Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast (Saccharomyces cerevisiae), and in vitro transportomic assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify Crocus sativus transporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug and toxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the gene encoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana leaves.

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 μM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.

L-Asparaginase (EC 3.5.1.1) was first used as a component of combination drug therapies to treat acute lymphoblastic leukemia (ALL), a cancer of the blood and bone marrow, almost 50 years ago. Administering this enzyme to reduce asparagine levels in the blood is a cornerstone of modern clinical protocols for ALL; indeed, this remains the only successful example of a therapy targeted against a specific metabolic weakness in any form of cancer. Three problems, however, constrain the clinical use of L-asparaginase. First, a type II bacterial variant of L-asparaginase is administered to patients, the majority of whom are children, which produces an immune response thereby limiting the time over which the enzyme can be tolerated. Second, L-asparaginase is subject to proteolytic degradation in the blood. Third, toxic side effects are observed, which may be correlated with the L-glutaminase activity of the enzyme. This Perspective will outline how asparagine depletion negatively impacts the growth of leukemic blasts, discuss the structure and mechanism of L-asparaginase, and briefly describe the clinical use of chemically modified forms of clinically useful L-asparaginases, such as Asparlas, which was recently given FDA approval for use in children (babies to young adults) as part of multidrug treatments for ALL. Finally, we review ongoing efforts to engineer L-asparaginase variants with improved therapeutic properties and briefly detail emerging, alternate strategies for the treatment of forms of ALL that are resistant to asparagine depletion.

The mechanisms of inflammation and cancer are intertwined by complex networks of signaling pathways. Dysregulations in the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway underlie several pathogenic conditions related to chronic inflammatory states, autoimmune diseases and cancer. Historically, the potential application of JAK inhibition has been thoroughly explored, thus triggering an escalation of favorable results in this field. So far, five JAK inhibitors have been approved by the Food and Drug Administration (FDA) for the treatment of different diseases. Considering the complexity of JAK-depending processes and their involvement in multiple disorders, JAK inhibitors are the perfect candidates for drug repurposing and for the assessment of multitarget strategies. Herein we reviewed the recent progress concerning JAK inhibition, including the innovations provided by the release of JAKs crystal structures and the improvement of synthetic strategies aimed to simplify of the industrial scale-up.

Campylobacter jejuni is a Gram-negative, pathogenic bacterium that causes campylobacteriosis, a form of gastroenteritis. C. jejuni is the most frequent cause of food-borne illness in the world, surpassing Salmonella and E. coli. Coating the surface of C. jejuni is a layer of sugar molecules known as the capsular polysaccharide that, in C. jejuni NCTC 11168, is composed of a repeating unit of d-glycero-l-gluco-heptose, d-glucuronic acid, d-N-acetyl-galactosamine, and d-ribose. The d-glucuronic acid moiety is further amidated with either serinol or ethanolamine. It is unknown how these modifications are synthesized and attached to the polysaccharide. Here, we report the catalytic activities of two previously uncharacterized, pyridoxal phosphate (PLP)-dependent enzymes, Cj1436 and Cj1437, from C. jejuni NCTC 11168. Using a combination of mass spectrometry and nuclear magnetic resonance, we determined that Cj1436 catalyzes the decarboxylation of l-serine phosphate to ethanolamine phosphate. Cj1437 was shown to catalyze the transamination of dihydroxyacetone phosphate to (S)-serinol phosphate in the presence of l-glutamate. The probable routes to the ultimate formation of the glucuronamide substructures in the capsular polysaccharides of C. jejuni are discussed.

Inflammatory states are among the most common and most treated medical conditions. Inflammation comes along with swelling, pain and uneasiness in using the affected area. Inflammation is not always a simple symptom; more often is part of a defensive response of the body to an external threat or is a sign that the damaged tissue has not healed yet and needs to rest. The management of the pain associated with an inflammatory state could be a tricky task. In fact, most remedies simply quench the pain, leaving the inflammatory state unaltered. This review focuses on sesquiterpene lactones, a class of natural compounds, that represents a future promise in the treatment of inflammation. Sesquiterpene lactones are efficient inhibitors of multiple targets of the inflammatory process. Their natural sources are often ancient remedies with relevant traditional uses in folk medicines. This work also aims to elucidate how these compounds may represent the starting material for the development of new anti-inflammatory drugs.

Artemisinin (ART) is an endoperoxide sesquiterpene lactone, commonly used in the treatment of malaria. Although it was isolated from Artemisia annua L., a plant widely applied in Chinese Traditional Medicine, its mechanism of action remains uncertain and its clinical use is still limited due to its low solubility, its poor bioavailability and short in vivo half-life. Over time, several studies have been aimed towards the discovery of potent ART derivatives that could overcome clinical drawbacks. In this review, we focus on the multifaced aspects of ART and on the efforts spent to improve its pharmacological profile that so far culminated in the discovery of more effective drugs. Lastly, we outline the new perspectives in the ART-derivatives scenario.

Ointments and lotions from natural extracts have a long tradition of being used in folk medicines against pain conditions. Monoterpenoids are among the major constituents of several natural topical remedies. The field of chronic pain is one of the most investigated for new active molecular entities. This review will discuss several molecular mechanisms against which monoterpenoids have been proven to be good candidates for the topical treatment of chronic pain.