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Immunotherapy has dramatically impacted cancer therapy, but it has been challenging to apply immunotherapy to estrogen receptor (ER) positive breast cancer and many other solid tumors that do not display neoantigens. One way to target these tumors is to induce necrosis, which robustly activates immune cells, inducing immunogenic cell death. However, anticancer therapy-induced necrosis was primarily characterized by morphological changes, and the molecular drivers of necrosis were largely obscure. To probe necrosis, we used our necrosis inducing anticancer agents, the small molecules BHPI and second-generation ErSO, which kill cancer cells by hyperactivating the anticipatory unfolded protein response (a-UPR). In orthotopic mouse xenografts, ErSO induces complete regression without recurrence of large, therapy-resistant primary ER positive breast tumors, of most lung, bone, and liver metastases, near complete regression of challenging breast cancer brain metastases and robust responses in PDX and patient derived organoids (PDOs) models. ErSO also induces complete or near complete regression in mouse xenograft models of ER positive ovarian and endometrial cancer. Using genome wide CRISPR-Cas9 screens with negative selection against our necrosis-inducing a-UPR hyperactivators, BHPI and ErSO, we identified the calcium-activated, ATP-inhibited, plasma membrane sodium channel, Transient Receptor Potential Melastatin Member 4 (TRPM4) as critical for anticancer therapy induced necrosis. TRPM4 knockout in multiple models abolished ErSO-induced ATP depletion, sustained UPR activation, cell swelling, necrotic cell death and increased migration of immune cells. Notably, knockout of TRPM4 completely abolished the ability of ErSO to induce regression of ER positive breast tumors in mice. Supporting a broad role for the TRPM4 pathway in anticancer therapy induced necrosis, rapid cancer cell death induced by four necrosis-inducing cancer therapies unrelated to ErSO, that range from FDA-approved to preclinical, is strongly reversed by TRPM4 knockout. ErSO treatment induces migration of macrophage into regressing tumors. Medium from cancer cells killed by necrosis-inducing ErSO, but not by an apoptosis inducer, dramatically increases macrophage migration and activation, as shown by induction of pro-inflammatory cytokines. This work identifies a protein that plays a pivotal role in the action of diverse anticancer therapies inducing immunogenic necrosis. Since increasing levels of TRPM4 increase sensitivity of breast cancer cells to killing by ErSO, TRPM4 is a novel biomarker whose levels can be used to identify patients most likely to benefit from ErSO and other necrosis-inducing cancer therapies. Citation Format: Santanu Ghosh, Rachel Yang, Darjan Duraki, Ji Eun Kim, Junyao Zhu, Mara Livezey, Matthew Boudreau, Ben H. Park, TImothy Fan, Erik R. Nelson, Paul J. Hergenrother, David J. Shapiro. The Executioner Protein for Immunogenic Anticancer Drug-induced Necrosis in ER Positive Breast Cancer is Transient Receptor Potential Melastatin Member 4 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-01.

Focal amplifications (FA) can mediate targeted therapy resistance in cancer. Understanding the structure and dynamics of FAs is critical for designing treatments that overcome plasticity-mediated resistance. We developed a melanoma model of dual MAPK inhibitor (MAPKi) resistance that bears BRAFV600 amplifications through either extrachromosomal DNA (ecDNA)/double minutes (DM) or intrachromosomal homogenously staining regions (HSR). Cells harboring BRAFV600E FAs displayed mode switching between DMs and HSRs, from both de novo genetic changes and selection of preexisting subpopulations. Plasticity is not exclusive to ecDNAs, as cells harboring HSRs exhibit drug addiction–driven structural loss of BRAF amplicons upon dose reduction. FA mechanisms can couple with kinase domain duplications and alternative splicing to enhance resistance. Drug-responsive amplicon plasticity is observed in the clinic and can involve other MAPK pathway genes, such as RAF1 and NRAS. BRAF FA-mediated dual MAPKi–resistant cells are more sensitive to proferroptotic drugs, extending the spectrum of ferroptosis sensitivity in MAPKi resistance beyond cases of dedifferentiation.Significance:Understanding the structure and dynamics of oncogene amplifications is critical for overcoming tumor relapse. BRAF amplifications are highly plastic under MAPKi dosage challenges in melanoma, through involvement of de novo genomic alterations, even in the HSR mode. Moreover, BRAF FA-driven, dual MAPKi–resistant cells extend the spectrum of resistance-linked ferroptosis sensitivity.This article is highlighted in the In This Issue feature, p. 873

Supplementary Table from Plasticity of Extrachromosomal and Intrachromosomal BRAF Amplifications in Overcoming Targeted Therapy Dosage Challenges

Supplementary Figure from Plasticity of Extrachromosomal and Intrachromosomal BRAF Amplifications in Overcoming Targeted Therapy Dosage Challenges

The supplementary information file contains details about primers used for this work, supplementary Figures S1-S8, supplementary Table S1, and results from cell line authentication and from mycoplasma testing.

A critical event in the apoptotic cascade is the proteolytic activation of procaspases to active caspases. The caspase autoactivating compound PAC-1 induces cancer cell apoptosis and exhibits antitumor activity in murine xenograft models when administered orally as a lipid-based formulation or implanted s.c. as a cholesterol pellet. However, high doses of PAC-1 were found to induce neurotoxicity, prompting us to design and assess a novel PAC-1 derivative called S-PAC-1. Similar to PAC-1, S-PAC-1 activated procaspase-3 and induced cancer cell apoptosis. However, S-PAC-1 did not induce neurotoxicity in mice or dogs. Continuous i.v. infusion of S-PAC-1 in dogs led to a steady-state plasma concentration of ∼10 μmol/L for 24 to 72 hours. In a small efficacy trial of S-PAC-1, evaluation of six pet dogs with lymphoma revealed that S-PAC-1 was well tolerated and that the treatments induced partial tumor regression or stable disease in four of six subjects. Our results support this canine setting for further evaluation of small-molecule procaspase-3 activators, including S-PAC-1, a compound that is an excellent candidate for further clinical evaluation as a novel cancer chemotherapeutic. Cancer Res; 70(18); 7232–41. ©2010 AACR.

PDF file - 7.1MB, Pharmacokinetics and metabolic stability of DNQ and �-lap in liver extracts. Supplemental Table 4. Table summarizing data in Supplemental Figure 8.

Agents, such as β-lapachone, that target the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to induce programmed necrosis in solid tumors have shown great promise, but more potent tumor-selective compounds are needed. Here, we report that deoxynyboquinone kills a wide spectrum of cancer cells in an NQO1-dependent manner with greater potency than β-lapachone. Deoxynyboquinone lethality relies on NQO1-dependent futile redox cycling that consumes oxygen and generates extensive reactive oxygen species (ROS). Elevated ROS levels cause extensive DNA lesions, PARP1 hyperactivation, and severe NAD+/ATP depletion that stimulate Ca2+–dependent programmed necrosis, unique to this new class of NQO1 “bioactivated” drugs. Short-term exposure of NQO1+ cells to deoxynyboquinone was sufficient to trigger cell death, although genetically matched NQO1− cells were unaffected. Moreover, siRNA-mediated NQO1 or PARP1 knockdown spared NQO1+ cells from short-term lethality. Pretreatment of cells with BAPTA-AM (a cytosolic Ca2+ chelator) or catalase (enzymatic H2O2 scavenger) was sufficient to rescue deoxynyboquinone-induced lethality, as noted with β-lapachone. Investigations in vivo showed equivalent antitumor efficacy of deoxynyboquinone to β-lapachone, but at a 6-fold greater potency. PARP1 hyperactivation and dramatic ATP loss were noted in the tumor, but not in the associated normal lung tissue. Our findings offer preclinical proof-of-concept for deoxynyboquinone as a potent chemotherapeutic agent for treatment of a wide spectrum of therapeutically challenging solid tumors, such as pancreatic and lung cancers. Cancer Res; 72(12); 3038–47. ©2012 AACR.

Supplementary Methods and Materials, Figure 1, Tables 1-2 from Discovery and Canine Preclinical Assessment of a Nontoxic Procaspase-3–Activating Compound

PDF file - 668K, LD50 values for DNQ versus �-lap in various cancer cell types. Supplemental Figure 3. NQO1-dependent oxygen consumption rates (OCRs) in NSCLC cells after various doses of DNQ.

PDF file - 990K, Structures of quinones used in the study, including DNQ and �-lap. Supplemental Figure 2. Minimum time determination for DNQ lethality and demonstration of NQO1-mediated DNA lesion formation by alkaline comet assays.

PDF file - 514K, NQO1-dependent nucleotide depletion in response to DNQ. Supplemental Figure 5. PARP1 hyperactivation is essential for NQO1-dependent ATP loss.

PDF file - 585K, NQO1 activity determinations using various quinone substrates used throughout this study. Supplemental Table 2. Relationship of NQO1 activities and doses required for equitoxicities, LD99 values.

PDF file - 133K, A detailed description of all Materials and Methods used in the Supplemental Figures and Tables.

PDF file - 533K, Intracellular ER Calcium release is required for NQO1-dependent lethality after DNQ. Supplemental Figure 7. NQO1-dependent therapeutic window in vitro of DNQ in NSCLC cells.

Focal amplifications (FA) can mediate targeted therapy resistance in cancer. Understanding the structure and dynamics of FAs is critical for designing treatments that overcome plasticity-mediated resistance. We developed a melanoma model of dual MAPK inhibitor (MAPKi) resistance that bears BRAFV600 amplifications through either extrachromosomal DNA (ecDNA)/double minutes (DM) or intrachromosomal homogenously staining regions (HSR). Cells harboring BRAFV600E FAs displayed mode switching between DMs and HSRs, from both de novo genetic changes and selection of preexisting subpopulations. Plasticity is not exclusive to ecDNAs, as cells harboring HSRs exhibit drug addiction–driven structural loss of BRAF amplicons upon dose reduction. FA mechanisms can couple with kinase domain duplications and alternative splicing to enhance resistance. Drug-responsive amplicon plasticity is observed in the clinic and can involve other MAPK pathway genes, such as RAF1 and NRAS. BRAF FA-mediated dual MAPKi–resistant cells are more sensitive to proferroptotic drugs, extending the spectrum of ferroptosis sensitivity in MAPKi resistance beyond cases of dedifferentiation.Significance:Understanding the structure and dynamics of oncogene amplifications is critical for overcoming tumor relapse. BRAF amplifications are highly plastic under MAPKi dosage challenges in melanoma, through involvement of de novo genomic alterations, even in the HSR mode. Moreover, BRAF FA-driven, dual MAPKi–resistant cells extend the spectrum of resistance-linked ferroptosis sensitivity.This article is highlighted in the In This Issue feature, p. 873

Novel classes of broad-spectrum antibiotics have been extremely difficult to discover, largely due to the impermeability of the Gram-negative membranes coupled with a poor understanding of the physicochemical properties a compound should possess to promote its accumulation inside the cell. To address this challenge, numerous methodologies for assessing intracellular compound accumulation in Gram-negative bacteria have been established, including classic radiometric and fluorescence-based methods. The recent development of accumulation assays that utilize liquid chromatography–tandem mass spectrometry (LC-MS/MS) have circumvented the requirement for labeled compounds, enabling assessment of a substantially broader range of small molecules. Our unbiased study of accumulation trends in Escherichia coli using an LC-MS/MS-based assay led to the development of the eNTRy rules, which stipulate that a compound is most likely to accumulate in E. coli if it has an ionizable Nitrogen, has low Three-dimensionality and is relatively Rigid. To aid in the implementation of the eNTRy rules, we developed a complementary web tool, eNTRyway, which calculates relevant properties and predicts compound accumulation. Here we provide a comprehensive protocol for analysis and prediction of intracellular accumulation of small molecules in E. coli using an LC-MS/MS-based assay (which takes ~2 d) and eNTRyway, a workflow that is readily adoptable by any microbiology, biochemistry or chemical biology laboratory. This protocol describes an LC-MS/MS assay and complementary web tool for analysis and prediction of intracellular accumulation of small molecules in E. coli based on their physiochemical properties, which could aid in future antibiotic discovery.

Developing effective therapies for the treatment of advanced head-and-neck squamous cell carcinoma (HNSCC) remains a major challenge, and there is a limited landscape of effective targeted therapies on the horizon. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a 2-electron reductase that is overexpressed in HNSCC and presents as a promising target for the treatment of HNSCC. Current NQO1-targeted drugs are hindered by their poor oxidative tolerability in human patients, underscoring a need for better preclinical screening for oxidative toxicities for NQO1-bioactivated small molecules. Herein, we describe our work to include felines and feline oral squamous cell carcinoma (FOSCC) patients in the preclinical assessment process to prioritize lead compounds with increased tolerability and efficacy prior to full human translation. Specifically, our data demonstrate that IB-DNQ, an NQO1-targeted small molecule, is well-tolerated in FOSCC patients and shows promising initial efficacy against FOSCC tumors in proof-of-concept single agent and radiotherapy combination cohorts. Furthermore, FOSCC tumors are amenable to evaluating a variety of target-inducible couplet hypotheses, evidenced herein with modulation of NQO1 levels with palliative radiotherapy. The use of felines and their naturally-occurring tumors provide an intriguing, often underutilized tool for preclinical drug development for NQO1-targeted approaches and has broader applications for the evaluation of other anticancer strategies.

BackgroundHigh-grade meningioma is an aggressive type of brain cancer that is often recalcitrant to surgery and radiotherapy, leading to poor overall survival. Currently, there are no FDA-approved drugs for meningioma, highlighting the need for new therapeutic options, but development is challenging due to the lack of predictive preclinical models.MethodsTo leverage the known overexpression of procaspase-3 in meningioma, PAC-1, a blood-brain barrier penetrant procaspase-3 activator, was evaluated for its ability to induce apoptosis in meningioma cells. To enhance the effects of PAC-1, combinations with either hydroxyurea or temozolomide were explored in cell culture. Both combinations were further investigated in small groups of canine meningioma patients and assessed by MRI, and the novel apoptosis tracer, [18F]C-SNAT4, was evaluated in patients treated with PAC-1 + HU.ResultsIn meningioma cell lines in culture, PAC-1 + HU are synergistic while PAC-1 + TMZ show additive-to-synergistic effects. In canine meningioma patients, PAC-1 + HU led to stabilization of disease and no change in apoptosis within the tumor, whereas PAC-1 + TMZ reduced tumor burden in all three canine patients treated.ConclusionsOur results suggest PAC-1 + TMZ as a potentially efficacious combination for the treatment of human meningioma, and also demonstrate the utility of including pet dogs with meningioma as a means to assess anticancer strategies for this common brain tumor.

Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the outer membrane, is an attractive target due to its surface location. Darobactins produced by Photorhabdus, a nematode gut microbiome symbiont, target BamA. We reasoned that a computational search for genes only distantly related to the darobactin operon may lead to novel compounds. Following this clue, we identified dynobactin A, a novel peptide antibiotic from Photorhabdus australis containing two unlinked rings. Dynobactin is structurally unrelated to darobactins, but also targets BamA. Based on a BamA-dynobactin co-crystal structure and a BAM-complex-dynobactin cryo-EM structure, we show that dynobactin binds to the BamA lateral gate, uniquely protruding into its β-barrel lumen. Dynobactin showed efficacy in a mouse systemic Escherichia coli infection. This study demonstrates the utility of computational approaches to antibiotic discovery and suggests that dynobactin is a promising lead for drug development.

Structurally complex natural products have been a fruitful source for the discovery and development of new drugs. In an effort to construct a compound collection populated by architecturally complex members with unique scaffolds, we have used the natural product limonin as a starting point. Limonin is an abundant triterpenoid natural product and, through alteration of its heptacyclic core ring system using short synthetic sequences, a collection of 98 compounds was created, including multiple members with novel ring systems. The reactions leveraged in the construction of these compounds include novel ring cleavage, rearrangements, and cyclizations, and this work is highlighted by the discovery of a novel B-ring cleavage reaction, a unique B/C-ring rearrangement, an atypical D-ring cyclization, among others. Computational analysis shows that 52 different scaffolds/ring systems were produced during the course of this work, of which 36 are unprecedented. Phenotypic screening and structure–activity relationships identified compounds with activity against a panel of cancer cell lines.

BackgroundTreatment of some blood cancers with T cells that express a chimeric antigen receptor (CAR) against CD19 have shown remarkable results. In contrast, CAR-T cell efficacy against solid tumors has been difficult to achieve.MethodsTo examine the potential of CAR-T cell treatments against ovarian cancers, we used the mouse ovarian cancer cell line ID8 in an intraperitoneal model that exhibits disseminated solid tumors in female C57BL/6J mice. The CAR contained a single-chain Fv from antibody 237 which recognizes a Tn-glycopeptide-antigen expressed by ID8 due to aberrant O-linked glycosylation in the absence of the transferase-dependent chaperoneCosmc. The efficacy of four Tn-dependent CARs with varying affinity to Tn antigen, and each containing CD28/CD3ζ cytoplasmic domains, were compared in vitro and in vivo in this study.ResultsIn line with many observations about the impact of aberrant O-linked glycosylation, the ID8Cosmcknock-out (ID8Cosmc-KO) exhibited more rapid tumor progression compared with wild-type ID8. Despite the enhanced tumor growth in vivo, 237 CAR and a mutant with 30-fold higher affinity, but not CARs with lower affinity, controlled advanced ID8Cosmc-KO tumors. Tumor regression could be achieved with a single intravenous dose of the CARs, but intraperitoneal administration was even more effective. The CAR-T cells persisted over a period of months, allowing CAR-treated mice to delay tumor growth in a re-challenge setting. The most effective CARs exhibited the highest affinity for antigen. Antitumor effects observed in vivo were associated with increased numbers of T cells and macrophages, and higher levels of cleaved caspase-3, in the tumor microenvironment. Notably, the least therapeutically effective CAR mediated tonic signaling leading to antigen-independent cytokine expression and it had higher levels of the immunosuppressive cytokine interleukin10.ConclusionThe findings support the development of affinity-optimized CAR-T cells as a potential treatment for established ovarian cancer, with the most effective CARs mediating a distinct pattern of inflammatory cytokine release in vitro. Importantly, the most potent Tn-dependent CAR-T cells showed no evidence of toxicity in tumor-bearing mice in a syngeneic, immunocompetent system.

Cancer is the second leading cause of death worldwide. Current treatments for solid cancers are associated with inherent resistance mechanisms and are ineffective against non-cycling cancer cells. Thus, there is a critical need to develop novel tumor-selective combination therapies to overcome resistance while optimally spare normal tissues. In this study, we investigate the mechanistic basis for tumor-selective potentiation of ionizing radiation (IR) in combination with IB-DNQ, a novel NQO1-bioactivatable agent. We found that combination of a low dose of ionizing radiation (IR, 2 Gy) with a sublethal dose of IB-DNQ dramatically increased Fe2+ leakage and induced elevated lipid peroxidation and ferroptosis in NQO1-positive cancer cells via upregulating the expression of ACSL4, a lipid metabolism enzyme required for ferroptosis. Knockdown of endogenous ACSL4 significantly inhibited lipid peroxidation via downregulating the expression of PTGS2 and dramatically inhibited the cell death that induced by IR or IB-DNQ alone or combination treatment. We further found that this combination treatment significantly decreased IR or IB-DNQ alone mediated upregulation of the expression of GPX4 that led to the accumulation of lipid peroxides, which suggests that IR or IB-DNQ-induced expression of GPX4 may function as a negative feedback loop to restore cell survival upon a sublethal dose of IR or IB-DNQ treatment. Finally, we demonstrated that this combination therapy synergistically suppressed tumor growth and dramatically extended mouse lifespan in orthotopic pancreatic and non-small-cell lung cancer xenograft models. Together, our study provides novel preclinical evidence for new combination therapy in NQO1-positive solid tumors that may overcome IR-induced resistance. Citation Format: Xiumei Huang, Xiaolin Su, Matthew W. Boudreau, Paul J. Hergenrother. IB-DNQ enhances radiation induced ferroptosis in NQO1-positive cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5052.

Breast cancer remains the second leading cause of cancer-related deaths among women. In recent years, immunotherapy has been tremendously successful in some metastatic cancers such as melanoma. However, a majority of breast cancer patients do not benefit from existing immunotherapy treatments, leaving many with an unmet need. Although undoubtedly multifactorial, one major obstacle to anti-cancer therapies, is the highly immunosuppressive breast tumor microenvironment. This phenomenon is strongly maintained by myeloid immune cells and immunosuppressive regulatory T cells (Tregs), which hinder anti-tumor immunosurveillance and promote tumor progression. Thus, strategies to ‘re-educate’ myeloid cells to inhibit Tregs is a potentially promising anti-cancer strategy. Mining clinical data, we have found that elevated mRNA expression of the nuclear receptor, NR0B2 within breast tumors is associated with an increased time to recurrence. Single cell RNA-sequencing indicates that NR0B2 is expressed within the macrophage populations of normal breast tissue, and various dendritic cell (DC) types in PBMCs. Overexpression of NR0B2 or activation with a small molecule agonist in murine bone marrow derived macrophages (BMDMs) or DCs resulted in a dichotomous T cell expansion - away from Tregs. Conversely, Treg expansion increased when NR0B2 was knocked-down. Tumor growth was markedly increased in mice lacking myeloid specific NR0B2 expression. We further investigated the downstream targets of NR0B2 mediating this anti-tumor phenotype and identified that NLRP3 inflammasome-IL1β activity is a likely modulator in re-educating myeloid cell-Treg function. Importantly, a putative small molecule agonist decreased established metastatic lesions and increased the efficacy of αPD-L1. Subsequent medicinal chemistry was used to develop a novel NR0B2 agonist with strong anti-metastatic properties when used as a single agent in a preclinical mouse model. Collectively, our data implicates NR0B2 within myeloid cells as a modulator of Tregs, a cell population that has thus far been therapeutically intractable. Therefore, NR0B2 may prove to be a promising therapeutic target to reshape the tumor microenvironment and improve breast cancer immunotherapy. This work was supported by the Era of Hope Scholar Award from the Department of Defense Breast Cancer Research Program grant (BC200206), National Cancer Institute (R01CA234025), and NIH Chemistry-Biology Interface Training Grant (T32-GM136629). Citation Format: Hashni Epa Vidana Gamage, Sayyed Hamed Shahoei, Tiffany Nguyen, Rachel Farmer, Samuel Albright, Erin Weisser, Rafael O. Bautista, Claire P. Schane, Yu Wang, Adam Nelczyk, Liqian Ma, Srishti Tiwari, Anasuya Das Gupta, Shruti Bendre, Lionel Apetoh, Paul J. Hergenrother, Erik R. Nelson. NR0B2 re-educates myeloid cells within the tumor microenvironment: Potential novel strategy for breast cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2358.

Fusidic acid (FA) is a potent steroidal antibiotic that has been used in Europe for more than 60 years to treat a variety of infections caused by Gram-positive pathogens. Despite its clinical success, FA requires significantly elevated dosing (3 g on the first day, 1.2 g on subsequent days) to minimize resistance, as FA displays a high resistance frequency, and a large shift in minimum inhibitory concentration is observed for resistant bacteria. Despite efforts to improve on these aspects, all previously constructed derivatives of FA have worse antibacterial activity against Gram-positive bacteria than the parent natural product. Here, we report the creation of a novel FA analogue that has equivalent potency against clinical isolates of Staphylococcus aureus (S. aureus) and Enterococcus faecium (E. faecium) as well as an improved resistance profile in vitro when compared to FA. Importantly, this new compound displays efficacy against an FA-resistant strain of S. aureus in a soft-tissue murine infection model. This work delineates the structural features of FA necessary for potent antibiotic activity and demonstrates that the resistance profile can be improved for this scaffold and target.

The isomalabaricanes comprise a large family of marine triterpenoids with fascinating structures that have been shown to be selective and potent apoptosis inducers in certain cancer cell lines. In this article, we describe the successful total syntheses of the isomalabaricanes stelletin A, stelletin E, and rhabdastrellic acid A, as well as the development of a general strategy to access other natural products within this unique family. High-throughput experimentation and computational chemistry methods were used in this endeavor. A preliminary structure-activity relationship study of stelletin A revealed the trans-syn-trans core motif of the isomalabaricanes to be critical for their cytotoxic activity.

Antibiotic resistance of Gram-negative bacteria is largely attributed to the low permeability of their outer membrane (OM). Recently, we disclosed the eNTRy rules, a key lesson of which is that the introduction of a primary amine enhances OM permeation in certain contexts. To understand the molecular basis for this finding, we perform an extensive set of molecular dynamics (MD) simulations and free energy calculations comparing the permeation of aminated and amine-free antibiotic derivatives through the most abundant OM porin of E. coli, OmpF. To improve sampling of conformationally flexible drugs in MD simulations, we developed a novel, Monte Carlo and graph theory based algorithm to probe more efficiently the rotational and translational degrees of freedom visited during the permeation of the antibiotic molecule through OmpF. The resulting pathways were then used for free-energy calculations, revealing a lower barrier against the permeation of the aminated compound, substantiating its greater OM permeability. Further analysis revealed that the amine facilitates permeation by enabling the antibiotic to align its dipole to the luminal electric field of the porin and form favorable electrostatic interactions with specific, highly-conserved charged residues. The importance of these interactions in permeation was further validated with experimental mutagenesis and whole cell accumulation assays. Overall, this study provides insights on the importance of the primary amine for antibiotic permeation into Gram-negative pathogens that could help the design of future antibiotics. We also offer a new computational approach for calculating free-energy of processes where relevant molecular conformations cannot be efficiently captured., A rapid pathway sampling method combining Monte Carlo and graph theory, developed to describe permeation pathways through outer membrane porins, can distinguish between structurally similar analogs with different permeabilities.

The blood–brain barrier (BBB) presents a major hurdle in the development of central nervous system (CNS) active therapeutics, and expression of the P-glycoprotein (P-gp) efflux transporter at the blood–brain interface further impedes BBB penetrance of most small molecules. Designing efflux liabilities out of compounds can be laborious, and there is currently no generalizable approach to directly transform periphery-limited agents to ones active in the CNS. Here, we describe a target-agnostic, prospective assessment of P-gp efflux using diverse compounds. Our results demonstrate that reducing the molecular size or appending a carboxylic acid in many cases enables evasion of P-gp efflux in cell-based experiments and in mice. These strategies were then applied to transform a periphery-limited (V600E)BRAF inhibitor, dabrafenib, into versions that possess potent and selective anti-cancer activity but now also evade P-gp-mediated efflux. When compared to dabrafenib, the compound developed herein (everafenib) has superior BBB penetrance and superior efficacy in an intracranial mouse model of metastatic melanoma, suggesting it as a lead candidate for the treatment of melanoma metastases to the brain and gliomas with BRAF mutation. More generally, the results described herein suggest the actionability of the trends observed in these target-agnostic efflux studies and provide guidance for the conversion of non-BBB-penetrant drugs into versions that are BBB-penetrant and efficacious.

Development of targeted anticancer modalities has prompted a new era in cancer treatment that is notably different from the age of radical surgery and highly toxic chemotherapy. Behind each effective compound is a rich and complex history from first identification of chemical matter, detailed optimization, and mechanistic investigations, ultimately leading to exciting molecules for drug development. Herein we review the history and on-going journey of one such anticancer scaffold, the 3-(4-hydroxyphenyl)indoline-2-ones. With humble beginnings in 19th century Bavaria, we review this scaffold's synthetic history and anticancer optimization, including its recent demonstration of tumor eradication of drug-resistant, estrogen receptor-positive breast cancer. Compounds containing the 3-(4-hydroxyphenyl)indoline-2-one pharmacophore are emerging as intriguing candidates for the treatment of cancer.

Multidrug-resistant Gram-negative bacterial infections are on the rise, and with no FDA approvals for new classes of broad-spectrum antibiotics in over 50 years, these infections constitute a major threat to human health. A significant challenge is the inability of most compounds to accumulate in Gram-negative bacteria. Recently developed predictive guidelines show that appending a primary amine to an appropriately shaped compound can enhance Gram-negative accumulation. Here, we report that other positively charged nitrogen functional groups, namely, N-alkyl guanidiniums and pyridiniums, can also facilitate compound uptake into Gram-negative bacteria. The accumulation of a set of 60 nonantibiotic compounds, consisting of 20 primary amines and their corresponding guanidiniums and pyridiniums, was assessed in Escherichia coli. We also installed these alternate functional groups onto antibiotic scaffolds and assessed their accumulation and antibacterial activity in Gram-negative bacteria. The results suggest that other positively-charged, nitrogen-containing functional groups should be considered when designing antibiotics with Gram-negative activity.

Glioblastoma (GBM) is the most lethal primary brain tumor. Currently, frontline treatment for primary GBM includes the DNA-methylating drug temozolomide (TMZ, of the imidazotetrazine class), while the optimal treatment for recurrent GBM remains under investigation. Despite its widespread use, a majority of GBM patients do not respond to TMZ therapy; expression of the O(6)-methylguanine DNA methyltransferase (MGMT) enzyme and loss of mismatch repair (MMR) function as the principal clinical modes of resistance to TMZ. Here, we describe a novel imidazotetrazine designed to evade resistance by MGMT while retaining suitable hydrolytic stability, allowing for effective prodrug activation and biodistribution. This dual-substituted compound, called CPZ, exhibits activity against cancer cells irrespective of MGMT expression and MMR status. CPZ has greater blood–brain barrier penetrance and comparable hematological toxicity relative to TMZ, while also matching its maximum tolerated dose in mice when dosed once-per-day over five days. The activity of CPZ is independent of the two principal mechanisms suppressing the effectiveness of TMZ, making it a promising new candidate for the treatment of GBM, especially those that are TMZ-resistant.

Approximately 75% of breast cancers are estrogen receptor alpha-positive (ERα+), and targeting ERα directly with ERα antagonists/degraders or indirectly with aromatase inhibitors is a successful therapeutic strategy. However, such treatments are rarely curative and development of resistance is universal. We recently reported ErSO, a compound that induces ERα-dependent cancer cell death through a mechanism distinct from clinically approved ERα drugs, via hyperactivation of the anticipatory unfolded protein response. ErSO has remarkable tumor-eradicative activity in multiple ERα+ tumor models. While ErSO has promise as a new drug, it has effects on ERα-negative (ERα−) cells in certain contexts. Herein, we construct modified versions of ErSO and identify variants with enhanced differential activity between ERα+ and ERα− cells. We report ErSO-DFP, a compound that maintains antitumor efficacy, has enhanced selectivity for ERα+ cancer cells, and is well tolerated in rodents. ErSO-DFP and related compounds represent an intriguing new class for the treatment of ERα+ cancers.

Significance Antibiotic resistance in Gram-negative pathogens has been identified as an urgent threat to human health by the World Health Organization. The major challenge with treating infections by these pathogens is developing antibiotics that can traverse the dense bacterial outer membrane (OM) formed by a mesh of lipopolysaccharides. Effective antibiotics permeate through OM porins, which have evolved for nutrient diffusion; however, the conformational states of these porins regulating permeation are still unclear. Here, we used molecular dynamics simulations, free energy calculations, Markov-state modeling, and whole-cell accumulation assays to provide mechanistic insight on how a porin shifts between open and closed states. We provide a mechanism of how Gram-negative bacteria confer resistance to antibiotics.

In Fall 2020, universities saw extensive transmission of SARS-CoV-2 among their populations, threatening health of the university and surrounding communities, and viability of in-person instruction. Here we report a case study at the University of Illinois at Urbana-Champaign, where a multimodal “SHIELD: Target, Test, and Tell” program, with other non-pharmaceutical interventions, was employed to keep classrooms and laboratories open. The program included epidemiological modeling and surveillance, fast/frequent testing using a novel low-cost and scalable saliva-based RT-qPCR assay for SARS-CoV-2 that bypasses RNA extraction, called covidSHIELD, and digital tools for communication and compliance. In Fall 2020, we performed >1,000,000 covidSHIELD tests, positivity rates remained low, we had zero COVID-19-related hospitalizations or deaths amongst our university community, and mortality in the surrounding Champaign County was reduced more than 4-fold relative to expected. This case study shows that fast/frequent testing and other interventions mitigated transmission of SARS-CoV-2 at a large public university.

Gram-negative bacteria pose a serious public health concern, primarily due to a higher frequency of antibiotic resistance conferred to them as a result of low permeability of their outer membrane (OM). Antibiotics capable of traversing the OM typically permeate through OM porins; thus, understanding the permeation properties of these porins is instrumental to the development of new antibiotics. A common macroscopic feature of many OM porins is their ability to transition between functionally distinct open and closed states that regulate transport properties and rate. To obtain a molecular basis for these processes, we performed tens of microseconds of molecular dynamics simulations of E. coli OM porin, OmpF. We observed that large-scale motion of the internal loop, L3, leads to widening and narrowing of the pore, suggesting its potential role in gating. Furthermore, Markov state analysis revealed multiple energetically stable conformations of L3 corresponding to open and closed states of the porin. Dynamics between these functional states occurs on the time scale of tens of microseconds and are mediated by the movement of highly conserved acidic residues of L3 to form H-bonds with opposing sides of the barrel wall of the pore. To validate our mechanism, we mutated key residues involved in the gating process that alter the H-bond pattern in the open/closed states and performed additional simulations. These mutations shifted the dynamic equilibrium of the pore towards open or closed states. Complementarily, the mutations favoring the open/closed states lead to increased/decreased accumulation of multiple antibiotics in our whole-cell accumulation assays. Notably, porins containing one of the mutations favoring the closed state has previously been found in antibiotic resistant bacterial strains. Overall, our 180 µs of simulation data (wild type and mutants) with concerted experiments suggests that regulation of the dynamic equilibrium between open and closed states of OM porins could be a mechanism by which Gram-negative bacteria acquire antibiotic resistance.