase 1/2, previously characterized molecular targets of naamidine A, nor does it require functional p53. Our studies support the continued study of naamidine A and its target(s) for the potential development of better clinical treatments for cancer.

A series of substituted quinoline-5,8-diones were synthesized and evaluated as inhibitors of the chaperone protein Hsp90 using two assays: competition for binding to C-terminal ATP-binding site and competition for binding to N-terminal ATP-binding site. In addition, the ability of the compounds to induce the heat shock response was determined using a reporter fibroblast cell line. Of all the compounds assayed, only 6-aziridinyl-2-biphenylquinoline-5,8-dione induced a heat shock response and did so without interacting at the ATP binding sites of Hsp90. COMPARE analysis was carried out on quinoline-5,8-diones active in the National Cancer Institute's 60-cell line screen with the goal of discovering quinoline-5,8-dione structures that interact with other cellular targets (molecular targets) important for cancer chemotherapy. COMPARE analysis led to the discovery of a combretastatin-like quinoline-5,8-dione structure that, in fact, inhibited angiogenesis.

Neoamphimedine, a pyridoacridine alkaloid from Xestospongia sp., is a potent antitumor agent both in vitro and in vivo. Neoamphimedine can efficiently induce topoisomerase II mediated catenation of plasmid DNA in vitro and is the only member of more than one hundred pyridoacridines thus far to have this mechanism of action. Herein we report the first total synthesis of neoamphimedine.

This report describes the synthesis and biological activity of imidazoquinoxalines, benzimidazole-based analogues of indole-based pyrroloiminoquinone marine natural products. Our analogues consist of series 1, which possesses the ethylene tether and extended amidine feature found in the pyrroloiminoquinone natural products, and series 2, which also has the ethylene tether but with an electrostatically stabilized iminoquinone rather than a resonance stabilized iminoquinone (i.e., extended amidine). The biological properties of series 1 analogues, bearing electron-rich side chain rings (indole and phenol), display cytostatic and cytotoxic properties similar to that of the pyrroloiminoquinone natural products. In contrast, COMPARE analysis suggests that analogues bearing benzyl and phenethyl side chains possess a different cytotoxicity mechanism. Hollow fiber assays of analogs of 1 indicate promising antitumor activity and acceptable levels of toxicity. One analogue of 2 is active only against breast cancer cell lines, but the cellular target is as yet unknown.

Fractionation of two Fijian specimens of the sponge Corticium sp. led to the isolation of the known active alkaloid steroid plakinamine A and two new halogenated cyclic peptides, corticiamide A (1) and cyclocinamide B (2). Structural elucidation of 1 and 2 was achieved by an extensive combination of high-field NMR and HRFT MS/MS experiments, and the absolute stereochemistry of 2 was determined by acid hydrolysis and Marfey's analysis. Corticiamide A (1) and cyclocinamide B (2) represent the first peptides to be described from the genus Corticium.

Chemical investigation of the cytotoxic and anti-tuberculosis active butanone extract obtained from the growth media of the marine-derived fungus Beauveria felina led to the isolation of two new destruxins, [beta-Me-Pro] destruxin E chlorohydrin (1) and pseudodestruxin C (3), along with five known cyclic depsipeptides. The structures of the new destruxin derivatives were established by analysis of spectroscopic data, while the absolute configuration of the common amino acid residues was established by Marfey's analysis. The absolute configuration of the 2(R),4(S)-5-chloro-2,4-dihydroxypentanoic acid residue in 1 could be established by application of a J-based configuration method followed by derivatization with R-MPA-Cl and NMR analysis.