Precise Readout of MEK1 Proteoforms upon MAPK Pathway Modulation by Individual Ion Mass Spectrometry.

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.

A catalytic process enables efficient and programmable access to precisely altered indole alkaloid scaffolds.

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.

Discovery of KRB-456, a KRAS G12D Switch-I/II Allosteric Pocket Binder That Inhibits the Growth of Pancreatic Cancer Patient-derived Tumors.

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.

Augmented Concentration of Isopentyl-Deoxynyboquinone in Tumors Selectively Kills NAD(P)H Quinone Oxidoreductase 1-Positive Cancer Cells through Programmed Necrotic and Apoptotic Mechanisms.

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. ß-Lapachone (ß-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.

IP-DNQ induces mitochondrial dysfunction and G2/M phase cell cycle arrest to selectively kill NQO1-positive pancreatic cancer cells.

Pancreatic cancer is among the top five leading causes of cancer-related deaths worldwide, with low 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 normal tissues, makes NQO1 bioactivatable drugs a potential therapy for selectively killing NQO1-positive cancer cells. Our previous studies have revealed that novel NQO1 bioactivatable drug deoxynyboquinone (DNQ) is ten-fold more potent than the prototypic NQO1 bioactivatable drug ß-lapachone in killing of NQO1-positive cancer cells. However, DNQ treatment results in high-grade methemoglobinemia, a significant side effect that limits clinical development. Here, we report for the first time on a DNQ derivative, isopentyl-deoxynboquinone (IP-DNQ), which selectively kills pancreatic ductal adenocarcinoma cells in an NQO1-dependent manner with equal potency to the parent DNQ. IP-DNQ evokes massive ROS production and oxidative DNA lesions that results in PARP1 hyperactivation, mitochondrial catastrophe and G2/M-phase arrest, leading to apoptotic and necrotic programmed cell death. Importantly, IP-DNQ treatment causes mild methemoglobinemia in vivo, with a three-fold improvement in the maximum tolerated dose compared to DNQ, while significantly suppresses tumor growth and extends the lifespan of mice in subcutaneous and orthotopic pancreatic cancer xenograft models. 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.

Porin-independent accumulation in Pseudomonas enables antibiotic discovery.

Gram-negative antibiotic development has been hindered by a poor understanding of the types of compounds that can accumulate within these bacteria1,2. The presence of efflux pumps and substrate-specific outer-membrane porins in Pseudomonas aeruginosa renders this pathogen particularly challenging3. As a result, there are few antibiotic options for P. aeruginosa infections4 and its many porins have made the prospect of discovering general accumulation guidelines seem unlikely5. 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.

The combination of PAC-1 and entrectinib for the treatment of metastatic uveal melanoma.

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.

Re-education of myeloid immune cells to reduce regulatory T cell expansion and impede breast cancer progression.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy but 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. We demonstrate that NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the NLRP3 inflammasome and reduce the expansion of immune-suppressive regulatory T cells (Treg). Loss of NR0B2 increased mammary tumor growth and metastasis. Small molecule agonists, including one developed here, reduced Treg expansion, reduced metastatic growth and improved the efficacy of ICB. This work identifies NR0B2 as a target to re-educate myeloid immune cells providing proof-of-principle that this cholesterol-homeostasis axis may have utility in enhancing ICB.

Phase I dose-escalation study of procaspase-activating compound-1 in combination with temozolomide in patients with recurrent high-grade astrocytomas.

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/m2/5 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, O6-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/m2/5 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.

Plasma Membrane Channel TRPM4 Mediates Immunogenic Therapy-Induced Necrosis.

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.

A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain.

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 (IC50 6.4 µM) and GS441524 (IC50 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, IC50 370 nM) and TFMU-ADPr (ADP-ribosylated trifluoromethyl umbelliferone, IC50 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.

Single CAR-T cell treatment controls disseminated ovarian cancer in a syngeneic mouse model.

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 ID8Cosmc knock-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. 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.

Phase I study of procaspase-activating compound-1 (PAC-1) in the treatment of advanced malignancies.

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 t1/2 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.

Correction to "An Iterative Approach Guides Discovery of the FabI Inhibitor Fabimycin, a Late-Stage Antibiotic Candidate with In Vivo Efficacy against Drug-Resistant Gram-Negative Infections".

[This corrects the article DOI: 10.1021/acscentsci.2c00598.].

An Iterative Approach Guides Discovery of the FabI Inhibitor Fabimycin, a Late-Stage Antibiotic Candidate with In Vivo Efficacy against Drug-Resistant Gram-Negative Infections.

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.

Evolution of 3-(4-hydroxyphenyl)indoline-2-one as a scaffold for potent and selective anticancer activity.

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.