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Triple negative breast cancer (TNBC) contains the highest proportion of cancer stem-like cells (CSCs), which display intrinsic resistance to currently available cancer therapies. This therapeutic resistance is partially mediated by an antioxidant defense coordinated by the transcription factor NRF2 and its downstream targets including NQO1. Here, we identified the antioxidant enzymes NQO1 and SOD1 as therapeutic vulnerabilities of ALDH+ epithelial-like CSCs and CD24-/loCD44+/hi mesenchymal-like CSCs in TNBC. Effective targeting of these CSC states was achieved by utilizing IB-DNQ, a potent and specific NQO1-bioactivatable futile redox cycling molecule, which generated large amounts of reactive oxygen species (ROS) including superoxide and hydrogen peroxide. Furthermore, the CSC killing effect was specifically enhanced by genetic or pharmacological inhibition of SOD1, a copper-containing superoxide dismutase highly expressed in TNBC. Mechanistically, a significant portion of NQO1 resided in the mitochondrial intermembrane space, catalyzing futile redox cycling from IB-DNQ to generate high levels of mitochondrial superoxide, and SOD1 inhibition markedly potentiated this effect resulting in mitochondrial oxidative injury, cytochrome c release, and activation of the caspase 3-mediated apoptotic pathway. Treatment with IB-DNQ alone or together with SOD1 inhibition effectively suppressed tumor growth, metastasis, and tumor-initiating potential in xenograft models of TNBC expressing different levels of NQO1. This futile oxidant-generating strategy, which targets CSCs across the epithelial-mesenchymal continuum, could be a promising therapeutic approach for treating TNBC patients.

Chemical inducers of apoptosis have been utilized for decades as tools to uncover steps of the apoptotic cascade and to treat various diseases, most notably cancer. While there are several useful compounds available, limitations in potency, universality, or speed of cell death of these pro-apoptotic agents have meant that no single compound is suitable for all (or most) purposes. Raptinal is a recently described small molecule that induces intrinsic pathway apoptosis rapidly and reliably, and consequently, has been utilized in cell culture and whole organisms for a wide range of biological studies. Its distinct mechanism of action complements the current arsenal of cytotoxic compounds, making it useful as a probe for the apoptosis pathway and other cellular processes. The rapid induction of cell death by Raptinal and its widespread commercial availability make it the pro-apoptotic agent of choice for many applications., (© 2024. The Author(s).)

Immune checkpoint blockade (ICB) has had limited utility in several solid tumors such as breast cancer, a major cause of cancer-related mortality in women. Therefore, there is considerable interest in alternate strategies to promote an anti-cancer immune response. A paper co-published in this issue describes how NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the inflammasome and reduce the expansion of immune-suppressive regulatory T cells (T reg ). Here, we develop NR0B2 as a potential therapeutic target. NR0B2 in tumors is associated with improved survival for several cancer types including breast. Importantly, NR0B2 expression is also prognostic of ICB success. Within breast tumors, NR0B2 expression is inversely associated with FOXP3, a marker of T regs . While a described agonist (DSHN) had some efficacy, it required high doses and long treatment times. Therefore, we designed and screened several derivatives. A methyl ester derivative (DSHN-OMe) emerged as superior in terms of (1) cellular uptake, (2) ability to regulate expected expression of genes, (3) suppression of T reg expansion using in vitro co-culture systems, and (4) efficacy against the growth of primary and metastatic tumors. This work identifies NR0B2 as a target to re-educate myeloid immune cells and a novel ligand with significant anti-tumor efficacy in preclinical models., Competing Interests: Declaration of competing interest ERN, PJN, SA, RF, HEVG and SHS have filed a provisional patent describing DSHN-OMe and its use targeting NR0B2., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Although survival from breast cancer has dramatically increased, many will develop recurrent, metastatic disease. Unfortunately, survival for this stage of disease remains very low. Activating the immune system has incredible promise since it has the potential to be curative. However, immune checkpoint blockade (ICB) which works through T cells has been largely disappointing for metastatic breast cancer. One reason for this is a suppressive myeloid immune compartment that is unaffected by ICB. Cholesterol metabolism and proteins involved in cholesterol homeostasis play important regulatory roles in myeloid cells. Here, we demonstrate that NR0B2, a nuclear receptor involved in negative feedback of cholesterol metabolism, works in several myeloid cell types to impair subsequent expansion of regulatory T cells (T regs ); T regs being a subset known to be highly immune suppressive and associated with poor therapeutic response. Within myeloid cells, NR0B2 serves to decrease many aspects of the inflammasome, ultimately resulting in decreased IL1β; IL1β driving T reg expansion. Importantly, mice lacking NR0B2 exhibit accelerated tumor growth. Thus, NR0B2 represents an important node in myeloid cells dictating ensuing T reg expansion and tumor growth, thereby representing a novel therapeutic target to re-educate these cells, having impact across different solid tumor types. Indeed, a paper co-published in this issue demonstrates the therapeutic utility of targeting NR0B2., Competing Interests: Declaration of competing interest ERN, PJN, SA, RF, HEVG and SHS have filed a patent describing DSHN-OMe and its use targeting NR0B2., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: Pancreatic cancer is among the top five leading causes of cancer-related deaths worldwide, with poor overall survival rates. Current therapies for pancreatic cancer lack tumor specificity, resulting in harmful effects on normal tissues. Therefore, developing tumor-specific agents for the treatment of pancreatic cancer is critical. NAD(P)H:quinone oxidoreductase 1 (NQO1), highly expressed in pancreatic cancers but not in associated normal tissues, makes NQO1 bioactivatable drugs a potential therapy for selectively killing NQO1 -positive cancer cells. Our previous studies have revealed that the novel NQO1 bioactivatable drug deoxynyboquinone (DNQ) is 10-fold more potent than the prototypic NQO1 bioactivatable drug β-lapachone in killing of NQO1 -positive cancer cells. However, DNQ treatment results in high-grade methemoglobinemia, a significant side effect that limits clinical development. Results: Here, we report for the first time on a DNQ derivative, isopentyl-deoxynboquinone (IP-DNQ), which selectively kills pancreatic ductal adenocarcinoma (PDAC) cells in an NQO1-dependent manner with equal potency to the parent DNQ. IP-DNQ evokes massive reactive oxygen species (ROS) production and oxidative DNA lesions that result in poly(ADP-ribose)polymerase-1 (PARP1) hyperactivation, mitochondrial catastrophe, and G2/M phase cell cycle arrest, leading to apoptotic and necrotic programmed cell death. Importantly, IP-DNQ treatment causes only mild methemoglobinemia in vivo , with a threefold improvement in the maximum tolerated dose (MTD) compared with DNQ, while it significantly suppresses tumor growth and extends the life span of mice in subcutaneous and orthotopic pancreatic cancer xenograft models. Innovation and Conclusion: Our study demonstrates that IP-DNQ is a promising therapy for NQO1 -positive pancreatic cancers and may enhance the efficacy of other anticancer drugs. IP-DNQ represents a novel approach to treating pancreatic cancer with the potential to improve patient outcomes.

The antibiotic fusidic acid (FA) is used to treat Staphylococcus aureus infections. It inhibits protein synthesis by binding to elongation factor G (EF-G) and preventing its release from the ribosome after translocation. While FA, due to permeability issues, is only effective against gram-positive bacteria, the available structures of FA-inhibited complexes are from gram-negative model organisms. To fill this knowledge gap, we solved cryo-EM structures of the S. aureus ribosome in complex with mRNA, tRNA, EF-G and FA to 2.5 Å resolution and the corresponding complex structures with the recently developed FA derivative FA-cyclopentane (FA-CP) to 2.0 Å resolution. With both FA variants, the majority of the ribosomal particles are observed in chimeric state and only a minor population in post-translocational state. As expected, FA binds in a pocket between domains I, II and III of EF-G and the sarcin-ricin loop of 23S rRNA. FA-CP binds in an identical position, but its cyclopentane moiety provides additional contacts to EF-G and 23S rRNA, suggesting that its improved resistance profile towards mutations in EF-G is due to higher-affinity binding. These high-resolution structures reveal new details about the S. aureus ribosome, including confirmation of many rRNA modifications, and provide an optimal starting point for future structure-based drug discovery on an important clinical drug target., (© 2024. The Author(s).)

Infections caused by Gram-negative pathogens are increasingly prevalent and are typically treated with broad-spectrum antibiotics, resulting in disruption of the gut microbiome and susceptibility to secondary infections 1-3 . There is a critical need for antibiotics that are selective both for Gram-negative bacteria over Gram-positive bacteria, as well as for pathogenic bacteria over commensal bacteria. Here we report the design and discovery of lolamicin, a Gram-negative-specific antibiotic targeting the lipoprotein transport system. Lolamicin has activity against a panel of more than 130 multidrug-resistant clinical isolates, shows efficacy in multiple mouse models of acute pneumonia and septicaemia infection, and spares the gut microbiome in mice, preventing secondary infection with Clostridioides difficile. The selective killing of pathogenic Gram-negative bacteria by lolamicin is a consequence of low sequence homology for the target in pathogenic bacteria versus commensals; this doubly selective strategy can be a blueprint for the development of other microbiome-sparing antibiotics., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

A compound's overall contour impacts its ability to elicit biological response, rendering access to distinctly shaped molecules desirable. A natural product's framework can be modified, but only if it is abundant and contains suitably modifiable functional groups. Here we introduce a programmable strategy for concise synthesis of precisely altered scaffolds of scarce bridged polycyclic alkaloids. Central to our approach is a scalable catalytic multi-component process that delivers diastereo- and enantiomerically enriched tertiary homoallylic alcohols bearing differentiable alkenyl moieties. We used one product to launch progressively divergent syntheses of a naturally occurring alkaloid and its precisely expanded, contracted and/or distorted framework analogues (average number of steps/scaffold of seven). In vitro testing showed that a skeleton expanded by one methylene in two regions is cytotoxic against four types of cancer cell line. Mechanistic and computational studies offer an account for several unanticipated selectivity trends., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Significance: Label-free quantitative phase imaging can potentially measure cellular dynamics with minimal perturbation, motivating efforts to develop faster and more sensitive instrumentation. We characterize fast, single-shot quantitative phase gradient microscopy (ss-QPGM) that simultaneously acquires multiple polarization components required to reconstruct phase images. We integrate a computationally efficient least squares algorithm to provide real-time, video-rate imaging (up to 75   frames / s ). The developed instrument was used to observe changes in cellular morphology and correlate these to molecular measures commonly obtained by staining., Aim: We aim to characterize a fast approach to ss-QPGM and record morphological changes in single-cell phase images. We also correlate these with biochemical changes indicating cell death using concurrently acquired fluorescence images., Approach: Here, we examine nutrient deprivation and anticancer drug-induced cell death in two different breast cell lines, viz. , M2 and MCF7. Our approach involves in-line measurements of ss-QPGM and fluorescence imaging of the cells biochemically labeled for viability., Results: We validate the accuracy of the phase measurement using a USAF1951 pattern phase target. The ss-QPGM system resolves 912.3    lp / mm , and our analysis scheme accurately retrieves the phase with a high correlation coefficient ( ∼ 0.99 ), as measured by calibrated sample thicknesses. Analyzing the contrast in phase, we estimate the spatial resolution achievable to be 0.55    μ m for this microscope. ss-QPGM time-lapse live-cell imaging reveals multiple intracellular and morphological changes during biochemically induced cell death. Inferences from co-registered images of quantitative phase and fluorescence suggest the possibility of necrosis, which agrees with previous findings., Conclusions: Label-free ss-QPGM with high-temporal resolution and high spatial fidelity is demonstrated. Its application for monitoring dynamic changes in live cells offers promising prospects., (© 2024 The Authors.)

The functions of proteins bearing multiple post-translational modifications (PTMs) are modulated by their modification patterns, yet precise characterization of them is difficult. MEK1 (also known as MAP2K1) is one such example that acts as a gatekeeper of the mitogen-activating protein kinase (MAPK) pathway and propagates signals via phosphorylation by upstream kinases. In principle, top-down mass spectrometry can precisely characterize whole MEK1 proteoforms, but fragmentation methods that would enable the site-specific characterization of labile modifications on 43 kDa protein ions result in overly dense tandem mass spectra. By using the charge-detection method called individual ion mass spectrometry, we demonstrate how complex mixtures of phosphoproteoforms and their fragment ions can be reproducibly handled to provide a "bird's eye" view of signaling activity through mapping proteoform landscapes in a pathway. Using this approach, the overall stoichiometry and distribution of 0-4 phosphorylations on MEK1 was determined in a cellular model of drug-resistant metastatic melanoma. This approach can be generalized to other multiply modified proteoforms, for which PTM combinations are key to their function and drug action.

Currently, there are no clinically approved drugs that directly thwart mutant KRAS G12D, a major driver of human cancer. Here, we report on the discovery of a small molecule, KRB-456, that binds KRAS G12D and inhibits the growth of pancreatic cancer patient-derived tumors. Protein nuclear magnetic resonance studies revealed that KRB-456 binds the GDP-bound and GCP-bound conformation of KRAS G12D by forming interactions with a dynamic allosteric binding pocket within the switch-I/II region. Isothermal titration calorimetry demonstrated that KRB-456 binds potently to KRAS G12D with 1.5-, 2-, and 6-fold higher affinity than to KRAS G12V, KRAS wild-type, and KRAS G12C, respectively. KRB-456 potently inhibits the binding of KRAS G12D to the RAS-binding domain (RBD) of RAF1 as demonstrated by GST-RBD pulldown and AlphaScreen assays. Treatment of KRAS G12D-harboring human pancreatic cancer cells with KRB-456 suppresses the cellular levels of KRAS bound to GTP and inhibits the binding of KRAS to RAF1. Importantly, KRB-456 inhibits P-MEK, P-AKT, and P-S6 levels in vivo and inhibits the growth of subcutaneous and orthotopic xenografts derived from patients with pancreatic cancer whose tumors harbor KRAS G12D and KRAS G12V and who relapsed after chemotherapy and radiotherapy. These results warrant further development of KRB-456 for pancreatic cancer., Significance: There are no clinically approved drugs directly abrogating mutant KRAS G12D. Here, we discovered a small molecule, KRB-456, that binds a dynamic allosteric binding pocket within the switch-I/II region of KRAS G12D. KRB-456 inhibits P-MEK, P-AKT, and P-S6 levels in vivo and inhibits the growth of subcutaneous and orthotopic xenografts derived from patients with pancreatic cancer. This discovery warrants further advanced preclinical and clinical studies in pancreatic cancer., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Lung and breast cancers rank as two of the most common and lethal tumors, accounting for a substantial number of cancer-related deaths worldwide. While the past two decades have witnessed promising progress in tumor therapy, developing targeted tumor therapies continues to pose a significant challenge. NAD(P)H quinone oxidoreductase 1 (NQO1), a two-electron reductase, has been reported as a promising therapeutic target across various solid tumors. β-Lapachone (β-Lap) and deoxynyboquinone (DNQ) are two NQO1 bioactivatable drugs that have demonstrated potent antitumor effects. However, their curative efficacy has been constrained by adverse effects and moderate lethality. To enhance the curative potential of NQO1 bioactivatable drugs, we developed a novel DNQ derivative termed isopentyl-deoxynyboquinone (IP-DNQ). Our study revealed that IP-DNQ treatment significantly increased reactive oxygen species generation, leading to double-strand break (DSB) formation, PARP1 hyperactivation, and catastrophic energy loss. Notably, we discovered that this novel drug induced both apoptosis and programmed necrosis events, which makes it entirely distinct from other NQO1 bioactivatable drugs. Furthermore, IP-DNQ monotherapy demonstrated significant antitumor efficacy and extended mice survival in A549 orthotopic xenograft models. Lastly, we identified that in mice IP-DNQ levels were significantly elevated in the plasma and tumor compared with IB-DNQ levels. This study provides novel preclinical evidence supporting IP-DNQ efficacy in NQO1 + NSCLC and breast cancer cells.

Gram-negative antibiotic development has been hindered by a poor understanding of the types of compounds that can accumulate within these bacteria 1,2 . The presence of efflux pumps and substrate-specific outer-membrane porins in Pseudomonas aeruginosa renders this pathogen particularly challenging 3 . As a result, there are few antibiotic options for P. aeruginosa infections 4 and its many porins have made the prospect of discovering general accumulation guidelines seem unlikely 5 . Here we assess the whole-cell accumulation of 345 diverse compounds in P. aeruginosa and Escherichia coli. Although certain positively charged compounds permeate both bacterial species, P. aeruginosa is more restrictive compared to E. coli. Computational analysis identified distinct physicochemical properties of small molecules that specifically correlate with P. aeruginosa accumulation, such as formal charge, positive polar surface area and hydrogen bond donor surface area. Mode of uptake studies revealed that most small molecules permeate P. aeruginosa using a porin-independent pathway, thus enabling discovery of general P. aeruginosa accumulation trends with important implications for future antibiotic development. Retrospective antibiotic examples confirmed these trends and these discoveries were then applied to expand the spectrum of activity of a gram-positive-only antibiotic, fusidic acid, into a version that demonstrates a dramatic improvement in antibacterial activity against P. aeruginosa. We anticipate that these discoveries will facilitate the design and development of high-permeating antipseudomonals., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The treatment of metastatic uveal melanoma remains a major clinical challenge. Procaspase-3, a proapoptotic protein and precursor to the key apoptotic executioner caspase-3, is overexpressed in a wide range of malignancies, and the drug PAC-1 leverages this overexpression to selectively kill cancer cells. Herein, we investigate the efficacy of PAC-1 against uveal melanoma cell lines and report the synergistic combination of PAC-1 and entrectinib. This preclinical activity, tolerability data in mice, and the known clinical effectiveness of these drugs in human cancer patients led to a small Phase 1b study in patients with metastatic uveal melanoma. The combination of PAC-1 and entrectinib was tolerated with no treatment-related grade ≥3 toxicities in these patients. The pharmacokinetics of entrectinib were not affected by PAC-1 treatment. In this small and heavily pretreated initial cohort, stable disease was observed in four out of six patients, with a median progression-free survival of 3.38 months (95% CI 1.6-6.5 months). This study is an initial demonstration that the combination of PAC-1 and entrectinib may warrant further clinical investigation. Clinical trial registration: Clinical Trials.gov: NCT04589832., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Several emerging therapies kill cancer cells primarily by inducing necrosis. As necrosis activates immune cells, potentially, uncovering the molecular drivers of anticancer therapy-induced necrosis could reveal approaches for enhancing immunotherapy efficacy. To identify necrosis-associated genes, we performed a genome-wide CRISPR-Cas9 screen with negative selection against necrosis-inducing preclinical agents BHPI and conducted follow-on experiments with ErSO. The screen identified transient receptor potential melastatin member 4 (TRPM4), a calcium-activated, ATP-inhibited, sodium-selective plasma membrane channel. Cancer cells selected for resistance to BHPI and ErSO exhibited robust TRPM4 downregulation, and TRPM4 reexpression restored sensitivity to ErSO. Notably, TRPM4 knockout (TKO) abolished ErSO-induced regression of breast tumors in mice. Supporting a broad role for TRPM4 in necrosis, knockout of TRPM4 reversed cell death induced by four additional diverse necrosis-inducing cancer therapies. ErSO induced anticipatory unfolded protein response (a-UPR) hyperactivation, long-term necrotic cell death, and release of damage-associated molecular patterns that activated macrophages and increased monocyte migration, all of which was abolished by TKO. Furthermore, loss of TRPM4 suppressed the ErSO-induced increase in cell volume and depletion of ATP. These data suggest that ErSO triggers initial activation of the a-UPR but that it is TRPM4-mediated sodium influx and cell swelling, resulting in osmotic stress, which sustains and propagates lethal a-UPR hyperactivation. Thus, TRPM4 plays a pivotal role in sustaining lethal a-UPR hyperactivation that mediates the anticancer activity of diverse necrosis-inducing therapies., Significance: A genome-wide CRISPR screen reveals a pivotal role for TRPM4 in cell death and immune activation following treatment with diverse necrosis-inducing anticancer therapies, which could facilitate development of necrosis-based cancer immunotherapies., (©2023 American Association for Cancer Research.)

Background: Procaspase-3 (PC-3) is overexpressed in various tumor types, including gliomas. Targeted PC-3 activation combined with chemotherapy is a novel strategy for treating patients with high-grade gliomas, with promising preclinical activity. This study aimed to define safety and tolerability of procaspase-activating compound-1 (PAC-1) in combination with temozolomide (TMZ) for patients with recurrent high-grade astrocytomas., Methods: A modified-Fibonacci dose-escalation 3 + 3 design was used. PAC-1 was administered at increasing dose levels (DL; DL1 = 375 mg) on days 1-21, in combination with TMZ 150 mg/m 2 /5 days, per 28-day cycle. Dose-limiting toxicity was assessed during the first 2 cycles. Neurocognitive function (NCF) testing was conducted throughout the study., Results: Eighteen patients were enrolled (13 GBM, IDH-wild type; 2 astrocytoma, IDH-mutant, grade 3; 3 astrocytoma, IDH-mutant, grade 4). Dose escalation was discontinued after DL3 (ie, PAC-1, 625 mg) due to lack of additional funding. Grade 3 toxicity was observed in 1 patient at DL1 (elevated liver transaminases) and 1 at DL 2 (headache). Two partial responses were observed at DL1 in patients with GBM, O 6 -methylguanine-DNA methyltransferase (MGMT) promoter methylated. Two patients had stable disease, and 11 experienced progression. NCF testing did not show a clear relationship between PAC-1 dose, treatment duration, and declines in NCF., Conclusions: Combination of PAC-1 and TMZ was well tolerated up to 625 mg orally daily and TMZ orally 150 mg/m 2 /5 days per 28-day cycle. The maximum tolerated dose was not reached. Further dose escalation of PAC-1 in combination with TMZ is advised before conducting a formal prospective efficacy study in this patient population., Competing Interests: The University of Illinois has filed a patent on PAC-1 in which T.M.T., T.F.M. and P.J.H. are inventors. The University of Illinois has licensed the patents on PAC-1 to Vanquish Oncology, Inc, and P.J.H. and T.M.F. serve as consultants for Vanquish Oncology, Inc. P.J.H. also serves as Chief Scientific Officer and has equity in Vanquish Oncology, Inc. T.M.T. serves as Chief Executive Officer and has equity in Vanquish Oncology, Inc. A.Z.D served as Chief Medical Officer in Vanquish Oncology. J.S.W. was a paid consultant to Vanquish Oncology, Inc. during this study. M.H. serves on a data safety monitoring board of Parexel and Advarra and a steering committee for Novartis., (© The Author(s) 2023. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Viral macrodomains, which can bind to and/or hydrolyze adenine diphosphate ribose (ADP-ribose or ADPr) from proteins, have been suggested to counteract host immune response and be viable targets for the development of antiviral drugs. Therefore, developing high-throughput screening (HTS) techniques for macrodomain inhibitors is of great interest. Herein, using a novel tracer TAMRA-ADPr , an ADP-ribose compound conjugated with tetramethylrhodamine, we developed a robust fluorescence polarization assay for various viral and human macrodomains including SARS-CoV-2 Macro1, VEEV Macro, CHIKV Macro, human MacroD1, MacroD2, and PARP9 Macro2. Using this assay, we validated Z8539 (IC 50 6.4 μM) and GS441524 (IC 50 15.2 μM), two literature-reported small-molecule inhibitors of SARS-CoV-2 Macro1. Our data suggest that GS441524 is highly selective for SARS-CoV-2 Macro1 over other human and viral macrodomains. Furthermore, using this assay, we identified pNP-ADPr (ADP-ribosylated p -nitrophenol, IC 50 370 nM) and TFMU-ADPr (ADP-ribosylated trifluoromethyl umbelliferone, IC 50 590 nM) as the most potent SARS-CoV-2 Macro1 binders reported to date. An X-ray crystal structure of SARS-CoV-2 Macro1 in complex with TFMU-ADPr revealed how the TFMU moiety contributes to the binding affinity. Our data demonstrate that this fluorescence polarization assay is a useful addition to the HTS methods for the identification of macrodomain inhibitors.

Background: Treatment 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., Methods: To 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 chaperone Cosmc . 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., Results: In line with many observations about the impact of aberrant O-linked glycosylation, the ID8 Cosmc knock-out (ID8 Cosmc -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 ID8 Cosmc -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., Conclusion: The 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., Competing Interests: Competing interests: DMK is a member of the scientific advisory board of Affini-T Therapeutics and a consultant for Tempus. PS, HS, KS, and DMK are co-inventors on a patent application related to these chimeric antigen receptors. No potential conflicts of interest were disclosed by the other authors., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Procaspase-3 (PC-3) is overexpressed in multiple tumour types and procaspase-activating compound 1 (PAC-1) directly activates PC-3 and induces apoptosis in cancer cells. This report describes the first-in-human, phase I study of PAC-1 assessing maximum tolerated dose, safety, and pharmacokinetics., Methods: Modified-Fibonacci dose-escalation 3 + 3 design was used. PAC-1 was administered orally at 7 dose levels (DL) on days 1-21 of a 28-day cycle. Dose-limiting toxicity (DLT) was assessed during the first two cycles of therapy, and pharmacokinetics analysis was conducted on days 1 and 21 of the first cycle. Neurologic and neurocognitive function (NNCF) tests were performed throughout the study., Results: Forty-eight patients were enrolled with 33 completing ≥2 cycles of therapy and evaluable for DLT. DL 7 (750 mg/day) was established as the recommended phase 2 dose, with grade 1 and 2 neurological adverse events noted, while NNCF testing showed stable neurologic and cognitive evaluations. PAC-1's t 1/2 was 28.5 h after multi-dosing, and systemic drug exposures achieved predicted therapeutic concentrations. PAC-1 clinical activity was observed in patients with neuroendocrine tumour (NET) with 2/5 patients achieving durable partial response., Conclusions: PAC-1 dose at 750 mg/day was recommended for phase 2 studies. Activity of PAC-1 in treatment-refractory NET warrants further investigation., Clinical Trial Registration: Clinical Trials.gov: NCT02355535., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Genomic studies and experiments with permeability-deficient strains have revealed a variety of biological targets that can be engaged to kill Gram-negative bacteria. However, the formidable outer membrane and promiscuous efflux pumps of these pathogens prevent many candidate antibiotics from reaching these targets. One such promising target is the enzyme FabI, which catalyzes the rate-determining step in bacterial fatty acid biosynthesis. Notably, FabI inhibitors have advanced to clinical trials for Staphylococcus aureus infections but not for infections caused by Gram-negative bacteria. Here, we synthesize a suite of FabI inhibitors whose structures fit permeation rules for Gram-negative bacteria and leverage activity against a challenging panel of Gram-negative clinical isolates as a filter for advancement. The compound to emerge, called fabimycin, has impressive activity against >200 clinical isolates of Escherichia coli , Klebsiella pneumoniae , and Acinetobacter baumannii , and does not kill commensal bacteria. X-ray structures of fabimycin in complex with FabI provide molecular insights into the inhibition. Fabimycin demonstrates activity in multiple mouse models of infection caused by Gram-negative bacteria, including a challenging urinary tract infection model. Fabimycin has translational promise, and its discovery provides additional evidence that antibiotics can be systematically modified to accumulate in Gram-negative bacteria and kill these problematic pathogens., Competing Interests: The authors declare the following competing financial interest(s): The University of Illinois and the Broad Institute have filed patents on some compounds described herein., (© 2022 The Authors. Published by American Chemical Society.)

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.

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., (© 2022. The Author(s).)

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., Competing Interests: The University of Illinois has filed patents on some compounds described herein on which M. W. B. and P. J. H. are inventors. This intellectual property has been licensed to Systems Oncology, LLC, and P. J. H. is a consultant and a member of the Scientific Advisory Board of Systems Oncology., (This journal is © The Royal Society of Chemistry.)

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., (©2021 American Association for Cancer Research.)

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.

Gram-negative bacteria pose a serious public health concern due to resistance to many antibiotics, caused by the low permeability of their outer membrane (OM). Effective antibiotics use porins in the OM to reach the interior of the cell; thus, understanding permeation properties of OM porins is instrumental to rationally develop broad-spectrum antibiotics. A functionally important feature of OM porins is undergoing open-closed transitions that modulate their transport properties. To characterize the molecular basis of these transitions, we performed an extensive set of molecular dynamics (MD) simulations of Escherichia coli OM porin OmpF. Markov-state analysis revealed that large-scale motion of an internal loop, L3, underlies the transition between energetically stable open and closed states. The conformation of L3 is controlled by H bonds between highly conserved acidic residues on the loop and basic residues on the OmpF β -barrel. Mutation of key residues important for the loop's conformation shifts the equilibrium between open and closed states and regulates translocation of permeants (ions and antibiotics), as observed in the simulations and validated by our whole-cell accumulation assay. Notably, one mutant system G119D, which we find to favor the closed state, has been reported in clinically resistant bacterial strains. Overall, our accumulated ∼200 µs of simulation data (the wild type and mutants) along with experimental assays suggest the involvement of internal loop dynamics in permeability of OM porins and antibiotic resistance in Gram-negative bacteria., Competing Interests: The authors declare no competing interest.

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.

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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background: High-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., Methods: To 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., Results: In 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., Conclusions: Our 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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

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., (Copyright © 2021. Published by Elsevier Inc.)

Metastatic estrogen receptor α (ERα)-positive breast cancer is presently incurable. Seeking to target these drug-resistant cancers, we report the discovery of a compound, called ErSO, that activates the anticipatory unfolded protein response (a-UPR) and induces rapid and selective necrosis of ERα-positive breast cancer cell lines in vitro. We then tested ErSO in vivo in several preclinical orthotopic and metastasis mouse models carrying different xenografts of human breast cancer lines or patient-derived breast tumors. In multiple orthotopic models, ErSO treatment given either orally or intraperitoneally for 14 to 21 days induced tumor regression without recurrence. In a cell line tail vein metastasis model, ErSO was also effective at inducing regression of most lung, bone, and liver metastases. ErSO treatment induced almost complete regression of brain metastases in mice carrying intracranial human breast cancer cell line xenografts. Tumors that did not undergo complete regression and regrew remained sensitive to retreatment with ErSO. ErSO was well tolerated in mice, rats, and dogs at doses above those needed for therapeutic responses and had little or no effect on normal ERα-expressing murine tissues. ErSO mediated its anticancer effects through activation of the a-UPR, suggesting that activation of a tumor protective pathway could induce tumor regression., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Acute kidney injury (AKI) is morphologically characterized by a synchronized plasma membrane rupture of cells in a specific section of a nephron, referred to as acute tubular necrosis (ATN). Whereas the involvement of necroptosis is well characterized, genetic evidence supporting the contribution of ferroptosis is lacking. Here, we demonstrate that the loss of ferroptosis suppressor protein 1 (Fsp1) or the targeted manipulation of the active center of the selenoprotein glutathione peroxidase 4 (Gpx4 cys/- ) sensitize kidneys to tubular ferroptosis, resulting in a unique morphological pattern of tubular necrosis. Given the unmet medical need to clinically inhibit AKI, we generated a combined small molecule inhibitor (Nec-1f) that simultaneously targets receptor interacting protein kinase 1 (RIPK1) and ferroptosis in cell lines, in freshly isolated primary kidney tubules and in mouse models of cardiac transplantation and of AKI and improved survival in models of ischemia-reperfusion injury. Based on genetic and pharmacological evidence, we conclude that GPX4 dysfunction hypersensitizes mice to ATN during AKI. Additionally, we introduce Nec-1f, a solid inhibitor of RIPK1 and weak inhibitor of ferroptosis., (© 2021. The Author(s).)

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., (© 2021 Wiley-VCH GmbH.)

It has been over half a century since the last class of antibiotics active against the most problematic Gram-negative bacteria was approved by the Food and Drug Administration (FDA). The major challenge with developing antibiotics to treat these infections is not drug-target engagement but rather the inability of most small molecules to traverse the Gram-negative membranes, be retained, and accumulate within the cell. Despite an abundance of lead compounds, limited understanding of the physicochemical properties needed for compound accumulation (or avoidance of efflux) in Gram-negative bacteria has precluded a generalizable approach for developing Gram-negative antibiotics. Indeed, in many instances, despite years of intensive derivatization efforts and the synthesis of hundreds of compounds aimed at building in Gram-negative activity, little or no progress has been made in expanding the spectrum of activity for many Gram-positive-only antibiotics. In this Account, we describe the discovery and successful applications of a promising strategy for enhancing the accumulation of Gram-positive-only antibiotics as a means of imbuing compounds with broad-spectrum activity.Utilizing a prospective approach examining the accumulation in Escherichia coli for more than 180 diverse compounds, we found that small molecules have an increased likelihood to accumulate in E. coli when they contain an ionizable N itrogen, have low T hree-dimensionality, and are R igid. Implementing these guidelines, codified as the "eNTRy rules" and assisted by web application www.entry-way.org, we have facilitated compound entry and systematically built Gram-negative activity into Gram-positive-only antibiotics. Though each antibiotic will have case-specific considerations, we describe a set of important criteria to consider when selecting candidate Gram-positive-only antibiotics for conversion to Gram-negative-active versions via the eNTRy rules. As detailed herein, using this blueprint the spectrum of activity was expanded for three antibiotic classes that engage three different biological targets: DNA gyrase inhibitor 6DNM, FabI inhibitor Debio-1452, and FMN riboswitch inhibitor Ribocil C. In each scenario, the eNTRy rules guided the synthesis of key analogues predisposed to accumulate in Gram-negative bacteria leading to compounds that display antibiotic activity (minimum inhibitory concentrations (MIC) ≤8 μg mL -1 ) against E. coli and other Gram-negative ESKAPE pathogens. While the eNTRy rules will continue to be refined and enhanced as more accumulation data is gathered, on the basis of these collective results and on other examples not covered herein it is clear that the eNTRy rules are actionable for the development of novel broad-spectrum antibiotics from Gram-positive-only compounds. By enabling the prediction of compound accumulation, the eNTRy rules should facilitate the process of discovering and developing novel antibiotics active against Gram-negative bacteria.

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. aureu s) 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.

Pantoea ananatis is the primary cause of onion center rot. Genetic data suggest that a phosphonic acid natural product is required for pathogenesis; however, the nature of the molecule is unknown. Here, we show that P. ananatis produces at least three phosphonates, two of which were purified and structurally characterized. The first, designated pantaphos, was shown to be 2-(hydroxy[phosphono]methyl)maleate; the second, a probable biosynthetic precursor, was shown to be 2-(phosphonomethyl)maleate. Purified pantaphos is both necessary and sufficient for the hallmark lesions of onion center rot. Moreover, when tested against mustard seedlings, the phytotoxic activity of pantaphos was comparable to the widely used herbicides glyphosate and phosphinothricin. Pantaphos was also active against a variety of human cell lines but was significantly more toxic to glioblastoma cells. Pantaphos showed little activity when tested against a variety of bacteria and fungi. IMPORTANCE Pantoea ananatis is a significant plant pathogen that targets a number of important crops, a problem that is compounded by the absence of effective treatments to prevent its spread. Our identification of pantaphos as the key virulence factor in onion center rot suggests a variety of approaches that could be employed to address this significant plant disease. Moreover, the general phytotoxicity of the molecule suggests that it could be developed into an effective herbicide to counter the alarming rise in herbicide-resistant weeds., (Copyright © 2021 Polidore et al.)

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.

Covering: up to 2020 Natural products have a long history in drug discovery, with their inherent biological activity often tailored by medicinal chemists to arrive at the final drug product. This process is illustrated by numerous examples, including the conversion of epothilone to ixabepilone, erythromycin to azithromycin, and lovastatin to simvastatin. However, natural products are also fruitful starting points for the creation of complex and diverse compounds, especially those that are markedly different from the parent natural product and accordingly do not retain the biological activity of the parent. The resulting products have physiochemical properties that differ considerably when compared to traditional screening collections, thus affording an opportunity to discover novel biological activity. The synthesis of new structural frameworks from natural products thus yields value-added compounds, as demonstrated in the last several years with multiple biological discoveries emerging from these collections. This Highlight details a handful of these studies, describing new compounds derived from natural products that have biological activity and cellular targets different from those evoked/engaged by the parent. Such re-engineering of natural products offers the potential for discovering compounds with interesting and unexpected biological activity.

Multidrug-resistant Gram-negative (GN) infections for which there are few available treatment options are increasingly common. The development of new antibiotics for these pathogens is challenging because of the inability of most small molecules to accumulate inside GN bacteria. Using recently developed predictive guidelines for compound accumulation in Escherichia coli , we have converted the antibiotic Ribocil C, which targets the flavin mononucleotide (FMN) riboswitch, from a compound lacking whole-cell activity against wild-type GN pathogens into a compound that accumulates to a high level in E. coli , is effective against Gram-negative clinical isolates, and has efficacy in mouse models of GN infections. This compound allows for the first assessment of the translational potential of FMN riboswitch binders against wild-type Gram-negative bacteria.

Diazomethane is one of the most versatile reagents in organic synthesis, but its utility is limited by its hazardous nature. Although alternative methods exist to perform the unique chemistry of diazomethane, these suffer from diminished reactivity and/or correspondingly harsher conditions. Herein, we describe the repurposing of imidazotetrazines (such as temozolomide, TMZ, the standard of care for glioblastoma) for use as synthetic precursors of alkyl diazonium reagents. TMZ was employed to conduct esterifications and metal-catalyzed cyclopropanations, and results show that methyl ester formation from a wide variety of substrates is especially efficient and operationally simple. TMZ is a commercially available solid that is non-explosive and non-toxic, and should find broad utility as a replacement for diazomethane., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Gram-negative bacterial infections are a significant public health concern, and the lack of new drug classes for these pathogens is linked to the inability of most drug leads to accumulate inside Gram-negative bacteria 1-7 . Here, we report the development of a web application-eNTRyway-that predicts compound accumulation (in Escherichia coli) from its structure. In conjunction with structure-activity relationships and X-ray data, eNTRyway was utilized to re-design Debio-1452-a Gram-positive-only antibiotic 8 -into versions that accumulate in E. coli and possess antibacterial activity against high-priority Gram-negative pathogens. The lead compound Debio-1452-NH3 operates as an antibiotic via the same mechanism as Debio-1452, namely potent inhibition of the enoyl-acyl carrier protein reductase FabI, as validated by in vitro enzyme assays and the generation of bacterial isolates with spontaneous target mutations. Debio-1452-NH3 is well tolerated in vivo, reduces bacterial burden in mice and rescues mice from lethal infections with clinical isolates of Acinetobacter baumannii, Klebsiella pneumoniae and E. coli. This work provides tools for the facile discovery and development of high-accumulating compounds in E. coli, and a general blueprint for the conversion of Gram-positive-only compounds into broad-spectrum antibiotics.