Drug-induced Metabolic Changes Research Articles

Combining CRISPRi and metabolomics for functional annotation of compound libraries.

Molecular profiling of small-molecules offers invaluable insights into the function of compounds and allows for hypothesis generation about small molecule direct targets and secondary effects. However, current profiling methods are either limited in the number of measurable parameters or throughput. Here, we developed a multiplexed, unbiased framework that, by linking genetic to drug-induced changes in nearly a thousand metabolites, allows for high-throughput functional annotation of compound libraries in Escherichia coli. First, we generated a reference map of metabolic changes from (CRISPR) interference with 352 genes in all major essential biological processes. Next, based on the comparison of genetic with 1342 drug-induced metabolic changes we made de novo predictions of compound functionality and revealed antibacterials with unconventional Modes of Action. We show that our framework, combining dynamic gene silencing with metabolomics, can be adapted as a general strategy for comprehensive high-throughput analysis of compound functionality, from bacteria to human cell lines.

From the Editor's Desk…

From the Editor's Desk…

Next-generation Metabolomics in the Development of New Antidepressants: Using Albiflorin as an Example.

Depression is a highly prevalent disorder that affects more than 300 million adults worldwide in 2015. Depression also frequently coexists with many other conditions such as osteoporosis and one-third of the Intensive Care Unit (ICU) survivors had depressive symptoms. Antidepressants have become the most commonly prescribed drugs in the United States. In addition to the regular process, drug discovery and development (R&D) for depression presents extra challenges because of the heterogeneity of the symptoms and various co-occurring disorders. Botanical medicine with multi-functional nature has been proposed to be more effective, providing rapid control of core and comorbid conditions of depression. With the technical advances in analytical instruments, metabolomics is entering into a "new generation". Next-generation metabolomics (NGM) has the capability to comprehensively characterize drug-induced metabolic changes in the biological systems. NGM has demonstrated great potential in all the stages of pharmaceutical R&D in the last 10 years. Albiflorin isolated from Peony roots is a promising drug candidate with multi-target for depression and is currently under development by Beijing Wonner Biotech. In this work, we summarized the common analytical platforms for NGM and its main applications in drug R&D. We used albiflorin as an example to illustrate how NGM improves our understanding of drug candidate actions and facilitates drug safety evaluation. Future directions on how to expand the use of NGM for new antidepressant development in pharmaceutical industry were also discussed.

Metabolic cofactors NAD(P)H and FAD as potential indicators of cancer cell response to chemotherapy with paclitaxel

Paclitaxel, a widely used antimicrotubular agent, predominantly eliminates rapidly proliferating cancer cells, while slowly proliferating and quiescent cells can survive the treatment, which is one of the main reasons for tumor recurrence and non-responsiveness to the drug. To improve the efficacy of chemotherapy, biomarkers need to be developed to enable monitoring of tumor responses. In this study we considered the auto-fluorescent metabolic cofactors NAD(P)H and FAD as possible indicators of cancer cell response to therapy with paclitaxel. It was found that, among the tested parameters (the fluorescence intensity-based redox ratio FAD/NAD(P)H, and the fluorescence lifetimes of NAD(P)H and FAD), the fluorescence lifetime of NAD(P)H is the most sensitive in tracking the drug response, and is capable of indicating heterogeneous cellular responses both in cell monolayers and in multicellular tumor spheroids. We observed that metabolic reorganization to a more oxidative state preceded the morphological manifestation of cell death and developed faster in cells that were more responsive to the drug. Our results suggest that noninvasive, label-free monitoring of the drug-induced metabolic changes by noting the NAD(P)H fluorescence lifetime is a valuable approach to characterize the responses of cancer cells to anti-cancer treatments and, therefore, to predict the effectiveness of chemotherapy.

Optical Imaging of Drug-Induced Metabolism Changes in Murine and Human Pancreatic Cancer Organoids Reveals Heterogeneous Drug Response.

ObjectivesThree-dimensional organoids derived from primary pancreatic ductal adenocarcinomas are an attractive platform for testing potential anticancer drugs on patient-specific tissue. Optical metabolic imaging (OMI) is a novel tool used to assess drug-induced changes in cellular metabolism, and its quantitative end point, the OMI index, is evaluated as a biomarker of drug response in pancreatic cancer organoids.MethodsOptical metabolic imaging is used to assess both malignant cell and fibroblast drug response within primary murine and human pancreatic cancer organoids.ResultsAnticancer drugs induce significant reductions in the OMI index of murine and human pancreatic cancer organoids. Subpopulation analysis of OMI data revealed heterogeneous drug response and elucidated responding and nonresponding cell populations for a 7-day time course. Optical metabolic imaging index significantly correlates with immunofluorescence detection of cell proliferation and cell death.ConclusionsOptical metabolic imaging of primary pancreatic ductal adenocarcinoma organoids is highly sensitive to drug-induced metabolic changes, provides a nondestructive method for monitoring dynamic drug response, and presents a novel platform for patient-specific drug testing and drug development.

18F-FDG-PET/CT imaging of drug-induced metabolic changes in genetically engineered mouse lung cancer models.

The most commonly used radiotracer for cancer imaging in humans and mice is 2-deoxy-2-((18)F)fluoro-d-glucose ((18)F-FDG). We have used FDG coupled with positron-emission tomography (PET) to assess the pharmacodynamic efficacy of a number of therapeutics in genetically engineered mouse lung cancer models. In this protocol, we present our approach for FDG-PET imaging of early tumor metabolic changes induced by drug treatment. Special consideration is given to animal preparation, anesthesia, and PET/computed tomography (CT) imaging of mice with lung tumors. Specifically, we recommend fasting the mice overnight to reduce background, using sevoflurane anesthesia and a "conscious" uptake period to minimize cardiac FDG uptake, adopting a relatively short duration of CT and PET scanning to facilitate serial imaging, and quantifying the comparison between the maximum standardized uptake values (SUVs) of lung tumors before and after treatment to determine treatment effects. Used in this manner, FDG-PET can rapidly assess tumor metabolism before and after treatment with an experimental therapeutic. In many cases, metabolic changes are apparent after just a single dose of treatment, helping to show target engagement and modulation by the drug (pharmacodynamic efficacy) within days of starting therapy.

Inhibition of monocarboxylate transporter-1 (MCT1) by AZD3965 enhances radiosensitivity by reducing lactate transport.

Inhibition of the monocarboxylate transporter MCT1 by AZD3965 results in an increase in glycolysis in human tumor cell lines and xenografts. This is indicated by changes in the levels of specific glycolytic metabolites and in changes in glycolytic enzyme kinetics. These drug-induced metabolic changes translate into an inhibition of tumor growth in vivo. Thus, we combined AZD3965 with fractionated radiation to treat small cell lung cancer (SCLC) xenografts and showed that the combination provided a significantly greater therapeutic effect than the use of either modality alone. These results strongly support the notion of combining MCT1 inhibition with radiotherapy in the treatment of SCLC and other solid tumors.

Edelfosine-induced metabolic changes in cancer cells that precede the overproduction of reactive oxygen species and apoptosis

BackgroundMetabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells.ResultsThe study of 13C distribution of Jukat cells exposed to low edelfosine concentration, which induces apoptosis in ≤5% of cells, revealed metabolic changes previous to the development of apoptotic program. Specifically, it was found that low dose of edelfosine stimulates the TCA cycle. These metabolic perturbations were coupled with an increase of nucleic acid synthesis de novo, which indicates acceleration of biosynthetic and reparative processes. The further increase of the TCA cycle fluxes, when higher doses of drug applied, eventually enhance reactive oxygen species (ROS) production and trigger apoptotic program.ConclusionThe application of Isodyn to the analysis of mechanism of edelfosine-induced apoptosis revealed primary drug-induced metabolic changes, which are important for the subsequent initiation of apoptotic program. Initiation of such metabolic changes could be exploited in anticancer therapy.

Asparagine synthetase expression alone is sufficient to induce l-asparaginase resistance in MOLT-4 human leukaemia cells

Childhood acute lymphoblastic leukaemia (ALL) is treated by combination chemotherapy with a number of drugs, always including the enzyme l-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance are a significant problem. In vitro studies have demonstrated increased asparagine synthetase (AS) expression in ASNase-resistant cells, which has led to the hypothesis that elevated AS activity permits drug-resistant survival. The data presented show that not only is elevated AS expression a property of ASNase-resistant MOLT-4 human leukaemia cells, but that short-term (12h) treatment of the cells with ASNase causes a relatively rapid induction of AS expression. The results also document that the elevated expression of AS in ASNase-resistant cells is not fully reversible, even 6 weeks after ASNase removal from the culture medium. Furthermore, ASNase resistance, assessed as both drug-insensitive cell growth rates and decreased drug-induced apoptosis, parallels this irreversible AS expression. Mimicking the elevated AS activity in ASNase-resistant cells by overexpression of the human AS protein by stable retroviral transformation of parental MOLT4 cells is sufficient to induce the ASNase-resistance phenotype. These data document that ASNase resistance in ALL cells is a consequence of elevated AS expression and that although other drug-induced metabolic changes occur, they are secondary to the increased asparagine biosynthetic rate.

Asparagine synthetase expression alone is sufficient to induce l-asparaginase resistance in MOLT-4 human leukaemia cells.

Childhood acute lymphoblastic leukaemia (ALL) is treated by combination chemotherapy with a number of drugs, always including the enzyme L-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance are a significant problem. In vitro studies have demonstrated increased asparagine synthetase (AS) expression in ASNase-resistant cells, which has led to the hypothesis that elevated AS activity permits drug-resistant survival. The data presented show that not only is elevated AS expression a property of ASNase-resistant MOLT-4 human leukaemia cells, but that short-term (12 h) treatment of the cells with ASNase causes a relatively rapid induction of AS expression. The results also document that the elevated expression of AS in ASNase-resistant cells is not fully reversible, even 6 weeks after ASNase removal from the culture medium. Furthermore, ASNase resistance, assessed as both drug-insensitive cell growth rates and decreased drug-induced apoptosis, parallels this irreversible AS expression. Mimicking the elevated AS activity in ASNase-resistant cells by overexpression of the human AS protein by stable retroviral transformation of parental MOLT4 cells is sufficient to induce the ASNase-resistance phenotype. These data document that ASNase resistance in ALL cells is a consequence of elevated AS expression and that although other drug-induced metabolic changes occur, they are secondary to the increased asparagine biosynthetic rate.

The study of neurotransmitter interactions using positron emission tomography and functional coupling.

Functional brain imaging with positron emission tomography (PET) has opened up new avenues for the investigation of possible functional disturbances related to psychiatric disease as well as pharmacodynamic assessment of drug treatment in vivo. Different strategies to study pharmacologic effects on the brain have been developed in recent years. The basic methods are to measure (a) blood flow or glucose metabolism, (b) parameters of specific receptor binding, or (c) neurotransmitter metabolism. Each of these can be performed either in a resting state or after perturbation with a pharmacologic challenge. Our group has developed a general strategy for investigating pharmacologic effects on brain function: (a) determining indirect drug-induced metabolic changes with fluorodeoxyglucose PET and (b) characterizing functional interactions of neurotransmitter systems by assaying drug-induced displacement of specific receptor ligands. These study designs reflect a paradigm shift where functional coupling of brain regions and interaction of different neurotransmitter systems are seen as the basis for a multitransmitter hypothesis of schizophrenia. In this view, any disturbance in the self-regulatory process is reflected in the loss of functional interaction between systems. An overview of recent studies and their possible clinical importance will be presented.

Coronary artery disease risk factor management in the hypertensive patient

Recent advances in understanding the relation of risk factors to coronary artery disease (CAD) have initiated a change in the approach to managing the hypertensive patient. Reduction of elevated blood pressure still remains a major therapeutic priority. However, the risk of cardiovascular morbidity is also related to hypercholesterolemia, hyperuricemia, hyperglycemia, hyperfibrinogenemia and obesity; all aggravate the risk of CAD in the patient with high blood pressure. Life-style is also important: cigarette smoking, high alcohol consumption and lack of physical exercise all predispose to precocious atheromatous CAD. Thus, the most favorable prognosis in terms of reducing CAD risk is accomplished by reducing elevated systemic arterial pressure while simultaneously improving all other risk factors. The method by which blood pressure is lowered is an important consideration. The ancillary metabolic activities of antihypertensive drugs now available differ widely. Diuretics and β blockers, for example, have potentially adverse metabolic effects, whereas agents such as selective α 1-adrenoceptor inhibitors appear to beneficially affect several metabolic cofactors influencing the CAD risk profile. The impact of such drug-induced metabolic changes on overall prognosis of the hypertensive patient remains to be clarified. In the absence of other contraindications, however, it is sensible to use drugs that do not increase the metabolic predilection to precocious CAD.

The Impact on Serum Lipids of Combinations of Diuretics and P-Blockers and of P-Blockers Alone

Treatment of hypertension with diuretics and beta-blockers is often associated with changes in lipids and uric acid levels. The clinical importance of these metabolic changes is still uncertain, but potentially they might counteract the beneficial effect of blood pressure reduction in some patients. As these drugs are widely used to prevent the cardiovascular complications of hypertension, it is imperative that we evaluate the effect of these drug-induced metabolic changes on the atherosclerotic process in humans.

Effects of Δ9-tetrahydrocannabinol administered subcutaneously to rabbits for 28 days

Subcutaneous (s.c.) administration of Δ 9-tetrahydrocannabinol ( Δ 9-THC) to rabbits produced dose-related cumulative toxicity. Five groups of three New Zealand albino rabbits each received 28 daily treatments with isotonic saline, sesame oil or 15.9, 45.0 or 153.4 mg/kg/day of Δ 9-THC dissolved in sesame oil. Dose-related dermal responses included erythema, edema, ulceration and nodule formation. Some of the granulomatous nodules contained an oily subtance and exhibited liquefactive necrosis. The severities of erythema and ulceration were generally maximal during the first week of treatment, but edema and nodule formation were most severe after days 12 and 14, respectively. All rabbits survived treatment, but body weights, liver weights and liver glycogen levels were decreased in a dose-related manner. Maximal body weight effects occurred after day 19. Hemochemical changes occurred only in rabbits treated with 153.4 mg/kg/day and included decreased blood sugar and alkaline phosphatase, and increased serum potassium. Hematology parameters were normal throughout the treatment period. No drug-related pathological lesions occurred in internal organs. The cumulative body weight changes, significantly decreased hepatic glycogen levels and reduced blood sugar and alkaline phosphatase values may have indicated drug-induced metabolic changes.