Structural Insights into Endobiotic Reactivation by Human Gut Microbiome-Encoded Sulfatases.

Phase II drug metabolism inactivates xenobiotics and endobiotics through the addition of either a glucuronic acid or sulfate moiety prior to excretion, often via the gastrointestinal tract. While the human gut microbial ß-glucuronidase enzymes that reactivate glucuronide conjugates in the intestines are becoming well characterized and even controlled by targeted inhibitors, the sulfatases encoded by the human gut microbiome have not been comprehensively examined. Gut microbial sulfatases are poised to reactivate xenobiotics and endobiotics, which are then capable of undergoing enterohepatic recirculation or exerting local effects on the gut epithelium. Here, using protein structure-guided methods, we identify 728 distinct microbiome-encoded sulfatase proteins from the 4.8 million unique proteins present in the Human Microbiome Project Stool Sample database and 1766 gut microbial sulfatases from the 9.9 million sequences in the Integrated Gene Catalogue. We purify a representative set of these sulfatases, elucidate crystal structures, and pinpoint unique structural motifs essential to endobiotic sulfate processing. Gut microbial sulfatases differentially process sulfated forms of the neurotransmitters serotonin and dopamine, and the hormones melatonin, estrone, dehydroepiandrosterone, and thyroxine in a manner dependent both on variabilities in active site architecture and on markedly distinct oligomeric states. Taken together, these data provide initial insights into the structural and functional diversity of gut microbial sulfatases, providing a path toward defining the roles these enzymes play in health and disease.

Ubiquitin chain-elongating enzyme UBE2S activates the RING E3 ligase APC/C for substrate priming.

The interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains. However, the potential coordination between these steps in ubiquitin chain formation remains undefined. While numerous studies have unveiled how RING E3s stimulate individual E2s for Ub transfer, here we change perspective to describe a case where the chain-elongating E2 UBE2S feeds back and directly stimulates the E3 APC/C to promote substrate priming and subsequent multiubiquitination by UBE2C. Our work reveals an unexpected model for the mechanisms of RING E3-dependent ubiquitination and for the diverse and complex interrelationship between components of the ubiquitination cascade.

Crystal structure of UDP-glucose pyrophosphorylase from Yersinia pestis, a potential therapeutic target against plague.

Yersinia pestis, the causative agent of bubonic plague, is one of the most lethal pathogens in recorded human history. Today, the concern is the possible misuse of Y. pestis as an agent in bioweapons and bioterrorism. Current therapies for the treatment of plague include the use of a small number of antibiotics, but clinical cases of antibiotic resistance have been reported in some areas of the world. Therefore, the discovery of new drugs is required to combat potential Y. pestis infection. Here, the crystal structure of the Y. pestis UDP-glucose pyrophosphorylase (UGP), a metabolic enzyme implicated in the survival of Y. pestis in mouse macrophages, is described at 2.17 Šresolution. The structure provides a foundation that may enable the rational design of inhibitors and open new avenues for the development of antiplague therapeutics.

Mechanisms and the clinical relevance of complex drug-drug interactions.

As a result of an increasing aging population, the number of individuals taking multiple medications simultaneously has grown considerably. For these individuals, taking multiple medications has increased the risk of undesirable drug-drug interactions (DDIs), which can cause serious and debilitating adverse drug reactions (ADRs). A comprehensive understanding of DDIs is needed to combat these deleterious outcomes. This review provides a synopsis of the pharmacokinetic (PK) and pharmacodynamic (PD) mechanisms that underlie DDIs. PK-mediated DDIs affect all aspects of drug disposition: absorption, distribution, metabolism and excretion (ADME). In this review, the cells that play a major role in ADME and have been investigated for DDIs are discussed. Key examples of drug metabolizing enzymes and drug transporters that are involved in DDIs and found in these cells are described. The effect of inhibiting or inducing these proteins through DDIs on the PK parameters is also reviewed. Despite most DDI studies being focused on the PK effects, DDIs through PD can also lead to significant and harmful effects. Therefore, this review outlines specific examples and describes the additive, synergistic and antagonistic mechanisms of PD-mediated DDIs. The effects DDIs on the maximum PD response (E max) and the drug dose or concentration (EDEC50) that lead to 50% of E max are also examined. Significant gaps in our understanding of DDIs remain, so innovative and emerging approaches are critical for overcoming them.

A Conformationally Gated Model of Methadone and Loperamide Transport by P-Glycoprotein.

P-glycoprotein (Pgp) is a multidrug resistance transporter that limits the penetration of a wide range of neurotherapeutics into the brain including opioids. The diphenylpropylamine opioids methadone and loperamide are structurally similar, but loperamide has about a 4-fold higher Pgp-mediated transport rate. In addition to these differences, they showed significant differences in their effects on Pgp-mediated adenosine triphosphate (ATP) hydrolysis. The activation of Pgp-mediated ATP hydrolysis by methadone was monophasic, whereas loperamide activation of ATP hydrolysis was biphasic implying methadone has a single binding site and loperamide has 2 binding sites on Pgp. Quenching of tryptophan fluorescence with these drugs and digoxin showed competition between the opioids and that loperamide does not compete for the digoxin-binding site. Acrylamide quenching of tryptophan fluorescence to probe Pgp conformational changes revealed that methadone- and loperamide-induced conformational changes were distinct. These results were used to develop a model for Pgp-mediated transport of methadone and loperamide where opioid binding and conformational changes are used to explain the differences in the opioid transport rates between methadone and loperamide.

Cooperativity between verapamil and ATP bound to the efflux transporter P-glycoprotein.

The P-glycoprotein (Pgp) transporter plays a central role in drug disposition by effluxing a chemically diverse range of drugs from cells through conformational changes and ATP hydrolysis. A number of drugs are known to activate ATP hydrolysis of Pgp, but coupling between ATP and drug binding is not well understood. The cardiovascular drug verapamil is one of the most widely studied Pgp substrates and therefore, represents an ideal drug to investigate the drug-induced ATPase activation of Pgp. As previously noted, verapamil-induced Pgp-mediated ATP hydrolysis kinetics was biphasic at saturating ATP concentrations. However, at subsaturating ATP concentrations, verapamil-induced ATPase activation kinetics became monophasic. To further understand this switch in kinetic behavior, the Pgp-coupled ATPase activity kinetics was checked with a panel of verapamil and ATP concentrations and fit with the substrate inhibition equation and the kinetic fitting software COPASI. The fits suggested that cooperativity between ATP and verapamil switched between low and high verapamil concentration. Fluorescence spectroscopy of Pgp revealed that cooperativity between verapamil and a non-hydrolyzable ATP analog leads to distinct global conformational changes of Pgp. NMR of Pgp reconstituted in liposomes showed that cooperativity between verapamil and the non-hydrolyzable ATP analog modulate each other's interactions. This information was used to produce a conformationally-gated model of drug-induced activation of Pgp-mediated ATP hydrolysis.