Discovery of Alternative Binding Poses through Fragment-Based Identification of DHODH Inhibitors.

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme that affects many aspects essential to cell proliferation and survival. Recently, DHODH has been identified as a potential target for acute myeloid leukemia therapy. Herein, we describe the identification of potent DHODH inhibitors through a scaffold hopping approach emanating from a fragment screen followed by structure-based drug design to further improve the overall profile and reveal an unexpected novel binding mode. Additionally, these compounds had low P-gp efflux ratios, allowing for applications where exposure to the brain would be required.

N-Heterocyclic 3-Pyridyl Carboxamide Inhibitors of DHODH for the Treatment of Acute Myelogenous Leukemia.

Acute myelogenous leukemia (AML), a disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway; however, small molecule DHODH inhibitors were recently shown to induce differentiation in multiple AML subtypes. Using virtual screening and structure-based drug design approaches, a new series of N-heterocyclic 3-pyridyl carboxamide DHODH inhibitors were discovered. Two lead compounds, 19 and 29, have potent biochemical and cellular DHODH activity, favorable physicochemical properties, and efficacy in a preclinical model of AML.

Quantification of In Vivo Target Engagement Using Microfluidic Activity-Based Protein Profiling.

Accurate measurement of drug-target interactions in vivo is critical for both preclinical development and translation to clinical studies, yet many assays rely on indirect measures such as biomarkers associated with target activity. Activity-based protein profiling (ABPP) is a direct method of quantifying enzyme activity using active site-targeted small-molecule covalent probes that selectively label active but not inhibitor-bound enzymes. Probe-labeled enzymes in complex proteomes are separated by polyacrylamide gel electrophoresis and quantified by fluorescence imaging. To accelerate workflows and avoid imaging artifacts that make conventional gels challenging to quantify, we adapted protocols for a commercial LabChip GXII microfluidic instrument to permit electrophoretic separation of probe-labeled proteins in tissue lysates and plasma, and quantification of fluorescence (probe/protein labeling ratio of 1:1). Electrophoretic separation on chips occurred in 40 s per sample, and instrument software automatically identified and quantified peaks, resulting in an overall time savings of 3-5 h per 96-well sample plate. Calculated percent inhibition was not significantly different between the two formats. Chip performance was consistent between chips and sample replicates. Conventional gel imaging was more sensitive but required five times higher sample volume than microfluidic chips. Microfluidic chips produced results comparable to those of gels but with much lower sample consumption, facilitating assay miniaturization for scarce biological samples. The time savings afforded by microfluidic electrophoresis and automatic quantification has allowed us to incorporate microfluidic ABPP early in the drug discovery workflow, enabling routine assessments of tissue distribution and engagement of targets and off-targets in vivo.

Monoacylglycerol Lipase Inhibition in Human and Rodent Systems Supports Clinical Evaluation of Endocannabinoid Modulators.

Monoacylglycerol lipase (MGLL) is the primary degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). The first MGLL inhibitors have recently entered clinical development for the treatment of neurologic disorders. To support this clinical path, we report the pharmacological characterization of the highly potent and selective MGLL inhibitor ABD-1970 [1,1,1,3,3,3-hexafluoropropan-2-yl 4-(2-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-4-chlorobenzyl)piperazine-1-carboxylate]. We used ABD-1970 to confirm the role of MGLL in human systems and to define the relationship between MGLL target engagement, brain 2-AG concentrations, and efficacy. Because MGLL contributes to arachidonic acid metabolism in a subset of rodent tissues, we further used ABD-1970 to evaluate whether selective MGLL inhibition would affect prostanoid production in several human assays known to be sensitive to cyclooxygenase inhibitors. ABD-1970 robustly elevated brain 2-AG content and displayed antinociceptive and antipruritic activity in a battery of rodent models (ED50 values of 1-2 mg/kg). The antinociceptive effects of ABD-1970 were potentiated when combined with analgesic standards of care and occurred without overt cannabimimetic effects. ABD-1970 also blocked 2-AG hydrolysis in human brain tissue and elevated 2-AG content in human blood without affecting stimulated prostanoid production. These findings support the clinical development of MGLL inhibitors as a differentiated mechanism to treat pain and other neurologic disorders.

Identification of ABX-1431, a Selective Inhibitor of Monoacylglycerol Lipase and Clinical Candidate for Treatment of Neurological Disorders.

The serine hydrolase monoacylglycerol lipase (MGLL) converts the endogenous cannabinoid receptor agonist 2-arachidonoylglycerol (2-AG) and other monoacylglycerols into fatty acids and glycerol. Genetic or pharmacological inactivation of MGLL leads to elevation in 2-AG in the central nervous system and corresponding reductions in arachidonic acid and eicosanoids, producing antinociceptive, anxiolytic, and antineuroinflammatory effects without inducing the full spectrum of psychoactive effects of direct cannabinoid receptor agonists. Here, we report the optimization of hexafluoroisopropyl carbamate-based irreversible inhibitors of MGLL, culminating in a highly potent, selective, and orally available, CNS-penetrant MGLL inhibitor, 28 (ABX-1431). Activity-based protein profiling experiments verify the exquisite selectivity of 28 for MGLL versus other members of the serine hydrolase class. In vivo, 28 inhibits MGLL activity in rodent brain (ED50 = 0.5-1.4 mg/kg), increases brain 2-AG concentrations, and suppresses pain behavior in the rat formalin pain model. ABX-1431 (28) is currently under evaluation in human clinical trials.

Pharmacokinetic and in vivo efficacy studies of the mycobactin biosynthesis inhibitor salicyl-AMS in mice.

Mycobactin biosynthesis in Mycobacterium tuberculosis facilitates iron acquisition, which is required for growth and virulence. The mycobactin biosynthesis inhibitor salicyl-AMS [5'-O-(N-salicylsulfamoyl)adenosine] inhibits M. tuberculosis growth in vitro under iron-limited conditions. Here, we conducted a single-dose pharmacokinetic study and a monotherapy study of salicyl-AMS with mice. Intraperitoneal injection yielded much better pharmacokinetic parameter values than oral administration did. Monotherapy of salicyl-AMS at 5.6 or 16.7 mg/kg significantly inhibited M. tuberculosis growth in the mouse lung, providing the first in vivo proof of concept for this novel antibacterial strategy.

Simultaneous structure-activity studies and arming of natural products by C-H amination reveal cellular targets of eupalmerin acetate.

Natural products have a venerable history of, and enduring potential for the discovery of useful biological activity. To fully exploit this, the development of chemical methodology that can functionalize unique sites within these complex structures is highly desirable. Here, we describe the use of rhodium(II)-catalysed C-H amination reactions developed by Du Bois to carry out simultaneous structure-activity relationship studies and arming (alkynylation) of natural products at 'unfunctionalized' positions. Allylic and benzylic C-H bonds in the natural products undergo amination while olefins undergo aziridination, and tertiary amine-containing natural products are converted to amidines by a C-H amination-oxidation sequence or to hydrazine sulfamate zwitterions by an unusual N-amination. The alkynylated derivatives are ready for conversion into cellular probes that can be used for mechanism-of-action studies. Chemo- and site-selectivity was studied with a diverse library of natural products. For one of these-the marine-derived anticancer diterpene, eupalmerin acetate-quantitative proteome profiling led to the identification of several protein targets in HL-60 cells, suggesting a polypharmacological mode of action.

Activity-based labeling of matrix metalloproteinases in living vertebrate embryos.

Extracellular matrix (ECM) remodeling is a physiologically and developmentally essential process mediated by a family of zinc-dependent extracellular proteases called matrix metalloproteinases (MMPs). In addition to complex transcriptional control, MMPs are subject to extensive post-translational regulation. Because of this, classical biochemical, molecular and histological techniques that detect the expression of specific gene products provide useful but limited data regarding the biologically relevant activity of MMPs. Using benzophenone-bearing hydroxamate-based probes that interact with the catalytic zinc ion in MMPs, active proteases can be covalently 'tagged' by UV cross-linking. This approach has been successfully used to tag MMP-2 in vitro in tissue culture supernatants, and we show here that this probe tags proteins with mobilities consistent with known MMPs and detectable gelatinolytic activity in homogenates of zebrafish embryos. Furthermore, because of the transparency of the zebrafish embryo, UV-photocroslinking can be accomplished in vivo, and rhodamated benzophenone probe is detected in striking spatial patterns consistent with known distributions of active matrix remodeling in embryos. Finally, in metamorphosing Xenopus tadpoles, this probe can be used to biotinylate active MMP-2 by injecting it and cross-linking it in vivo, allowing the protein to be subsequently extracted and biochemically identified.

Fully functionalized small-molecule probes for integrated phenotypic screening and target identification.

Phenotypic screening offers a powerful approach to identify small molecules that perturb complex biological processes in cells and organisms. The tendency of small molecules, however, to interact with multiple protein targets, often with moderate to weak affinities, along with the lack of straightforward technologies to characterize these interactions in living systems, has hindered efforts to understand the mechanistic basis for pharmacological activity. Here we address this challenge by creating a fully functionalized small-molecule library whose membership is endowed with: (1) one or more diversity elements to promote interactions with different protein targets in cells, (2) a photoreactive group for UV light-induced covalent cross-linking to interacting proteins, and (3) an alkyne handle for reporter tag conjugation to visualize and identify cross-linked proteins. A library member was found to inhibit cancer cell proliferation selectively under nutrient-limiting (low glucose) conditions. Quantitative chemoproteomics identified MT-ND1, an integral membrane subunit of the ∼1 MDa NADH:ubiquinone oxidoreductase (complex 1) involved in oxidative phosphorylation, as a specific target of the active probe. We further demonstrated that the active probe inhibits complex 1 activity in vitro (IC(50) = 720 nM), an effect that is known to induce cell death in low-glucose conditions. Based on this proof of principle study, we anticipate that the generation and integration of fully functionalized compound libraries into phenotypic screening programs should facilitate the discovery of bioactive probes that are amenable to accelerated target identification and mechanistic characterization using advanced chemoproteomic technologies.

Metabolomics annotates ABHD3 as a physiologic regulator of medium-chain phospholipids.

All organisms, including humans, possess a huge number of uncharacterized enzymes. Here we describe a general cell-based screen for enzyme substrate discovery by untargeted metabolomics and its application to identify the protein α/ß-hydrolase domain-containing 3 (ABHD3) as a lipase that selectively cleaves medium-chain and oxidatively truncated phospholipids. Abhd3(-/-) mice possess elevated myristoyl (C14)-phospholipids, including the bioactive lipid C14-lysophosphatidylcholine, confirming the physiological relevance of our substrate assignments.

Designed semisynthetic protein inhibitors of Ub/Ubl E1 activating enzymes.

Semisynthetic, mechanism-based protein inhibitors of ubiquitin (Ub) and ubiquitin-like modifier (Ubl) activating enzymes (E1s) have been developed to target E1-catalyzed adenylation and thioesterification of the Ub/Ubl C-terminus during the processes of protein SUMOylation and ubiquitination. The inhibitors were generated by intein-mediated expressed protein ligation using a truncated Ub/Ubl protein (SUMO residues 1-94; Ub residues 1-71) with a C-terminal thioester and synthetic tripeptides having a C-terminal adenosine analogue and an N-terminal cysteine residue. SUMO-AMSN (4a) and Ub-AMSN (4b) contain a sulfamide group as a nonhydrolyzable mimic of the phosphate group in the cognate Ub/Ubl-AMP adenylate intermediate in the first half-reaction, and these constructs selectively inhibit SUMO E1 and Ub E1, respectively, in a dose-dependent manner. SUMO-AVSN (5a) and Ub-AVSN (5b) contain an electrophilic vinyl sulfonamide designed to trap the incoming E1 cysteine nucleophile (Uba2 Cys173 in SUMO E1; Uba1 Cys593 in Ub E1) in the second half-reaction, and these constructs selectively, covalently, and stably cross-link to SUMO E1 and Ub E1, respectively, in a cysteine nucleophile-dependent manner. These inhibitors are powerful tools to probe outstanding mechanistic questions in E1 function and can also be used to study the biological functions of E1 enzymes.

Small molecule inhibition of microbial natural product biosynthesis-an emerging antibiotic strategy.

A variety of natural products modulate critical biological processes in the microorganisms that produce them. Thus, inhibition of the corresponding natural product biosynthesis pathways represents a promising avenue to develop novel antibiotics. In this tutorial review, we describe several recent examples of designed small molecule inhibitors of microbial natural product biosynthesis and their use in evaluating this emerging antibiotic strategy.

Functional self-assembling bolaamphiphilic polydiacetylenes as colorimetric sensor scaffolds.

Conjugated polymers capable of responding to external stimuli by changes in optical, electrical, or electrochemical properties can be used for the construction of direct sensing devices. Polydiacetylene-based systems are attractive for sensing applications due to their colorimetric response to changes in the local environment. Here we present the design, preparation, and characterization of self-assembling functional bolaamphiphilic polydiacetylenes (BPDAs) inspired by nature's strategy for membrane stabilization. We show that by placing polar headgroups on both ends of the diacetylene lipids in a transmembranic fashion and by altering the chemical nature of the polar surface residues, the conjugated polymers can be engineered to display a range of radiation-, thermal-, and pH-induced colorimetric responses. We observed dramatic nanoscopic morphological transformations accompanying charge-induced chromatic transitions, suggesting that both side-chain disordering and main-chain rearrangement play important roles in altering the effective conjugation lengths of the poly(ene-yne). These results establish the foundation for further development of BPDA-based colorimetric sensors.