Toward Understanding the Chemistry and Biology of 1-Deoxy-d-xylulose 5-Phosphate (DXP) Synthase: A Unique Antimicrobial Target at the Heart of Bacterial Metabolism.

Antibiotics are the cornerstone of modern healthcare. The 20th century discovery of sulfonamides and ß-lactam antibiotics altered human society immensely. Simple bacterial infections were no longer a leading cause of morbidity and mortality, and antibiotic prophylaxis greatly reduced the risk of infection from surgery. The current healthcare system requires effective antibiotics to function. However, antibiotic-resistant infections are becoming increasingly prevalent, threatening the emergence of a postantibiotic era. To prevent this public health crisis, antibiotics with novel modes of action are needed. Currently available antibiotics target just a few cellular processes to exert their activity: DNA, RNA, protein, and cell wall biosynthesis. Bacterial central metabolism is underexploited, offering a wealth of potential new targets that can be pursued toward expanding the armamentarium against microbial infections. Discovered in 1997 as the first enzyme in the methylerythritol phosphate (MEP) pathway, 1-deoxy-d-xylulose 5-phosphate (DXP) synthase is a thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the decarboxylative condensation of pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) to form DXP. This five-carbon metabolite feeds into three separate essential pathways for bacterial central metabolism: ThDP synthesis, pyridoxal phosphate (PLP) synthesis, and the MEP pathway for isoprenoid synthesis. While it has long been identified as a target for the development of antimicrobial agents, limited progress has been made toward developing selective inhibitors of the enzyme. This Account highlights advances from our lab over the past decade to understand this important and unique enzyme. Unlike all other known ThDP-dependent enzymes, DXP synthase uses a random-sequential mechanism that requires the formation of a ternary complex prior to decarboxylation of the lactyl-ThDP intermediate. Its large active site accommodates a variety of acceptor substrates, lending itself to a number of alternative activities, such as the production of α-hydroxy ketones, hydroxamates, amides, acetolactate, and peracetate. Knowledge gained from mechanistic and substrate-specificity studies has guided the development of selective inhibitors with antibacterial activity and provides a biochemical foundation toward understanding DXP synthase function in bacterial cells. Although it is a promising drug target, the centrality of DXP synthase in bacterial metabolism imparts specific challenges to assessing antibacterial activity of DXP synthase inhibitors, and the susceptibility of most bacteria to current DXP synthase inhibitors is remarkably culture-medium-dependent. Despite these challenges, the study of DXP synthase is poised to reveal the role of DXP synthase in bacterial metabolic adaptability during infection, ultimately providing a more complete picture of how inhibiting this crucial enzyme can be used to develop novel antibiotics.

Pharmacological activation of myosin II paralogs to correct cell mechanics defects.

Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human myosin IIB (MYH10) and human myosin IIC (MYH14), but not human myosin IIA (MYH9). We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof of concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior.

Bisphosphonamidate clodronate prodrug exhibits selective cytotoxic activity against melanoma cell lines.

Bisphosphonates are used clinically to treat disorders of calcium metabolism and malignant bone disease and are known to inhibit cancer cell growth, adhesion, and invasion. However, clinical use of these agents for the treatment of extraskeletal disease is limited because of low cell permeability. We recently described a bisphosphonamidate prodrug strategy for efficient intracellular release of bisphosphonates, including clodronate (CLO), in non-small cell lung cancer cells. To evaluate anticancer activity of this prodrug class across many cancer cell types, the bisphosphonamidate clodronate prodrug (CLO prodrug) was screened against the NCI-60 cell line panel, and was found to exhibit selectivity toward melanoma cell lines. Here, we confirm efficient cellular uptake and intracellular activation of this prodrug class in melanoma cells. We further demonstrate inhibition of melanoma cell proliferation, induction of apoptosis, and an antitumor effect of CLO prodrug in a xenograft model. These data suggest a novel therapeutic application for the CLO prodrug and potential to selectively target melanoma cells.

Bisphosphonamidate clodronate prodrug exhibits potent anticancer activity in non-small-cell lung cancer cells.

Bisphoshonates are used clinically to treat disorders of calcium metabolism, hypercalcemia and osteoporosis, and malignant bone disease. Although these agents are commonly used in cancer patients and have potential direct anticancer effects, their use for the treatment of extraskeletal disease is limited as a result of poor cellular uptake. We have designed and synthesized bisphosphonamidate prodrugs that undergo intracellular activation to release the corresponding bisphosphonate and require only two enzymatic activation events to unmask multiple negative charges. We demonstrate efficient bisphosphonamidate activation and significant enhancement in anticancer activity of two bisphosphonamidate prodrugs in vitro compared to the parent bisphosphonate. These data suggest a novel approach to optimizing the anticancer activities of commonly used bisphosphonates.

Versatile (1)H-(31)P-(31)P COSY 2D NMR techniques for the characterization of polyphosphorylated small molecules.

Di- and triphosphorylated small molecules represent key intermediates in a wide range of biological and chemical processes. The importance of polyphosphorylated species in biology and medicine underscores the need to develop methods for the detection and characterization of this compound class. We have reported two-dimensional HPP-COSY spectroscopy techniques to identify diphosphate-containing metabolic intermediates at submillimolar concentrations in the methylerythritol phosphate (MEP) isoprenoid biosynthetic pathway. (1) In this work, we explore the scope of HPP-COSY-based techniques to characterize a diverse group of small organic molecules bearing di- and triphosphorylated moieties. These include molecules containing P-O-P and P-C-P connectivities, multivalent P(III)-O-P(V) phosphorus nuclei with widely separated chemical shifts, as well as virtually overlapping (31)P resonances exhibiting strong coupling effects. We also demonstrate the utility of these experiments to rapidly distinguish between mono- and diphosphates. A detailed protocol for optimizing these experiments to achieve best performance is presented.

The crystal structure of the novobiocin biosynthetic enzyme NovP: the first representative structure for the TylF O-methyltransferase superfamily.

NovP is an S-adenosyl-l-methionine-dependent O-methyltransferase that catalyzes the penultimate step in the biosynthesis of the aminocoumarin antibiotic novobiocin. Specifically, it methylates at 4-OH of the noviose moiety, and the resultant methoxy group is important for the potency of the mature antibiotic: previous crystallographic studies have shown that this group interacts directly with the target enzyme DNA gyrase, which is a validated drug target. We have determined the high-resolution crystal structure of NovP from Streptomyces spheroides as a binary complex with its desmethylated cosubstrate S-adenosyl-l-homocysteine. The structure displays a typical class I methyltransferase fold, in addition to motifs that are consistent with a divalent-metal-dependent mechanism. This is the first representative structure of a methyltransferase from the TylF superfamily, which includes a number of enzymes implicated in the biosynthesis of antibiotics and other therapeutics. The NovP structure reveals a number of distinctive structural features that, based on sequence conservation, are likely to be characteristic of the superfamily. These include a helical 'lid' region that gates access to the cosubstrate binding pocket and an active center that contains a 3-Asp putative metal binding site. A further conserved Asp likely acts as the general base that initiates the reaction by deprotonating the 4-OH group of the noviose unit. Using in silico docking, we have generated models of the enzyme-substrate complex that are consistent with the proposed mechanism. Furthermore, these models suggest that NovP is unlikely to tolerate significant modifications at the noviose moiety, but could show increasing substrate promiscuity as a function of the distance of the modification from the methylation site. These observations could inform future attempts to utilize NovP for methylating a range of glycosylated compounds.

Crystallization and preliminary X-ray analysis of the O-methyltransferase NovP from the novobiocin-biosynthetic cluster of Streptomyces spheroides.

Crystals of recombinant NovP (subunit MW = 29 967 Da; 262 amino acids), an S-adenosyl-L-methionine-dependent O-methyltransferase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in space group P2, with unit-cell parameters a = 51.81, b = 46.04, c = 61.22 A, beta = 104.97 degrees. Native data to a maximum resolution of 1.4 A were collected from a single crystal at the synchrotron. NovP is involved in the biosynthesis of the aminocoumarin antibiotic novobiocin that targets the essential bacterial enzyme DNA gyrase.