DNA-encoded chemistry technology yields expedient access to SARS-CoV-2 Mpro inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration-approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (Mpro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [Ki] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of Mpro (Ki = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (Ki = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.

Synthesis of 5-substituted tetrazoles via DNA-conjugated nitrile.

A zinc bromide-catalyzed synthesis of 5-substituted tetrazoles via DNA-conjugated nitriles using sodium azide has been developed. The protocol offered moderate to excellent yields of tetrazoles with a broad range of substrates, including a variety of functionalized aromatic, heterocyclic, and aliphatic nitriles. In addition, the electronic effect within the substrate scope was evaluated. DNA fidelity was assessed by ligation efficiency and amplifiability analysis. The ability to generate tetrazoles expands the diversity of heterocycles in the preparation of DNA-encoded chemical libraries.

Multistep Synthesis of 1,2,4-Oxadiazoles via DNA-Conjugated Aryl Nitrile Substrates.

A multistep protocol for the synthesis of 3,5-disubstituted 1,2,4-oxadiazoles on DNA-chemical conjugates has been developed. A set of six DNA-connected aryl nitriles were converted to corresponding amidoximes with hydroxylamine followed by the O-acylation with a series of aryl and aliphatic carboxylic acids. After cyclodehydration of the O-acyl amidoximes by heating at 90 °C in pH 9.5 borate buffer for 2 h, the desired oxadiazole products were observed in 51-92% conversion with the cleavage of O-acylamidoximes as the major side-product. The reported protocol paves the way for the synthesis of oxadiazole core-focused DNA-encoded chemical libraries.

DNA-Compatible Nitro Reduction and Synthesis of Benzimidazoles.

DNA-encoded chemical libraries have emerged as a cost-effective alternative to high-throughput screening (HTS) for hit identification in drug discovery. A key factor for productive DNA-encoded libraries is the chemical diversity of the small molecule moiety attached to an encoding DNA oligomer. The library structure diversity is often limited to DNA-compatible chemical reactions in aqueous media. Herein, we describe a facile process for reducing aryl nitro groups to aryl amines. The new protocol offers simple operation and circumvents the pyrophoric potential of the conventional method (Raney nickel). The reaction is performed in aqueous solution and does not compromise DNA structural integrity. The utility of this method is demonstrated by the versatile synthesis of benzimidazoles on DNA.

DNA-Compatible Nitro Reduction and Synthesis of Benzimidazoles.

A key factor for productive DNA-encoded libraries is the chemical diversity of the small molecule moiety attached to an encoding DNA oligomer. The library structure diversity is often limited to DNA-compatible chemical reactions in aqueous media. Herein, we describe a facile process for reducing aryl nitro groups to aryl amines by using sodium dithionite (Na2S2O4). The new protocol offers simple operation and circumvents the pyrophoric potential of the conventional method (Raney nickel). The utility of this method is demonstrated by the versatile synthesis of benzimidazoles on DNA.

A Mild, DNA-Compatible Nitro Reduction Using B2(OH)4.

A hypodiboric acid system for the reduction of nitro groups on DNA-chemical conjugates has been developed. This transformation provided good to excellent yields of the reduced amine product for a variety of functionalized aromatic, heterocyclic, and aliphatic nitro compounds. DNA tolerance to reaction conditions, extension to decigram scale reductions, successful use in a DNA-encoded chemical library synthesis, and subsequent target selection are also described.

In Vivo and In Vitro Analyses of Novel Peptidomimetic Disruptors for the Serotonin 5-HT2C Receptor Interaction With Phosphatase and Tensin Homolog.

Hypofunction of the serotonin (5-HT) 5-HT2C receptor (5-HT2CR) has been implicated in a variety of disorders including substance use disorders. As such, approaches to enhance 5-HT2CR signaling display therapeutic potential. In the present study, we show that disruption of the 5-HT2CR interaction with the protein phosphatase and tensin homolog (PTEN) via peptidomimetics enhances 5-HT2CR-mediating signaling in vitro and potentiates selective 5-HT2CR agonists in behavioral rodent models. Overall, the present study provides further evidence that 5-HT2CR activity can be modulated through an allosteric protein-protein interaction. This work provides the groundwork for the continued exploration of protein-protein interactions that can allosterically modulate this critical receptor and other important G protein-coupled receptors (GPCRs) for new therapeutic development through mechanisms that may display clinical utility.

Evaluation of organophosphorus chemicals-degrading enzymes: a comparison of Escherichia coli and human cytosolic aminopeptidase P.

An enzyme capable of hydrolyzing organophosphate compounds is of biological as well as environmental significance. We evaluated the possibility of human cytosolic aminopeptidase P (hcAMPP) as an attractive bioscavenger candidate by measuring the enzymatic rates of hydrolysis for a wide variety of organophosphorus compounds. The comparison of substrate specificity exhibited by hcAMPP and E. coli aminopeptidase P (E. coli AMPP) was studied. We cloned, expressed, and purified hcAMPP from HeLa cells and AMPP from Escherichia coli. The pH-rate profiles of hcAMPP were measured in the presence of organophosphate compound 3 or 5. All of the organophosphorus compounds, 1-19, were synthesized by using the approach of phosphorus chemistry described in a previous publication. The relative activity of hcAMPP and E. coli AMPP in hydrolyzing a series of organophosphorus analogues, 1-17, was evaluated in a spectrophotometric assay by monitoring the difference of accumulation of 4-nitrophenol at 400 nm. The overall substrate preference of hcAMPP is as follows: methylphosphonates>ethylphosphonates> or =organophosphates. Interestingly, the observed enhancement in the activity of hcAMPP with methyl phosphonates, 8, 10, 12, and 13, suggests that there is particularly special about the substructure of both methyl moiety and P=O ligand, since the values of specific activity with hcAMPP for the methylphosphonates 8, 10, 12, and 13 are 2- to 73-fold greater than those for the ethylphosphonates 14-17 and the organophosphates 1-7. Similarly, in E. coli AMPP toward ethylphosphonates 14-17, the results indicate that the regions of both MeO moiety and P=O ligand may be located in the vicinity of the substrate-binding site, which have not been altered within the active site of enzyme upon mutation of Trp88, Arg153, and Arg370. These studies demonstrate that E. coli AMPP and hcAMPP display different substrate preference toward organophosphorus compounds. Evidence here, therefore, represents the first example of hcAMPP that might serve as a valuable bioscavenger candidate as E. coli AMPP due to the promise from the hydrolysis of these toxic chemicals.