Mycobacterium tuberculosis drug resistance is emerging and new drug targets are needed. Tryptophan biosynthesis is necessary for M. tuberculosis replication and virulence. Indole-3-glycerol phosphate synthase (IGPS) catalyzes a step in M. tuberculosis tryptophan biosynthesis and has been suggested as a potential anti-infective target, but our understanding of this enzyme is limited. To aid in inhibitor design and gain a greater mechanistic picture of this enzyme, there is a need to understand the roles of active site amino acids in ligand binding and catalysis. In this work, we explored the roles of conserved active site amino acids Glu57, Lys59, Lys119, Glu168, and Glu219. Mutation of each to Ala results in loss of all detectable activity. The Glu57Gln, Lys59Arg, Lys119Arg, Glu168Gln, and Glu219Asp mutations result in large activity losses, while Glu219Gln has enhanced activity. Analysis of the enzymatic data yields the following main conclusions: (A) Lys119 is the likely catalytic acid in the CdRP ring closure step. (B) Glu168 stabilizes a charged reaction intermediate and may also be the catalytic base. (C) Glu57, Glu219, and Lys119 form a closely arranged triad in which Glu57 and Glu219 modulate the pKa of Lys119, and thus overall activity. This increased understanding of inter- and intramolecular interactions and demonstration of the highly coordinated nature of the M. tuberculosis IGPS active site provide new mechanistic information and guidance for future work with this potential new drug target.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a class of exponentially increased natural products with characteristic chemical structures, topologies, and biosynthetic mechanisms as well as exceptional bioactivities including antibacteria, antitumors, and antiviruses. The biosynthesis of RiPP proceeds via a ribosomally assembled precursor peptide that undergoes varied post-translational modifications to generate a mature peptide. Cytochrome P450 (CYP or P450) monooxygenases are a superfamily of heme-containing enzymes that span a wide range of secondary metabolite biosynthetic pathways due to their broad substrate scopes and excellent catalytic versatility. In contrast to the enormous quantities of RiPPs and P450s, the P450 associated RiPP biosynthesis is comparatively limited, with most of their functions and timings remaining mysterious. Herein, this Review aims to provide an overview on the striking roles of P450s in RiPP biosyntheses uncovered to date and to illustrate their remarkable functions, mechanisms, as well as remaining challenges. This will shed light on novel P450 discovery and characterizations in RiPP biosyntheses.

Hydrogen sulfide (H2S) is one of the critical gasotransmitters, which play important roles in regular physiological processes, especially in vital signaling pathways. However, fluctuations in endogenous H2S concentration can be linked to serious health problems, such as neurodegenerative diseases, cancer, diabetes, inflammation, cardiovascular diseases, and hypertension. Thus, it has attracted a great deal of attention in therapeutic applications, specifically in the field of phototherapy. Photodynamic therapy (PDT) and photothermal therapy (PTT) are two subclasses of phototherapy, which utilize either reactive oxygen species (ROS) or local temperature increase upon irradiation of a photosensitizer (PS) to realize the therapeutic action. Phototherapies offer unique advantages compared to conventional methods; thus, they are highly promising and popular. One of the design principles followed in new generation PSs is to build activity-based PSs, which stay inactive before getting activated by disease-associated stimuli. These activatable PSs dramatically improve the selectivity and efficacy of the therapy. In this review, we summarize small molecule and nanomaterial-based PDT and PTT agents that are activated selectively by H2S to initiate their cytotoxic effect. We incorporate single mode PDT and PTT agents along with synergistic and/or multimodal photosensitizers that can combine more than one therapeutic approach. Additionally, H2S-responsive theranostic agents, which offer therapy and imaging at the same time, are highlighted. Design approaches, working principles, and biological applications for each example are discussed in detail.

Diabetic foot ulcers are challenging to treat. Current strategies to treat these wounds focus on preventing infection and promoting tissue regrowth but are ineffective in many individuals. Low-grade chronic inflammation is present in individuals with diabetes, and altering the inflammatory responses at the wound site could be an alternate approach to promote healing. We hypothesized that immunomodulation of the wound microenvironment would result in accelerated healing. To test this hypothesis, we began by characterizing the changes in the myeloid cell phenotype in a mouse model [leptin receptor knockout (KO) mouse] that closely mimics the type 2 diabetes condition observed in humans. We observed increased numbers of monocytes and neutrophils in the circulation of the KO mice compared to that in wild-type control mice. We also observed several phenotypic changes in neutrophils from the KO diabetic mice, suggesting low-grade systemic inflammation. Hence, we developed a rapamycin-loaded chitosan scaffold that may be used to modulate immune responses. The use of these immunomodulatory scaffolds at a wound site resulted in accelerated healing compared to the healing using blank scaffolds. In summary, our data suggest that immunomodulation may be a viable strategy to promote the healing of wounds in individuals with diabetes.

Alanine racemase (Alr) is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the racemization of l-alanine to d-alanine. Alr is one of the two targets of the broad-spectrum antibiotic d-cycloserine (DCS), a structural analogue of d-alanine. Despite being an essential component of regimens used to treat multi- and extensively drug-resistant tuberculosis for almost seven decades, resistance to DCS has not been observed in patients. We previously demonstrated that DCS evades resistance due to an ultralow rate of emergence of mutations. Yet, we identified a single polymorphism (converting Asp322 to Asn) in the alr gene, which arose in 8 out of 11 independent variants identified and that confers resistance. Here, we present the crystal structure of the Alr variant D322N in both the free and DCS-inactivated forms and the characterization of its DCS inactivation mechanism by UV–visible and fluorescence spectroscopy. Comparison of these results with those obtained with wild-type Alr reveals the structural basis of the 240-fold reduced inhibition observed in Alr D322N.

There are many severe bacterial infections notorious for their ability to become resistant to clinically relevant antibiotics. Indeed, antibiotic resistance is a growing threat to human health, further exacerbated by the lack of new antibiotics. We now describe the practical synthesis of a series of substituted long linear polyamines that produce rapid antibacterial activity against both Gram-positive and Gram-negative bacteria, including meticillin-resistant Staphylococcus aureus. These compounds also reduce biofilm formation in Pseudomonas aeruginosa. The most potent analogues are thermine, spermine, and 1,12-diaminododecane homo- and heterodimeric polyamine succinic acid amides. They are of the order of activity of the aminoglycoside antibiotics kanamycin and tobramycin as positive controls. Their low human cell toxicity is demonstrated in ex vivo hemolytic assays where they did not produce even 5% hemolysis of human erythrocytes. These long, linear polyamines are a new class of broad-spectrum antibacterials active against drug-resistant pathogens.

We describe a small molecule ligand ACA-14 (2-hydroxy-5-{[(2-phenylcyclopropyl) carbonyl] amino} benzoic acid) as an initial lead for the development of direct inhibitors of KRAS, a notoriously difficult anticancer drug target. We show that the compound binds to KRAS near the switch regions with affinities in the low micromolar range and exerts different effects on KRAS interactions with binding partners. Specifically, ACA-14 impedes the interaction of KRAS with its effector Raf and reduces both intrinsic and SOS-mediated nucleotide exchange rates. Likely as a result of these effects, ACA-14 inhibits signal transduction through the MAPK pathway in cells expressing mutant KRAS and inhibits the growth of pancreatic and colon cancer cells harboring mutant KRAS. We thus propose compound ACA-14 as a useful initial lead for the development of broad-acting inhibitors that target multiple KRAS mutants and simultaneously deplete the fraction of GTP-loaded KRAS while abrogating the effector-binding ability of the already GTP-loaded fraction.

Methyllycaconitine (MLA), 1, is a naturally occurring norditerpenoid alkaloid that is a highly potent (IC50 = 2 nM) selective antagonist of α7 nicotinic acetylcholine receptors (nAChRs). Several structural factors affect its activity such as the neopentyl ester side-chain and the piperidine ring N-side-chain. The synthesis of simplified AE-bicyclic analogues 14–21 possessing different ester and nitrogen side-chains was achieved in three steps. The antagonist effects of synthetic analogues were examined on human α7 nAChRs and compared to that of MLA 1. The most efficacious analogue (16) reduced α7 nAChR agonist responses [1 nM acetylcholine (ACh)] to 53.2 ± 1.9% compared to 3.4 ± 0.2% for MLA 1. This demonstrates that simpler analogues of MLA 1 possess antagonist effects on human α7 nAChRs but also indicates that further optimization may be possible to achieve antagonist activity comparable to that of MLA 1.

β-lactamase production with vast catalytic divergence in the pathogenic strain limits the antibiotic spectrum in the clinical environment. Class A carbapenemase shares significant sequence similarities, structural features, and common catalytic mechanisms although their resistance spectrum differs from class A β-lactamase in carbapenem and monobactam hydrolysis. In other words, it limited the antibiotic treatment option against infection, causing carbapenemase-producing superbugs. Ftu-1 is a class A β-lactamase expressed by the Francisella tularensis strain, a potent causative organism of tularemia. The chromosomally encoded class A β-lactamase shares two conserved cysteine residues, a common characteristic of a carbapenemase, and a distinctive class in the phylogenetic tree. Complete biochemical and biophysical characterization of the enzyme was performed to understand the overall stability and environmental requirements to perform optimally. To comprehend the enzyme–drug interaction and its profile toward various chemistries of β-lactam and β-lactamase inhibitors, comprehensive kinetic and thermodynamic analyses were conducted using various β-lactam drugs. The dynamic property of Ftu-1 β-lactamase was also predicted using molecular dynamics (MD) simulation to compare its loop flexibility and ligand binding with other related class A β-lactamases. Overall, this study fosters a comprehensive understanding of Ftu-1, proposed to be an intermediate class by characterizing its kinetic profiling, stability by biochemical and biophysical methodologies, and susceptibility profiling. This understanding would be beneficial for the design of new-generation therapeutics.

All aerobic organisms require O2 for survival. When their O2 is limited (hypoxia), a response is required to reduce demand and/or improve supply. A hypoxic response mechanism has been identified in flowering plants: the stability of certain proteins with N-terminal cysteine residues is regulated in an O2-dependent manner by the Cys/Arg branch of the N-degron pathway. These include the Group VII ethylene response factors (ERF-VIIs), which can initiate adaptive responses to hypoxia. Oxidation of their N-terminal cysteine residues is catalyzed by plant cysteine oxidases (PCOs), destabilizing these proteins in normoxia; PCO inactivity in hypoxia results in their stabilization. Biochemically, the PCOs are sensitive to O2 availability and can therefore act as plant O2 sensors. It is not known whether oxygen-sensing mechanisms exist in other phyla from the plant kingdom. Known PCO targets are only conserved in flowering plants, however PCO-like sequences appear to be conserved in all plant species. We sought to determine whether PCO-like enzymes from the liverwort, Marchantia polymorpha (MpPCO), and the freshwater algae, Klebsormidium nitens (KnPCO), have a similar function as PCO enzymes from Arabidopsis thaliana. We report that MpPCO and KnPCO show O2-sensitive N-terminal cysteine dioxygenase activity toward known AtPCO ERF-VII substrates as well as a putative endogenous substrate, MpERF-like, which was identified by homology to the Arabidopsis ERF-VIIs transcription factors. This work confirms functional and O2-dependent PCOs from Bryophyta and Charophyta, indicating the potential for PCO-mediated O2-sensing pathways in these organisms and suggesting PCO O2-sensing function could be important throughout the plant kingdom.

Lipoic acid is an eight-carbon sulfur-containing biomolecule that functions primarily as a cofactor in several multienzyme complexes. It is biosynthesized as an attachment to a specific lysyl residue on one of the subunits of these multienzyme complexes. In Escherichia coli and many other organisms, this biosynthetic pathway involves two dedicated proteins: octanoyltransferase (LipB) and lipoyl synthase (LipA). LipB transfers an n-octanoyl chain from the octanoyl-acyl carrier protein to the target lysyl residue, and then, LipA attaches two sulfur atoms (one at C6 and one at C8) to give the final lipoyl cofactor. All classical lipoyl synthases (LSs) are radical S-adenosylmethionine (SAM) enzymes, which use an [Fe4S4] cluster to reductively cleave SAM to generate a 5′-deoxyadenosyl 5′-radical. Classical LSs also contain a second [Fe4S4] cluster that serves as the source of both appended sulfur atoms. Recently, a novel pathway for generating the lipoyl cofactor was reported. This pathway replaces the canonical LS with two proteins, LipS1 and LipS2, which act together to catalyze formation of the lipoyl cofactor. In this work, we further characterize LipS1 and LipS2 biochemically and spectroscopically. Although LipS1 and LipS2 were previously annotated as biotin synthases, we show that both proteins, unlike E. coli biotin synthase, contain two [Fe4S4] clusters. We identify the cluster ligands to both iron–sulfur clusters in both proteins and show that LipS2 acts only on an octanoyl-containing substrate, while LipS1 acts only on an 8-mercaptooctanoyl-containing substrate. Therefore, similarly to E. coli biotin synthase and in contrast to E. coli LipA, sulfur attachment takes place initially at the terminal carbon (C8) and then at the C6 methylene carbon.

Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that interacts with five native G-protein coupled receptors (S1P1–5) to regulate cell growth, survival, and proliferation. S1P has been implicated in a variety of pathologies including cancer, kidney fibrosis, and multiple sclerosis. As key mediators in the synthesis of S1P, sphingosine kinase (SphK) isoforms 1 and 2 have attracted attention as viable targets for pharmacologic intervention. In this report, we describe the design, synthesis, and biological evaluation of sphingosine kinase 2 (SphK2) inhibitors with a focus on systematically introducing rigid structures in the aliphatic lipid tail present in existing SphK2 inhibitors. Experimental as well as molecular modeling studies suggest that conformationally restricted “lipophilic tail” analogues bearing a bulky terminal moiety or an internal phenyl ring are useful to complement the “J”-shaped sphingosine binding pocket of SphK2. We identified 14c (SLP9101555) as a potent SphK2 inhibitor (Ki = 90 nM) with 200-fold selectivity over SphK1. Molecular docking studies indicated key interactions: the cyclohexyl ring binding in the cleft deep in the pocket, a trifluoromethyl group fitting in a small side cavity, and a hydrogen bond between the guanidino group and Asp308 (amino acid numbering refers to human SphK2 (isoform c) orthologue). In vitro studies using U937 human histiocytic lymphoma cells showed marked decreases in extracellular S1P levels in response to our SphK2 inhibitors. Administration of 14c (dose: 5 mg/kg) to mice resulted in a sustained increase of circulating S1P levels, suggesting target engagement.

Lipoyl synthase (LS) catalyzes the last step in the biosynthesis of the lipoyl cofactor, which is the attachment of sulfur atoms at C6 and C8 of an n-octanoyllysyl side chain of a lipoyl carrier protein (LCP). The protein is a member of the radical S-adenosylmethionine (SAM) superfamily of enzymes, which use SAM as a precursor to a 5′-deoxyadenosyl 5′-radical (5′-dA·). The role of the 5′-dA· in the LS reaction is to abstract hydrogen atoms from C6 and C8 of the octanoyl moiety of the substrate to initiate subsequent sulfur attachment. All radical SAM enzymes have at least one [4Fe–4S] cluster that is used in the reductive cleavage of SAM to generate the 5′-dA·; however, LSs contain an additional auxiliary [4Fe–4S] cluster from which sulfur atoms are extracted during turnover, leading to degradation of the cluster. Therefore, these enzymes catalyze only 1 turnover in the absence of a system that restores the auxiliary cluster. In Escherichia coli, the auxiliary cluster of LS can be regenerated by the iron–sulfur (Fe–S) cluster carrier protein NfuA as fast as catalysis takes place, and less efficiently by IscU. NFU1 is the human ortholog of E. coli NfuA and has been shown to interact directly with human LS (i.e., LIAS) in yeast two-hybrid analyses. Herein, we show that NFU1 and LIAS form a tight complex in vitro and that NFU1 can efficiently restore the auxiliary cluster of LIAS during turnover. We also show that BOLA3, previously identified as being critical in the biosynthesis of the lipoyl cofactor in humans and Saccharomyces cerevisiae, has no direct effect on Fe–S cluster transfer from NFU1 or GLRX5 to LIAS. Further, we show that ISCA1 and ISCA2 can enhance LIAS turnover, but only slightly.

Targeted protein degradation (TPD) is emerging as one of the most innovative strategies to tackle infectious diseases. Particularly, proteolysis-targeting chimera (PROTAC)-mediated protein degradation may offer several benefits over classical anti-infective small-molecule drugs. Because of their peculiar and catalytic mechanism of action, anti-infective PROTACs might be advantageous in terms of efficacy, toxicity, and selectivity. Importantly, PROTACs may also overcome the emergence of antimicrobial resistance. Furthermore, anti-infective PROTACs might have the potential to (i) modulate “undruggable” targets, (ii) “recycle” inhibitors from classical drug discovery approaches, and (iii) open new scenarios for combination therapies. Here, we try to address these points by discussing selected case studies of antiviral PROTACs and the first-in-class antibacterial PROTACs. Finally, we discuss how the field of PROTAC-mediated TPD might be exploited in parasitic diseases. Since no antiparasitic PROTAC has been reported yet, we also describe the parasite proteasome system. While in its infancy and with many challenges ahead, we hope that PROTAC-mediated protein degradation for infectious diseases may lead to the development of next-generation anti-infective drugs.

The amazing thing about art is that there are truly no limits. If you can see an image in your mind clearly enough, the image can be transferred to paper using just seven colors. The opposite is true as well. If you can see an image on paper, it becomes easier to picture it in real life. After all, life imitates art. There is nothing quite like the inspiration that comes from looking at an illustration that you see yourself in and wanting it to come to life, to take up as much space in the 3-D world as it does in the 2-D one. This cover art depicts an African American woman who is also an intelligent and feminine chemist. It is important to me because these are titles African American women do not often get the luxury of being called. Creation is important to me because I believe that God is the original creator, and so everything I am able to create comes from Him. Hebrews 3:4 says, “For every house is built by someone, but God is the builder of everything.” In other words, I believe humans are able to create and discover only because God created everything first. Jayde Frederick. (Image courtesy of Jayde Frederick.) Increasing the visibility of Black women and minorities in STEM (science, technology, engineering, and math) is a lifelong passion of mine. As a child, when I was not studying, I was reading or creating anything and everything. I would make bracelets, bookmarks, paper fashions, and drawings. As I grew, I discovered my love for the sciences as well as art. The endless combinations of versatile chemicals and elements and the beauty and intricacy of the human body fascinated me. I found connections between the medical side of my goals and the artistic side. As a child, I had more than enough time to explore the creative side of myself, often from a hospital bed, as someone diagnosed with sickle cell anemia. Not being able to play intense sports, heavily exert myself, or spend hours outside to avoid getting pain crises, I found other ways to entertain myself with quieter activities. Through books, I could see the imaginary worlds of others, and through my art, I could create my own. There came a time in my life when I seemed to be going to endless doctor’s visits for problems that were heartbreaking and out of my control. Through that experience, I saw firsthand the impact that science has on people’s lives through the healing I was blessed to receive. Additionally, during that time, I remember wishing that just one of the health professionals I saw could be a black woman. Although the professionals I saw were amazing and talented, it saddened me that the profession I had always been interested in had so little representation of people who looked like me in it. After a conversation with my parents about a fairly new concept called STEAM, which stands for STEM + Art, I decided to start my organization xxSTEAM, with the two x’s representing the female chromosomes. I wanted to use my art to inspire and empower young women and girls of color to pursue their dreams in STEAM no matter what the world was telling them. I have been blessed to be able to explore both my artistic passions and my scientific ones. Family members in healthcare have shown me what a privilege it is to serve and heal others, and my teachers have always pushed me to explore my passions: my high school art teacher, Ms. DeAmbrose, and my college science professors such as Dr. Kenneth Lai Hing, Dr. Steven Lai Hing, Dr. Vanterpool, and Dr. Durant, just to name a few. I am privileged to be a minority who has had a great support system around me, but not every young woman of color can say the same. So through xxSTEAM and my art, I aim to introduce others to STEAM fields while also providing representation to inspire women and minorities to pursue these careers. For each piece, character, and story I create, I ask God to lead me and use the talents He gifted me with for His glory. I desire for others to see my work and feel empowered to be the chemist, physician, biologist, engineer, or technologist they were meant to become. This article has not yet been cited by other publications. Jayde Frederick. (Image courtesy of Jayde Frederick.)

The devastating human pathogenMycobacterium tuberculosis (Mtb) is able to parasitize phagosomal compartments within alveolar macrophage cells due, in part, to the activity of its cell-surface lipids. Prominent among these is 1-tuberculosinyl-adenosine (1-TbAd), a derivative of the diterpenoid tuberculosinyl (halima-5,13-dienyl) diphosphate produced by the class II diterpene cyclase encoded by Rv3377c, termed here MtHPS. Given the demonstrated ability of 1-TbAd to act as a virulence factor for Mtb and the necessity for Rv3377c for its production, there is significant interest in MtHPS activity. Class II diterpene cyclases catalyze a general acid–base-mediated carbocation cascade reaction initiated by protonation of the terminal alkene in the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate and terminated by deprotonation of the final cyclized (and sometimes also rearranged) intermediate. Here, structure-guided mutagenesis was applied to characterize the various residues contributing to activation of the enzymatic acid, as well as identify the enzymatic base in MtHPS. Particularly given the ability of conservative substitution for the enzymatic base (Y479F) to generate an alternative product (labda-7,13-dienyl diphosphate) via deprotonation of an earlier unrearranged intermediate, further mutational analysis was carried out to introduce potential alternative catalytic bases. The results were combined with mechanistic molecular modeling to elucidate how these mutations affect the catalytic activity of this important enzyme. This not only provided detailed structure–function insight into MtHPS but also further emphasized the inert nature of the active site of MtHPS and class II diterpene cyclases more generally.

The mycobacterial cell envelope has spatially resolved inner and outer membrane layers with distinct compositions and membrane properties. However, the functional implication and relevance of this organization remain unknown. Using membrane biophysics and molecular simulations, we reveal a varied interaction profile of these layers with antibiotic Rifabutin, underlined by the structural and chemical makeup of the constituent lipids. The mycobacterial inner membrane displayed the highest partitioning of Rifabutin, which was located exclusively in the lipid head group/interfacial region. In contrast, the drug exhibited specific interaction sites in the head group/interfacial and hydrophobic acyl regions within the outer membrane. Altogether, we show that the design of membrane-active agents that selectively disrupt the mycobacterial outer membrane structure can increase drug uptake and enhance intracellular drug concentrations. Exploiting the mycobacterium-specific membrane–drug interaction profiles, chemotypes consisting of outer membrane-disruptive agents and antitubercular drugs can offer new opportunities for combinational tuberculosis (TB) therapy.

Protease-activated receptors (PARs) comprise a family of four G protein-coupled receptors (GPCRs) that have broad functions in health and disease. Unlike most GPCRs, PARs are uniquely activated by proteolytic cleavage of their extracellular N termini. To fully understand PAR activation and function in vivo, it is critical to also study the proteases that activate them. As proteases are heavily regulated at the post-translational level, measures of total protease abundance have limited utility. Measures of protease activity are instead required to inform their function. This review will introduce several classes of chemical probes that have been developed to measure the activation of PAR-cleaving proteases. Their strengths, weaknesses, and applications will be discussed, especially as applied to image protease activity at the whole organism, tissue, and cellular level.

Rapid cell division and reprogramming of energy metabolism are two crucial hallmarks of cancer cells. In humans, hexose trafficking into cancer cells is mainly mediated through a family of glucose transporters (GLUTs), which are facilitative transmembrane hexose transporter proteins. In several breast cancers, fructose can functionally substitute glucose as an alternative energy supply supporting rapid proliferation. GLUT5, the principal fructose transporter, is overexpressed in human breast cancer cells, providing valuable targets for breast cancer detection as well as selective targeting of anticancer drugs using structurally modified fructose mimics. Herein, a novel fluorescence assay was designed aiming to screen a series of C-3 modified 2,5-anhydromannitol (2,5-AM) compounds as d-fructose analogues to explore GLUT5 binding site requirements. The synthesized probes were evaluated for their ability to inhibit the uptake of the fluorescently labeled d-fructose derivative 6-NBDF into EMT6 murine breast cancer cells. A few of the compounds screened demonstrated highly potent single-digit micromolar inhibition of 6-NBDF cellular uptake, which was substantially more potent than the natural substrate d-fructose, at a level of 100-fold or more. The results of this assay are consistent with those obtained from a previous study conducted for some selected compounds against 18F-labeled d-fructose-based probe 6-[18F]FDF, indicating the reproducibility of the current non-radiolabeled assay. These highly potent compounds assessed against 6-NBDF open avenues for the development of more potent probes targeting GLUT5-expressing cancerous cells.

Neuromedin-U (NMU) mediates several physiological functions via its two cognate receptors, NMUR1 and NMUR2. Disentangling the individual roles of each receptor has largely been undertaken through the use of transgenic mice bearing a deletion in one of the two receptors or by testing native molecules (NMU or its truncated version NMU-8) in a tissue-specific manner, in effect, taking advantage of the distinct receptor expression profiles. These strategies have proved quite useful despite the inherent limitations of overlapping receptor roles and potential compensatory influences of germline gene deletion. With these considerations in mind, the availability of potent, selective NMU compounds with appropriate pharmacokinetic profiles would advance the capabilities of investigators undertaking such efforts. Here, we evaluate a recently reported NMUR2-selective peptide (compound 17) for its in vitro potency (mouse and human), binding affinity, murine pharmacokinetic properties, and in vivo effects. Despite being designed as an NMUR2 agonist, our results show compound 17 unexpectedly binds but does not have functional activity on NMUR1, thereby acting as an R1 antagonist while simultaneously being a potent NMUR2 agonist. Furthermore, evaluation of compound 17 across all known and orphan G-protein-coupled receptors demonstrates multiple receptor partners beyond NMUR2/R1 binding. These properties need to be appreciated for accurate interpretation of results generated using this molecule and may limit the broader ability of this particular entity in disentangling the physiological role of NMU receptor biology.