Increased throughput in methods for simulating protein ligand binding and unbinding

By incorporating full flexibility and enabling the quantification of crucial parameters such as binding free energies and residence times, methods for investigating protein-ligand binding and unbinding via molecular dynamics provide details on the involved mechanisms at the molecular level. While these advancements hold promise for impacting drug discovery, a notable drawback persists: their relatively time-consuming nature limits throughput. Herein, we survey recent implementations which, employing a blend of enhanced sampling techniques, a clever choice of collective variables, and often machine learning, strive to enhance the efficiency of new and previously reported methods without compromising accuracy. Particularly noteworthy is the validation of these methods that was often performed on systems mirroring real-world drug discovery scenarios

Improving the treatment of acute lymphoblastic leukemia

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

Functional characterization of two PLP-dependent enzymes involved in capsular polysaccharide biosynthesis from campylobacter jejuni

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

High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity

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

Monoterpenoids: The Next Frontier in the Treatment of Chronic Pain?

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

ABCC transporters mediate the vacuolar accumulation of crocins in saffron stigmas

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