We have developed a new mass spectrometry (MS) technology, the Single-probe MS, capable of real-time, in situ metabolomic analysis individual living cells. The is miniaturized multifunctional sampling and ionization device that directly coupled to spectrometer. With tip smaller than eukaryotic cells (<10 μm), can be inserted into single sample intracellular compounds for real-time MS analysis. used detect several cellular metabolites anticancer small molecules paclitaxel, doxorubicin, OSW-1...

The remarkable action of an IIII species on a tripeptide containing acyclic tyrosine/tryptophan is the key step in total synthesis (−)-diazonamide A. completed preparation this new antimitotic concise, multiply convergent, and amenable to diversification intermediates.

Unable to bear the weight of scrutiny, original structure proposed for (−)-diazonamide A (1) must be revised. convergent, stereocontrolled total synthesis provided 1, which shows altered physical and spectroscopic characteristics relative those a sample natural product. Reinterpretation reported data new insight indicate that actual diazonamide is aminal-containing (S)-α-hydroxy isovaleric acid conjugate 2. Gratifyingly, synthetic C11 acetal congener 2 is, by all biological measures...

We have studied a naturally occurring small-molecule antimitotic called diazonamide A. Diazonamide A is highly effective at blocking spindle assembly in mammalian cell culture and does so through unique mechanism. biotinylated form of affinity purifies ornithine delta-amino transferase (OAT), mitochondrial enzyme, from HeLa Xenopus egg extracts. In the latter system, interaction between OAT regulated by RanGTP. find that specific knockdown human cervical carcinoma osteosarcoma cells RNA...

Proteomics analysis of mass-limited samples has become increasingly important for understanding biological systems in physiologically relevant contexts such as patient samples, multicellular organoids, spheroids, and single cells. However, relatively low sensitivity top-down proteomics methods makes their application to challenging. Capillary electrophoresis (CE) emerged an ideal separation method due its high resolution, ultralow detection limit, minimal sample volume requirements....

γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression essential in cysteine homeostasis, its induction has been implicated the pathology of asthma, reperfusion injury, cancer. In this study, we report four new crystal structures human (hGGT1) show conformational changes within active site as enzyme progresses from free to inhibitor-bound tetrahedral transition states finally...

Existing single cell mass spectrometry (SCMS) sampling platforms are largely designed to work only with immobilized cells and not the suspended isolated from patient samples. Here, we present a novel method that integrates commercially available manipulation system commonly used for in vitro fertilization Single-probe SCMS technology. The combined SCMS/cell manipulating platform is capable of rapidly analyzing intracellular species real time suspension leukemia line. A broad range molecular...

Analyzing cellular constituents on the single-cell level through mass spectrometry (MS) allows for a wide range of compounds to be studied simultaneously. However, there is need quantitative (qSCMS) methods fully characterize drug efficacy from individual cells within cell populations. In this study, qSCMS experiments were carried out using Single-probe MS technique. The method was successfully used perform rapid absolute quantifications anticancer irinotecan in mammalian cancer under...

Blocking cell division through the inhibition of mitosis is one most successful clinical strategies for treatment cancer. Taxanes and vinca alkaloids are in widespread use have demonstrated substantive therapeutic efficacy. Both classes compounds bind directly to tubulin, a structural component mitotic spindle. The ubiquitous utilization tubulin both cancerous normal cells, however, tempers broad spectrum activity currently used antimitotics by significant toxicities dividing tissue....

Der durch eine IIII-Spezies vermittelte Ringschluss eines acyclischen Tripeptids ist der Schlüsselschritt hier vorgestellten Totalsynthese von (−)-Diazonamid A. Dieser Zugang nicht nur schnell, sondern bietet auch etliche Möglichkeiten zur Diversifizierung.

In clinical cancer medicine, the current inability to quantify intracellular chemotherapy drug concentrations in individual human cells limits personalization and overall effectiveness of administration. New bioanalytical methods capable real-time measurement levels live single would allow for more adaptive personalized administration drugs, potentially leading better outcomes with fewer side effects. this study, we report development a new quantitative cell mass spectrometry (qSCMS) method...

Aromatic prenyltransferases from natural product biosynthetic pathways display relaxed specificity for their aromatic substrates. While a growing body of evidence suggests to be more tolerant towards alkyl-donor substrates, most studies aimed at probing donor-substrate are limited only small set alkyl pyrophosphate donors, restricting broader utility as biocatalysts synthetic applications. Here, we assess the donor substrate an l-tryptophan C4-prenyltransferase, also known...

The development of precision drugs for the selective treatment ovarian cancer will require targeting proliferative factors selectively expressed in tumors or unique physiological microenvironments specific tumors. Here, we report that oxysterol-binding protein (OSBP)-related 4 (ORP4) is a potential druggable target cells. ORP4 has limited expression normal tissues and was recently recognized to be cancer-specific driver cellular proliferation, including patient-isolated leukemias. We...

Oxysterol-binding protein (OSBP) and OSBP-related 4 (ORP4) have emerged as potentially druggable targets in antiviral precision cancer drug development. Multiple structurally diverse small molecules function through targeting the OSBP/ORP family of proteins, including steroidal compounds OSW-1 T-00127-HEV2. Here, structure–activity relationships oxysterols related compound binding to human OSBP ORP4 are characterized. Oxysterols with hydroxylation at various side chain positions (i.e., C-20,...

Einer genauen Prüfung konnte sie nicht standhalten, die ursprünglich dem antimitotisch wirksamen Naturstoff (−)-Diazonamid A 1 zugeordnete Struktur. Das durch eine konvergente, stereokontrollierte Totalsynthese erhaltene Produkt zeigt andere physikalische und spektroskopische Eigenschaften als natürliche Probe. Neuinterpretationen publizierter Daten neue Erkenntnisse lassen nun den Schluss zu, dass Diazonamid tatsächlich Struktur 2 mit einer Aminal-Einheit...

Oxysterol-binding protein (OSBP) is a lipid transport and regulatory required for the replication of Enterovirus genus viruses, which includes many significant human pathogens. Short-term exposure (i.e., 1–6 h) to low dose 1 nM) natural product compound OSW-1 induces reduction cellular OSBP levels by ∼90% in multiple different cell lines with no measurable cytotoxicity, defect proliferation, or global proteome reduction. Interestingly, persists days after low-dose, transient treatment ended...

One bond and a water molecule separate title compound 1 from the potently bioactive peptide metabolite diazonamide A. Compositionally similar, yet topographically distinct, A are both toxic towards cultured human cancer cells although mechanisms underlying their actions likely differ. The quest completely synthetic diazonamides continues.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), a global pandemic characterized by an exaggerated immune response and respiratory illness. Age (&gt;60 years) significant risk factor for developing severe COVID-19. To better understand host aged airway epithelium to SARS-CoV-2 infection, we performed in vitro study using primary human bronchial epithelial cells from donors &gt;67 years age differentiated on air–liquid...

Tryprostatin A and B are prenylated, tryptophan-containing, diketopiperazine natural products, displaying cytotoxic activity through different mechanisms of action. The presence the 6-methoxy substituent on indole moiety tryprostatin was shown to be essential for dual inhibition topoisomerase II tubulin polymerization. However, inability perform late-stage modification ring has limited structure–activity relationship studies this class products. Herein, we describe an efficient...

Overexpression of γ-glutamyl transpeptidase (GGT1) has been implicated in an array human diseases including asthma, reperfusion injury, and cancer. Inhibitors are needed for therapy, but development potent, specific inhibitors GGT1 hampered by a lack structural information regarding substrate binding cleavage. To enhance our understanding the molecular mechanism cleavage, we have solved crystal structures (hGGT1) with glutathione (a substrate) phosphate-glutathione analog (an irreversible...

The retinaldehyde dehydrogenase (RALDH) enzymes, RALDH1, RALDH2, and RALDH3, catalyze the irreversible oxidation of to all-trans-retinoic acid (ATRA). Despite importance RALDH enzymes in embryonic development, postnatal growth differentiation, several disease states, there are no commercially available inhibitors that specifically target these isozymes. We report here development characterization a small molecule inhibitor dichloro-all-trans-retinone (DAR) (Summers et al., 2017) is an 2, 3...

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges cellular species on the individual-cell level. The single-probe, a microscale sampling ionization device, can be coupled with spectrometer for on-line, rapid SCMS constituents under ambient conditions. Previously, single-probe technique was primarily used to measure cells immobilized onto substrate, limiting types studies. In current study, technology has been integrated manipulation...

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges cellular species on the individual-cell level. The single-probe, a microscale sampling ionization device, can be coupled with spectrometer for on-line, rapid SCMS constituents under ambient conditions. Previously, single-probe technique was primarily used to measure cells immobilized onto substrate, limiting types studies. In current study, technology has been integrated manipulation...