Mammalian carboxylesterases (CEs) are key enzymes from the serine hydrolase superfamily. In the human body, two predominant carboxylesterases (CES1 and CES2) have been identified and extensively studied over the past decade. These two enzymes play crucial roles in the metabolism of a wide variety of endogenous esters, ester-containing drugs and environmental toxicants. The key roles of CES in both human health and xenobiotic metabolism arouse great interest in the discovery of potent CES modulators to regulate endobiotic metabolism or to improve the efficacy of ester drugs. This review covers the structural and catalytic features of CES, tissue distributions, biological functions, genetic polymorphisms, substrate specificities and inhibitor properties of CES1 and CES2, as well as the significance and recent progress on the discovery of CES modulators. The information presented here will help pharmacologists explore the relevance of CES to human diseases or to assign the contribution of certain CES in xenobiotic metabolism. It will also facilitate medicinal chemistry efforts to design prodrugs activated by a given CES isoform, or to develop potent and selective modulators of CES for potential biomedical applications.

Cytochrome P450 1A (CYP1A), one of the most important phase I drug-metabolizing enzymes in humans, plays a crucial role in the metabolic activation of procarcinogenic compounds to their ultimate carcinogens. Herein, we reported the development of a ratiometric two-photon fluorescent probe NCMN that allowed for selective and sensitive detection of CYP1A for the first time. The probe was designed on the basis of substrate preference of CYP1A and its high capacity for O-dealkylation, while 1,8-naphthalimide was selected as fluorophore because of its two-photon absorption properties. To achieve a highly selective probe for CYP1A, a series of 1,8-naphthalimide derivatives were synthesized and used to explore the potential structure-selectivity relationship, by using a panel of human CYP isoforms for selectivity screening. After screening and optimization, NCMN displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following CYP1A-catalyzed O-demetylation. Furthermore, the probe can be used to real-time monitor the enzyme activity of CYP1A in complex biological systems, and it has the potential for rapid screening of CYP1A modulators using tissue preparation as enzyme sources. NCMN has also been successfully used for two-photon imaging of intracellular CYP1A in living cells and tissues, and showed high ratiometric imaging resolution and deep-tissue imaging depth. In summary, a two-photon excited ratiometric fluorescent probe NCMN has been developed and well-characterized for sensitive and selective detection of CYP1A, which holds great promise for bioimaging of endogenous CYP1A in living cells and for further investigation on CYP1A associated biological functions in complex biological systems.

Cytosolic RNA/DNA sensing elicits primary defense against viral pathogens. Interferon regulatory factor 3 (IRF3), a key signal mediator/transcriptional factor of the antiviral-sensing pathway, is indispensible for interferon production and antiviral defense. However, how the status of IRF3 activation is controlled remains elusive. Through a functional screen of the human kinome, we found that mammalian sterile 20-like kinase 1 (Mst1), but not Mst2, profoundly inhibited cytosolic nucleic acid sensing. Mst1 associated with IRF3 and directly phosphorylated IRF3 at Thr75 and Thr253. This Mst1-mediated phosphorylation abolished activated IRF3 homodimerization, its occupancy on chromatin, and subsequent IRF3-mediated transcriptional responses. In addition, Mst1 also impeded virus-induced activation of TANK-binding kinase 1 (TBK1), further attenuating IRF3 activation. As a result, Mst1 depletion or ablation enabled an enhanced antiviral response and defense in cells and mice. Therefore, the identification of Mst1 as a novel physiological negative regulator of IRF3 activation provides mechanistic insights into innate antiviral defense and potential antiviral prevention strategies.

Proton-linked monocarboxylate transporters (MCTs) must transport monocarboxylate efficiently to facilitate monocarboxylate efflux in glycolytically active cells, and transport monocarboxylate slowly or even shut down to maintain a physiological monocarboxylate concentration in glycolytically inactive cells. To discover how MCTs solve this fundamental aspect of intracellular monocarboxylate homeostasis in the context of multicellular organisms, we analyzed pyruvate transport activity of human monocarboxylate transporter 2 (MCT2). Here we show that MCT2 transport activity exhibits steep dependence on substrate concentration. This property allows MCTs to turn on almost like a switch, which is physiologically crucial to the operation of MCTs in the cellular context. We further determined the cryo-electron microscopy structure of the human MCT2, demonstrating that the concentration sensitivity of MCT2 arises from the strong inter-subunit cooperativity of the MCT2 dimer during transport. These data establish definitively a clear example of evolutionary optimization of protein function.