Discovery of Proline-Based p300/CBP Inhibitors Using DNA-Encoded Library Technology in Combination with High-Throughput Screening.

E1A binding protein (p300) and CREB binding protein (CBP) are two highly homologous and multidomain histone acetyltransferases. These two proteins are involved in many cellular processes by acting as coactivators of a large number of transcription factors. Dysregulation of p300/CBP has been found in a variety of cancers and other diseases, and inhibition has been shown to decrease Myc expression. Herein, we report the identification of a series of highly potent, proline-based small-molecule p300/CBP histone acetyltransferase (HAT) inhibitors using DNA-encoded library technology in combination with high-throughput screening. The strategy of reducing ChromlogD and fluorination of metabolic soft spots was explored to improve the pharmacokinetic properties of potent p300 inhibitors. Fluorination of both cyclobutyl and proline rings of 22 led to not only reduced clearance but also improved cMyc cellular potency.

An Opportunistic Survey Reveals an Unexpected Coronavirus Diversity Hotspot in North America.

In summer 2020, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was detected on mink farms in Utah. An interagency One Health response was initiated to assess the extent of the outbreak and included sampling animals from on or near affected mink farms and testing them for SARS-CoV-2 and non-SARS coronaviruses. Among the 365 animals sampled, including domestic cats, mink, rodents, raccoons, and skunks, 261 (72%) of the animals harbored at least one coronavirus. Among the samples that could be further characterized, 127 alphacoronaviruses and 88 betacoronaviruses (including 74 detections of SARS-CoV-2 in mink) were identified. Moreover, at least 10% (n = 27) of the coronavirus-positive animals were found to be co-infected with more than one coronavirus. Our findings indicate an unexpectedly high prevalence of coronavirus among the domestic and wild free-roaming animals tested on mink farms. These results raise the possibility that mink farms could be potential hot spots for future trans-species viral spillover and the emergence of new pandemic coronaviruses.

Discovery of soluble epoxide hydrolase inhibitors through DNA-encoded library technology (ELT).

Inhibition of soluble epoxide hydrolase (sEH) has recently emerged as a new approach to treat cardiovascular disease and respiratory disease. Inhibitors based on 1,3,5-triazine chemotype were discovered through affinity selection against two triazine-based DNA-encoded libraries. The structure and activity relationship study led to the expansion of the original 1,4-cycloalkyl series to related aniline, piperidine, quinoline, aryl-ether and benzylic series. The 1,3-cycloalkyl chemotype led to the discovery of a clinical candidate (GSK2256294) for COPD.

DNA-Encoded Library Technology-Based Discovery, Lead Optimization, and Prodrug Strategy toward Structurally Unique Indoleamine 2,3-Dioxygenase-1 (IDO1) Inhibitors.

We report the discovery of a novel indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor class through the affinity selection of a previously unreported indole-based DNA-encoded library (DEL). The DEL exemplar, spiro-chromane 1, had moderate IDO1 potency but high in vivo clearance. Series optimization quickly afforded a potent, low in vivo clearance lead 11. Although amorphous 11 was highly bio-available, crystalline 11 was poorly soluble and suffered disappointingly low bio-availability because of solubility-limited absorption. A prodrug approach was deployed and proved effective in discovering the highly bio-available phosphonooxymethyl 31, which rapidly converted to 11 in vivo. Obtaining crystalline 31 proved problematic, however; thus salt screening was performed in an attempt to circumvent this obstacle and successfully delivered greatly soluble and bio-available crystalline tris-salt 32. IDO1 inhibitor 32 is characterized by a low calculated human dose, best-in-class potential, and an unusual inhibition mode by binding the IDO1 heme-free (apo) form.

Design and Application of a DNA-Encoded Macrocyclic Peptide Library.

A DNA-encoded macrocyclic peptide library was designed and synthesized with 2.4 × 1012 members composed of 4-20 natural and non-natural amino acids. Affinity-based selection was performed against two therapeutic targets, VHL and RSV N protein. On the basis of selection data, some peptides were selected for resynthesis without a DNA tag, and their activity was confirmed.

Discovery and Characterization of a Class of Pyrazole Inhibitors of Bacterial Undecaprenyl Pyrophosphate Synthase.

Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in bacterial cell wall synthesis. Here we report the discovery of Staphylococcus aureus UppS inhibitors from an Encoded Library Technology screen and demonstrate binding to the hydrophobic substrate site through cocrystallography studies. The use of bacterial strains with regulated uppS expression and inhibitor resistant mutant studies confirmed that the whole cell activity was the result of UppS inhibition, validating UppS as a druggable antibacterial target.

Discovery and Optimization of Potent, Selective, and in Vivo Efficacious 2-Aryl Benzimidazole BCATm Inhibitors.

To identify BCATm inhibitors suitable for in vivo study, Encoded Library Technology (ELT) was used to affinity screen a 117 million member benzimidazole based DNA encoded library, which identified an inhibitor series with both biochemical and cellular activities. Subsequent SAR studies led to the discovery of a highly potent and selective compound, 1-(3-(5-bromothiophene-2-carboxamido)cyclohexyl)-N-methyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamide (8b) with much improved PK properties. X-ray structure revealed that 8b binds to the active site of BACTm in a unique mode via multiple H-bond and van der Waals interactions. After oral administration, 8b raised mouse blood levels of all three branched chain amino acids as a consequence of BCATm inhibition.

A Simple Hydraulic Analog Model of Oxidative Phosphorylation.

Mitochondrial oxidative phosphorylation is the primary source of cellular energy transduction in mammals. This energy conversion involves dozens of enzymatic reactions, energetic intermediates, and the dynamic interactions among them. With the goal of providing greater insight into the complex thermodynamics and kinetics ("thermokinetics") of mitochondrial energy transduction, a simple hydraulic analog model of oxidative phosphorylation is presented. In the hydraulic model, water tanks represent the forward and back "pressures" exerted by thermodynamic driving forces: the matrix redox potential (ΔGredox), the electrochemical potential for protons across the mitochondrial inner membrane (ΔGH), and the free energy of adenosine 5'-triphosphate (ATP) (ΔGATP). Net water flow proceeds from tanks with higher water pressure to tanks with lower pressure through "enzyme pipes" whose diameters represent the conductances (effective activities) of the proteins that catalyze the energy transfer. These enzyme pipes include the reactions of dehydrogenase enzymes, the electron transport chain (ETC), and the combined action of ATP synthase plus the ATP-adenosine 5'-diphosphate exchanger that spans the inner membrane. In addition, reactive oxygen species production is included in the model as a leak that is driven out of the ETC pipe by high pressure (high ΔGredox) and a proton leak dependent on the ΔGH for both its driving force and the conductance of the leak pathway. Model water pressures and flows are shown to simulate thermodynamic forces and metabolic fluxes that have been experimentally observed in mammalian skeletal muscle in response to acute exercise, chronic endurance training, and reduced substrate availability, as well as account for the thermokinetic behavior of mitochondria from fast- and slow-twitch skeletal muscle and the metabolic capacitance of the creatine kinase reaction.

Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor.

DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

Discovery of Potent and Selective Inhibitors for ADAMTS-4 through DNA-Encoded Library Technology (ELT).

The aggrecan degrading metalloprotease ADAMTS-4 has been identified as a novel therapeutic target for osteoarthritis. Here, we use DNA-encoded Library Technology (ELT) to identify novel ADAMTS-4 inhibitors from a DNA-encoded triazine library by affinity selection. Structure-activity relationship studies based on the selection information led to the identification of potent and highly selective inhibitors. For example, 4-(((4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-6-(((4-methylpiperazin-1-yl)methyl)amino)-1,3,5-triazin-2-yl)amino)methyl)-N-ethyl-N-(m-tolyl)benzamide has IC50 of 10 nM against ADAMTS-4, with >1000-fold selectivity over ADAMT-5, MMP-13, TACE, and ADAMTS-13. These inhibitors have no obvious zinc ligand functionality.

Discovery, SAR, and X-ray Binding Mode Study of BCATm Inhibitors from a Novel DNA-Encoded Library.

As a potential target for obesity, human BCATm was screened against more than 14 billion DNA encoded compounds of distinct scaffolds followed by off-DNA synthesis and activity confirmation. As a consequence, several series of BCATm inhibitors were discovered. One representative compound (R)-3-((1-(5-bromothiophene-2-carbonyl)pyrrolidin-3-yl)oxy)-N-methyl-2'-(methylsulfonamido)-[1,1'-biphenyl]-4-carboxamide (15e) from a novel compound library synthesized via on-DNA Suzuki-Miyaura cross-coupling showed BCATm inhibitory activity with IC50 = 2.0 µM. A protein crystal structure of 15e revealed that it binds to BCATm within the catalytic site adjacent to the PLP cofactor. The identification of this novel inhibitor series plus the establishment of a BCATm protein structure provided a good starting point for future structure-based discovery of BCATm inhibitors.

Design, synthesis and selection of DNA-encoded small-molecule libraries.

Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

Pyruvate and citric acid cycle carbon requirements in isolated skeletal muscle mitochondria.

Carbohydrate depletion precipitates fatigue in skeletal muscle, but, because pyruvate provides both acetyl-CoA for mainline oxidation and anaplerotic carbon to the citric acid cycle (CAC), the mechanism remains obscure. Thus pyruvate and CAC kinetic parameters were independently quantified in mitochondria isolated from rat mixed skeletal muscle. Mitochondrial oxygen consumption rate (Jo) was measured polarographically while either pyruvate or malate was added stepwise in the presence of a saturating concentration of the other substrate. These substrate titrations were carried out across a physiological range of fixed extramitochondrial ATP free energy states (DeltaGP), established with a creatine kinase energy clamp, and also at saturating [ADP]. The apparent Km,malate for mitochondrial Jo ranged from 21 to 32 microM, and the apparent Km,pyruvate ranged from 12 to 26 microM, with both substrate Km values increasing as DeltaGP declined. Vmax for both substrates also increased as DeltaGP fell, reflecting thermodynamic control of Jo. Reported in vivo skeletal muscle [malate] are >10-fold greater than the Km,malate determined in this study. In marked contrast, the K(m,pyruvate) determined is near the [pyruvate] reported in muscle approaching exhaustion associated with glycogen depletion. When data were evaluated in the context of a linear thermodynamic force-flow (DeltaGP-Jo) relationship, the DeltaGP-Jo slope was essentially insensitive to changes in [malate] in the range observed in vivo but decreased markedly with declining [pyruvate] across the physiological range. Mitochondrial respiration is particularly sensitive to variations in [pyruvate] in the physiological range. In contrast, physiological [malate] exerts very little, if any, influence on mitochondrial pyruvate oxidation measured in vitro.