European Journal of Medicinal Chemistry

Pyrazolo[3,4-b]pyridine is a medicinally privileged structure. We have achieved a new and facile synthesis of a combinatorial library of its tetra- and persubstituted derivatives by trifluoracetic acid catalysed condensation of a group of 5-aminopyrazoles and a group of -oxoketene dithioacetals. Furthermore, we demonstrated structural modification of the products via reductive desulfurization, hydrolysis of the ester and Suzuki coupling of the bromo derivative with aryl bornoic acids. Some of the products were subjected to in vitro MABA assay against M. tuberclulosis H37Rv strain and in silico analysis by binding to pantothenate synthetase from M. tuberclulosis (MTBPS). The results indicated that the pyazolo[3,4-b]pyridine with N(1)CH3, C(3)C6H5, C(4) pCH3C6H5, C(5)CO2Et, C(6)SMe substitutions exhibits promising antituberculotic activity.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed to combat additional SARS-CoV-2 variants or novel CoVs. Here, we describe small molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation mediated innate immune responses. The compounds inhibiting Mac1 were discovered through high-throughput screening (HTS) using a protein FRET-based competition assay and the best hit compound had an IC50 of 14 {micro}M. Three validated HTS hits have the same 2-amide-3-methylester thiophene scaffold and the scaffold was selected for structure-activity relationship (SAR) studies through commercial and synthesized analogs. We studied the compound binding mode in detail using X-ray crystallography and this allowed us to focus on specific features of the compound and design analogs. Compound 27 (MDOLL-0229) had an IC50 of 2.1 {micro}M and was generally selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human ADP-ribose binding proteins. The improved potency allowed testing of its effect on virus replication and indeed, 27 inhibited replication of both MHVa prototype CoV, and SARS-CoV-2. Furthermore, sequencing of a drug-resistant MHV identified mutations in Mac1, further demonstrating the specificity of 27. Compound 27 is the first Mac1 targeted small molecule demonstrated to inhibit coronavirus replication in a cell model. This, together with its well-defined binding mode, makes 27 a good candidate for further hit/lead-optimization efforts.

SARS-CoV-2 is the etiological agent responsible for the COVID-19 outbreak in Wuhan. Specific antiviral drug are urgently needed to treat COVID-19 infections. The main protease (Mpro) of SARS-CoV-2 is a key CoV enzyme that plays a pivotal role in mediating viral replication and transcription, which makes it an attractive drug target. In an effort to rapidly discover lead compounds targeting Mpro, two compounds (11a and 11b) were designed and synthesized, both of which exhibited excellent inhibitory activity with an IC50 value of 0.05 M and 0.04 M respectively. Significantly, both compounds exhibited potent anti-SARS-CoV-2 infection activity in a cell-based assay with an EC50 value of 0.42 M and 0.33 M, respectively. The X-ray crystal structures of SARS-CoV-2 Mpro in complex with 11a and 11b were determined at 1.5 [A] resolution, respectively. The crystal structures showed that 11a and 11b are covalent inhibitors, the aldehyde groups of which are bound covalently to Cys145 of Mpro. Both compounds showed good PK properties in vivo, and 11a also exhibited low toxicity which is promising drug leads with clinical potential that merits further studies.

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued structure-based design of peptidomimetic -ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease:inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the -ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against Middle East Respiratory Syndrome coronavirus.

Adenovirus infections of immunocompromised humans are a significant source of morbidity and mortality. At present, no drug has been approved by FDA for the treatment of adenovirus infections. A current treatment of such infections is off-label use of an antiviral acyclic nucleotide phosphonate, cidofovir (CDV, (S)-HPMPC), which requires i.v. administration and has dose-limiting kidney toxicity. We recently reported that USC-093, a homoserinamide analogue of the tyrosinamide (S)- HPMPA prodrug USC-087, was orally effective at a 10 mg/kg against disseminated human adenovirus infection (HAdV-C6) in a Syrian hamster model, although their efficacy was marginal after respiratory infection. Neither prodrug manifested GI toxicity. Unlike USC-087, USC-093 showed no significant nephrotoxicity at the effective dose. Here, we describe in detail the synthesis of USC-093 and also its D-homoserinamide analogue, USC-093D, in four steps (20-40% overall yield) starting from Boc-protected L-homoserine or D-homoserine lactone, respectively. The two stereoisomeric prodrugs had EC50 30-70 nM vs. Ad5 or 1-6 nM vs. Ad6 in HFF cells, with USC-093D giving the lower values. The prodrugs were 30-59x more potent vs. Ad5 and 82-332x more potent than Ad6 relative to the positive control, CDV. To ascertain whether D-chirality in the promoiety could enhance the performance of the prodrug in vivo, USC-093D and USC-093 were compared in the Syrian hamster model (treated from day 1 q.d at an experimentally determined maximum tolerated oral dose of 20 mg/kg)). In this study, the hamsters were instilled i.n. with vehicle or 4X1010 PFU/kg of HAdV-C6 to promote lung infection. Oral valganciclovir (VGCV) at 200 mg/kg b.i.d. was used as the positive control. The body weights were recorded daily, and at 3 days post challenge, gross pathological observation was performed. Lung samples were collected, and the virus burden was determined by TCID50 assay. The results show that altering homoserine stereochemistry did not markedly improve the efficacy of the orally administered prodrug, consistent with the premise that its mechanism of transport is likely not dependent on stereoselective pathways, such as hPEPT1-mediated uptake.

Macrocyclization of acyclic compounds is a powerful strategy for improving inhibitor potency and selectivity. Here, we developed a 2-aminopyrimidine-based macrocyclic dual EPHA2/GAK kinase inhibitor as a chemical tool to study the role of these two kinases in viral entry and assembly. Starting with a promiscuous macrocyclic inhibitor, 6, we performed a structure-guided activity relationship and selectivity study using a panel of over 100 kinases. The crystal structure of EPHA2 in complex with the developed macrocycle 23 provided a basis for further optimization by specifically targeting the back pocket, resulting in compound 55 as a potent dual EPHA2/GAK inhibitor. Subsequent front-pocket derivatization resulted in an interesting in cellulo selectivity profile, favoring EPHA4 over the other ephrin receptor kinase family members. The dual EPHA2/GAK inhibitor 55 prevented dengue virus infection of Huh7 liver cells, mainly via its EPHA2 activity, and is therefore a promising candidate for further optimization of its activity against dengue virus.

We previously reported potent ligands and inhibitors of Mycobacterium tuberculosis dethiobiotin synthetase (MtDTBS), a promising target for antituberculosis drug development (Schumann et al., ACS Chem Biol. 2021, 16, 2339-2347); here the unconventional origin of the fragment compound they were derived from is described for the first time. Compound 1 (9b-hydroxy-6b,7,8,9,9a,9b-hexahydrocyclopenta[3,4]cyclobuta[1,2-c]chromen-6(6aH)-one), identified by in silico fragment screen, was subsequently shown by surface plasmon resonance to have dose-responsive binding (KD 0.6 mM). Clear electron density was revealed in the DAPA substrate binding pocket, when 1 was soaked into MtDTBS crystals, but the density was inconsistent with the structure of 1. Here we show the lactone of 1 hydrolyses to carboxylic acid 2 under basic conditions, including those of the crystallography soak, with subsequent ring-opening of the component cyclobutane ring to form cyclopentylacetic acid 3. Crystals soaked directly with authentic 3 produced electron density that matched that of crystals soaked with presumed 1, confirming the identity of the bound ligand. The synthetic utility of fortuitously formed 3 enabled subsequent compound development into nanomolar inhibitors. Our findings represent an example of chemical modification within drug discovery assays and demonstrate the value of high-resolution structural data in the fragment hit validation process. SynopsisA molecule flagged in an in silico docking screen against MtDTBS, was inadvertently hydrolysed in the crystal conditions used for hit validation. The resulting fragment-sized molecule bound to the DAPA substrate binding pocket of the target enzyme (MtDTBS) with millimolar affinity, as measured by surface plasmon resonance, but was later modified to a highly potent (nanomolar) ligand and promising lead for the development of novel tuberculosis treatments. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

Molecular glues are small molecules that engage their target and presenter proteins cooperatively. FKBP12 molecular glues (FK506 and rapamycin) were discovered several decades ago and have been used clinically, but our understanding of the breadth of FKBP12 molecular glues and targets has yet to be fully revealed. To identify novel targets of FKBP12 molecular glues, we constructed and screened a multi-million-member non-macrocyclic FKBP12-ligand DNA-encoded library using 25 structurally distinct proteins. Synthesis and validation of selected hits in biophysical and cell-based assays confirm FKBP12-dependent molecular-glue recruitment to bromodomain-containing protein 9 (BRD9) and quinoid dihydropteridine reductase (QDPR). One glue showed no measurable binding to QDPR alone but had appreciable binding in the presence of FKBP12 using either purified proteins or intact cells. The sites of recruitment were characterized with mutational analysis, competition-based methods and X-ray crystallography. The results of this study confirm that FKBP12-binding DELs can yield novel molecular glues generating highly selective FKBP12-target protein interactions.

Salt-inducible kinases (SIKs) are key metabolic regulators. Imbalance of SIK function is associated with the development of diverse cancers, including breast, gastric and ovarian cancer. Chemical tools to clarify the roles of SIK in different diseases are, however, sparse and are generally characterized by poor kinome-wide selectivity. Here, we have adapted the pyrido[2,3-d]pyrimidin-7-one-based PAK inhibitor G-5555 for the targeting of SIK, by exploiting differences in the back-pocket region of these kinases. Optimization was supported by high-resolution crystal structures of G-5555 bound to the known off-targets MST3 and MST4, leading to a chemical probe, MRIA9, with dual SIK/PAK activity and excellent selectivity over other kinases. Furthermore, we show that MRIA9 sensitizes ovarian cancer cells to treatment with the mitotic agent paclitaxel, confirming earlier data from genetic knockdown studies and suggesting a combination therapy with SIK inhibitors and paclitaxel for the treatment of paclitaxel-resistant ovarian cancer.

STK10 (serine/threonine kinase 10, LOK), is an important regulator of diverse cellular processes, such as cell cycle progression or lymphocyte migration. STK10 has emerged as a potential therapeutic target for diseases associated with impaired cell migration and cell division. Here we present a late-stage optimization of a macrocyclic pyrazolo[1,5-a]pyrimidine scaffold that led to a urea-based lead series targeting the back-pocket of STK10. Co-crystal structure analysis of 23 revealed that the optimized macrocycles adopted a unique binding mode that protrudes deep into the back pocket of STK10. Compound 23 exhibited potent on-target activity in biophysical and activity assays and displayed nanomolar activity for STK10 in cells. In addition, 23 shows good selectivity against the kinome and remarkably also against the closely related kinase SLK (STE20-like kinase). Therefore, we propose that targeting the unique and largely extended pocket in STK10 represents an opportunity to develop highly selective STK10 inhibitors. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/666149v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@36f039org.highwire.dtl.DTLVardef@d55025org.highwire.dtl.DTLVardef@80c007org.highwire.dtl.DTLVardef@bf2471_HPS_FORMAT_FIGEXP M_FIG C_FIG

The opioid crisis is a catastrophic health emergency catalyzed by the misuse of opioids that target and activate the mu opioid receptor. Traditional radioligands used to study the mu opioid receptor are often tightly regulated owing to their abuse and respiratory depression potential. In the present study, we sought to design and characterize a library of 24 non-agonist ligands for the mu opioid receptor. Ligands were evaluated for the binding affinity, intrinsic activity, and predicted blood-brain barrier permeability. Several ligands demonstrated single-digit nM binding affinity for the mu opioid receptor while also demonstrating selectivity over the delta and kappa opioid receptors. The antagonist behavior of 1A and 3A at the mu opioid receptor indicate that these ligands would likely not induce opioid-dependent respiratory depression. Therefore, these ligands can enable a safer means to interrogate the endogenous opioid system. Based on binding affinity, selectivity, and potential off-target binding, [11C]1A was prepared via metallophotoredox of the aryl-bromide functional group to [11C]methyl iodide. The nascent radiotracer demonstrated brain uptake in a rhesus macaque model and accumulation in the caudate and putamen. Naloxone was able to reduce [11C]1A binding, though the interactions were not as pronounced as naloxones ability to displace [11C]carfentanil. These results suggest that GSK1521498 and related congeners are amenable to radioligand design and can offer a safer way to query opioid neurobiology.

Proteases encoded by SARS-CoV-2 constitute a promising target for new therapies against COVID-19. SARS-CoV-2 main protease (Mpro, 3CLpro) and papain-like protease (PLpro) are responsible for viral polyprotein cleavage - a process crucial for viral survival and replication. Recently it was shown that 2-phenylbenzisoselenazol-3(2H)-one (ebselen), an organoselenium anti-inflammatory small-molecule drug, is a potent, covalent inhibitor of both the proteases and its potency was evaluated in enzymatic and anti-viral assays. In this study, we screened a collection of 23 ebselen derivatives for SARS-CoV-2 PLpro and Mpro inhibitors. Our studies revealed that ebselen derivatives are potent inhibitors of both the proteases. We identified three PLpro and four Mpro inhibitors superior to ebselen. Our work shows that ebselen constitutes a promising platform for development of new antiviral agents targeting both SARS-CoV-2 PLpro and Mpro.

We aimed to prepare novel dibenzosuberane derivatives that act on N-methyl-D-aspartate (NMDA) receptors with potential neuroprotective effects. Our approach involved modifying the tropane moiety of MK-801, a potent open-channel blocker known for its psychomimetic side effects, by introducing a seven-membered ring with substituted base moieties specifically to alleviate these undesirable effects. Our in silico analyses showed that these derivatives should have high gastrointestinal absorption and cross the blood-brain barrier (BBB). Our pharmacokinetic studies in rats supported this conclusion and confirmed the ability of leading compounds 3l and 6f to penetrate the BBB. Electrophysiological experiments showed that all compounds exhibited different inhibitory activity towards the two major NMDA receptor subtypes, GluN1/GluN2A and GluN1/GluN2B. Of the selected compounds intentionally differing in the inhibitory efficacy, 6f showed high relative inhibition ([~]90% for GluN1/GluN2A), while 3l showed moderate inhibition ([~]50%). An in vivo toxicity study determined that compounds 3l and 6f were safe at 10 mg/kg doses with no adverse effects. Behavioral studies demonstrated that these compounds did not induce hyperlocomotion or impair prepulse inhibition of startle response in rats. Neuroprotective assays using a model of NMDA-induced hippocampal neurodegeneration showed that compound 3l at a concentration of 30 M significantly reduced hippocampal damage in rats. These results suggest that these novel dibenzosuberane derivatives are promising candidates for developing NMDA receptor-targeted therapies with minimal psychotomimetic side effects.

N-methyl-D-aspartate receptors (NMDARs) are crucial therapeutic targets, modulated by endogenous neurosteroids like pregnenolone sulfate (PES). This study investigates a novel structure-activity relationship approach focusing on the steroidal D-ring, employing the bioisosteric replacement of C-17 or C-20 keto groups with oximes and oxime ethers. We synthesized a series of pregn-5-ene and androst-5-ene derivatives (11-23) and evaluated their positive allosteric modulator (PAM) activity on recombinant rat GluN1/GluN2B receptors via patch-clamp in HEK293 cells. Our study revealed that pregnenolone-derived C-20 oxime ethers are potent and efficacious PAMs of NMDAR. Several analogues have been demonstrated as more potent than PES (Emax = 116%; EC50 = 21.7 {micro}M). Compound 12 (C-20 ethyl oxime ether, C-3 hemiglutarate) displayed the highest efficacy, potentiating NMDAR currents over 6-fold more than PES (Emax = 673 {+/-} 121%; EC50 = 8.7 {+/-} 1.1 {micro}M). Compound 17 (C-20 methyl oxime ether analogue) exhibited the highest potency, being over 3.5-fold more potent than PES (Emax = 503 {+/-} 68%; EC50 = 6.1 {+/-} 0.4 {micro}M). In contrast, some C-17 analogues and derivatives with bulkier C-20 oxime substituents showed complex modulatory behavior. Promisingly, key compounds demonstrated favorable in vitro ADME profiles, including high metabolic stability and, for 12, excellent thermodynamic solubility. These results validate C-20 oxime ether modification of the pregnenolone scaffold as an effective strategy for generating potent NMDAR PAMs with potentially superior efficacy and drug-like properties compared to endogenous modulators.

Boceprevir is an HCV NSP3 inhibitor that has been explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (MPro) and contains an -ketoamide warhead, a P1 {beta}-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert-butyl-glycine, and a P4 N-terminal tert-butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based MPro inhibitors including PF-07321332 and characterized their MPro inhibition potency in test tubes (in vitro) and human host cells (in cellulo). Crystal structures of MPro bound with 10 inhibitors and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a {beta}-(S-2-oxopyrrolidin-3-yl)-alanyl (opal) residue and the warhead with an aldehyde leads to high in vitro potency. The original moieties at P2, P3 and the P4 N-terminal cap positions in boceprevir are better than other tested chemical moieties for high in vitro potency. In crystal structures, all inhibitors form a covalent adduct with the MPro active site cysteine. The P1 opal residue, P2 dimethylcyclopropylproline and P4 N-terminal tert-butylcarbamide make strong hydrophobic interactions with MPro, explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains an P4 N-terminal isovaleramide. In its MPro complex structure, the P4 N-terminal isovaleramide is tucked deep in a small pocket of MPro that originally recognizes a P4 alanine side chain in a substrate. Although all inhibitors show high in vitro potency, they have drastically different in cellulo potency in inhibiting ectopically expressed MPro in human 293T cells. All inhibitors including PF-07321332 with a P4 N-terminal carbamide or amide have low in cellulo potency. This trend is reversed when the P4 N-terminal cap is changed to a carbamate. The installation of a P3 O-tert-butyl-threonine improves in cellulo potency. Three molecules that contain a P4 N-terminal carbamate were advanced to antiviral tests on three SARS-CoV-2 variants. They all have high potency with EC50 values around 1 M. A control compound with a nitrile warhead and a P4 N-terminal amide has undetectable antiviral potency. Based on all observations, we conclude that a P4 N-terminal carbamate in a boceprevir derivative is key for high antiviral potency against SARS-CoV-2.

In all of the clinical trials for COVID-19 conducted thus far and among those ongoing involving chloroquine or hydroxychloroquine, the drug substance used has invariably been chloroquine (CQ) diphosphate or hydroxychloroquine (HCQ) sulfate, i.e., the phosphoric or sulfuric acid salt of a racemic mixture of R- and S-enantiomer (50/50), respectively. As a result, the clinical outcome from previous CQ or HCQ trials were, in fact, the collective manifestation of both R and S- enantiomers with inherent different pharmacodynamic and pharmacokinetic properties, and toxicity liabilities. Our data for the first time demonstrated the stereoselective difference of CQ and HCQ against live SARS-CoV-2 virus in a Biosafety Level 3 laboratory. S-chloroquine (S-CQ) and S-hydroxychloroquine (S-HCQ) significantly more active against SARS-CoV-2, as compared to R-CQ and R-HCQ, respectively. In addition, Mpro, as one of the critical enzymes for viral transcription and replication, also exhibited an enantioselective binding affinity toward the S-enantiomers. The most significant finding from this study is the pronounced difference of the two enantiomers of CQ and HCQ observed in hERG inhibition assay. The IC50 value of S-HCQ was higher than 20 M against hERG channel, which was much less active over all tested CQ and HCQ compounds. Moreover, S-HCQ alone did not prolong QT interval in guinea pigs after 3 days and 6 days of administration, indicating a much lower cardiac toxicity potential. With these and previous findings on the enantio-differentiated metabolism, we recommend that future clinical studies should employ S-HCQ, substantially free of the R-enantiomer, to potentially improve the therapeutic index for the treatment of COVID-19 over the racemic CQ and HCQ.

Methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive bacterial pathogen, continues to pose a serious threat to the current public health system in our society. The high level of resistance to {beta}-lactam antibiotics in MRSA is attributed to the expression of penicillin-binding protein 2a (PBP2a), which catalyzes cell wall cross-linking. According to numerous research reports, the activity of the PBP2a protein is known to be regulated by an allosteric site distinct from the active site where cell wall cross-linking occurs. Here, we conducted a screening of 113 compounds containing a 1,3,4-oxadiazole core to design new covalent inhibitors targeting the allosteric site of PBP2a and establish their structural-activity relationship. The stereochemically selective synthesis of sulfonyl oxadiazole compounds identified in the initial screening resulted in a maximum eightfold enhancement in cell inhibition activity. The sulfonyl oxadiazole-based compounds formulated as PEG-based ointments, with low toxicity test results on human cells (CC50: >78M), demonstrated potent antimicrobial effects not only in a mouse skin wound infection model but also against oxacillin-resistant clinical isolate MRSA (IC50 {approx} 1M), as evidenced by the results. Furthermore, additional studies utilizing LC-MS/MS and in-silico approaches clearly support the allosteric site covalent binding mechanism through the nucleophilic aromatic substitution (SNAr) reaction, as well as its association with the closure of the major active site of PBP2a.

Insulin-Regulated Aminopeptidase (IRAP) is an enzyme with important biological functions and the target of several drug-discovery efforts although no clinically useful inhibitors have been reported yet. We combined in silico screening with a medicinal chemistry optimization campaign to discover a nanomolar inhibitor of IRAP based on a pyrazolylpyrimidine scaffold. This compound displays an excellent selectivity profile versus homologous aminopeptidases and kinetic analysis suggests it utilizes an uncompetitive mechanism of action when inhibiting the cleavage of a typical dipeptidic substrate. Surprisingly, the compound is a poor inhibitor of the processing of the physiological cyclic peptide substrate oxytocin and a 10mer antigenic epitope precursor but displays a biphasic inhibition profile for the trimming of a 9mer antigenic peptide and is active in blocking IRAP-dependent cross-presentation of an 8mer epitope. To better understand the mechanism of action and the basis for the unusual substrate selectivity of this inhibitor, we solved the crystal structure of the compound in complex with IRAP. The structure indicated direct zinc(II) engagement by the pyrazolylpyrimidine scaffold and revealed that the compound binds to an open conformation of the enzyme in a pose that should block the conformational transition to the closed conformation previously observed with other low molecular weight inhibitors and hypothesized to be important for catalysis. This compound constitutes the first IRAP inhibitor targeting the active site that utilizes a conformation-specific mechanism of action, provides insight into the intricacies of the IRAP catalytic cycle, and highlights a novel approach to regulating IRAP activity by blocking its conformational rearrangements.

The excitatory neurotransmitter glutamate triggers a Ca2+ rise and the extension of processes in astrocytes. Our results suggest that kainic acid receptors (KAR) can independently initiate glutamate signaling in astrocytoma U118-MG cells. The natural product kainic acid triggered glioexcitablity in cells and was inhibited by the KAR antagonist CNQX, but its activity was lower than glutamate on KARs. We created a new heteroaryl kainoid based on rational design: aminooxadiazolyl kainic acid 1 (AODKA). AODKA induced a larger calcium influx and a faster processes extension than kainic acid in U118-MG cells. AODKA is a new tool to study KAR activity in the nervous system. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/426948v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1b63f72org.highwire.dtl.DTLVardef@1ff92c4org.highwire.dtl.DTLVardef@1fcb212org.highwire.dtl.DTLVardef@10ed76e_HPS_FORMAT_FIGEXP M_FIG C_FIG

A series of amino acid based 7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp22 and the amide backbone NH of Ile23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe157 and Asp156, part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low M Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/482176v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@167aceorg.highwire.dtl.DTLVardef@1d88c47org.highwire.dtl.DTLVardef@1e1b34borg.highwire.dtl.DTLVardef@c2321a_HPS_FORMAT_FIGEXP M_FIG C_FIG