Coronaviruses (CoVs) can emerge from zoonotic sources and cause severe diseases in humans and animals. CoVs encode for a macrodomain (Mac1) that binds to and removes ADP-ribose from target proteins. SARS-CoV-2 Mac1 promotes virus replication in the presence of interferon (IFN) and blocks the production of IFN, although the mechanisms by which it mediates these functions remain unknown. Mac1 inhibitors could help elucidate these mechanisms and serve as therapeutic agents against CoV-induced diseases. We previously identified compound 4a (a.k.a. MCD-628), a pyrrolo-pyrimidine that inhibited Mac1 activity in vitro at low micromolar levels. Here, we determined the binding mode of 4a by crystallography, further defining its interaction with Mac1. However, 4a did not reduce CoV replication, which we hypothesized was due to its acidic side chain limiting permeability. To test this hypothesis, we developed several hydrophobic derivatives of 4a. We identified four compounds that both inhibited Mac1 in vitro and inhibited murine hepatitis virus (MHV) replication: 5a, 5c, 6d, and 6e. Furthermore, 5c and 6e inhibited SARS-CoV-2 replication only in the presence of IFNγ, similar to a Mac1 deletion virus. To confirm their specificity, we passed MHV in the presence of 5a to identify drug-resistant mutations and identified an alanine-to-threonine and glycine-to-valine double mutation in Mac1. Recombinant virus with these mutations had enhanced replication compared with the WT virus when treated with 5a, demonstrating the specificity of these compounds during infection. However, this virus is highly attenuated in vivo, indicating that drug resistance emerged at the expense of viral fitness.IMPORTANCECoronaviruses (CoVs) present significant threats to human and animal health, as evidenced by recent outbreaks of MERS-CoV and SARS-CoV-2. CoVs encode for a highly conserved macrodomain protein (Mac1) that binds to and removes ADP-ribose from proteins, which promotes virus replication and blocks IFN production, although the exact mechanisms remain unclear. Inhibiting Mac1 could provide valuable insights into these mechanisms and offer new therapeutic avenues for CoV-induced diseases. We have identified several unique pyrrolo-pyrimidine-based compounds as Mac1 inhibitors. Notably, at least two of these compounds inhibited both murine hepatitis virus (MHV) and SARS-CoV-2 replication. Furthermore, we identified a drug-resistant mutation in Mac1, confirming target specificity during infection. However, this mutant is highly attenuated in mice, indicating that drug resistance appears to come at a fitness cost. These results emphasize the potential of Mac1 as a drug target and the promise of structure-based inhibitor design in combating CoV infections.

Cancers reprogram macrophages (MΦs) to a tumor-growth-promoting TAM (tumor-associated MΦ) phenotype that is similar to the anti-inflammatory M2 phenotype. Poly(ADP-ribose) polymerase (PARP) enzymes regulate various aspects of MΦ biology, but their role in the development of TAM phenotype has not yet been investigated. Here, we show that the multispectral PARP inhibitor (PARPi) PJ34 and the PARP14 specific inhibitor MCD113 suppress the expression of M2 marker genes in IL-4-polarized primary murine MΦs, in THP-1 monocytic human MΦs, and in primary human monocyte-derived MΦs. MΦs isolated from PARP14 knockout mice showed a limited ability to differentiate to M2 cells. In a murine model of TAM polarization (4T1 breast carcinoma cell supernatant transfer to primary MΦs) and in a human TAM model (spheroids formed from JIMT-1 breast carcinoma cells and THP-1-MΦs), both PARPis and the PARP14 KO phenotype caused weaker TAM polarization. Increased JIMT-1 cell apoptosis in co-culture spheroids treated with PARPis suggested reduced functional TAM reprogramming. Protein profiling arrays identified lipocalin-2, macrophage migration inhibitory factor, and plasminogen activator inhibitor-1 as potential (ADP-ribosyl)ation-dependent mediators of TAM differentiation. Our data suggest that PARP14 inhibition might be a viable anticancer strategy with a potential to boost anticancer immune responses by reprogramming TAMs.

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 IC 50 of 14 µM. 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 IC 50 of 2.1 µM 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.

Supplementary Methods from Inhibition of Glutaminase Preferentially Slows Growth of Glioma Cells with Mutant IDH1

Supplementary Figure 2 from Inhibition of Glutaminase Preferentially Slows Growth of Glioma Cells with Mutant IDH1

Non-structural protein 3 (nsp3) from all coronaviruses (CoVs) contains a conserved macrodomain, known as Mac1, that has been proposed as a potential therapeutic target for CoVs due to its critical role in viral pathogenesis. Mac1 is an ADP-ribose binding protein and ADP-ribosylhydrolase that promotes replication and blocks IFN responses, though the precise mechanisms it uses to carry out these functions remain unknown. Over the past 3 years following the onset of COVID-19, several groups have used high-throughput screening with multiple assays and chemical modifications to create unique chemical inhibitors of the SARS-CoV-2 Mac1 protein. Here, we summarize the current efforts to identify selective and potent inhibitors of SARS-CoV-2 Mac1.

Supplementary Figure Legends 1-7 from Inhibition of Glutaminase Preferentially Slows Growth of Glioma Cells with Mutant IDH1

Glutamate carboxypeptidase-II (GCPII) is a zinc-dependent metalloenzyme implicated in numerous neurological disorders. The pharmacophoric requirements of active-site GCPII inhibitors makes them highly charged, manifesting poor pharmacokinetic (PK) properties. Herein, we describe the discovery and characterization of catechol-based inhibitors including L-DOPA, D-DOPA, and caffeic acid, with sub-micromolar potencies. Of these, D-DOPA emerged as the most promising compound, with good metabolic stability, and excellent PK properties. Orally administered D-DOPA yielded high plasma exposures (AUCplasma = 72.7 nmol·h/mL) and an absolute oral bioavailability of 47.7%. Unfortunately, D-DOPA brain exposures were low with AUCbrain = 2.42 nmol/g and AUCbrain/plasma ratio of 0.03. Given reports of isomeric inversion of D-DOPA to L-DOPA via D-amino acid oxidase (DAAO), we evaluated D-DOPA PK in combination with the DAAO inhibitor sodium benzoate and observed a >200% enhancement in both plasma and brain exposures (AUCplasma = 185 nmol·h/mL; AUCbrain = 5.48 nmol·h/g). Further, we demonstrated GCPII target engagement; orally administered D-DOPA with or without sodium benzoate caused significant inhibition of GCPII activity. Lastly, mode of inhibition studies revealed D-DOPA to be a noncompetitive, allosteric inhibitor of GCPII. To our knowledge, this is the first report of D-DOPA as a distinct scaffold for GCPII inhibition, laying the groundwork for future optimization to obtain clinically viable candidates.

The emergence of several zoonotic viruses in the last twenty years, especially the pandemic outbreak of SARS-CoV-2, has exposed a dearth of antiviral drug therapies for viruses with pandemic potential. Developing a diverse drug portfolio will be critical for our ability to rapidly respond to novel coronaviruses (CoVs) and other viruses with pandemic potential. Here we focus on the SARS-CoV-2 conserved macrodomain (Mac1), a small domain of non-structural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that cleaves mono-ADP-ribose (MAR) from target proteins, protects the virus from the anti-viral effects of host ADP-ribosyltransferases, and is critical for the replication and pathogenesis of CoVs. In this study, a luminescent-based high-throughput assay was used to screen ∼38,000 small molecules for those that could inhibit Mac1-ADP-ribose binding. We identified 5 compounds amongst 3 chemotypes that inhibit SARS-CoV-2 Mac1-ADP-ribose binding in multiple assays with IC 50 values less than 100 µ M, inhibit ADP-ribosylhydrolase activity, and have evidence of direct Mac1 binding. These chemotypes are strong candidates for further derivatization into highly effective Mac1 inhibitors.

Background: The clinical outcomes of patients with Acute Myeloid Leukemia (AML) remain unsatisfactory. Therefore the development of more efficacious and better-tolerated therapy for AML is critical. We have previously reported anti-leukemic activity of synthetic halohydroxyl dimeric naphthoquinones (BiQ) and aziridinyl BiQ. Objective: This study aimed to improve the potency and bioavailability of BiQ compounds and investigate anti leukemic activity of the lead compound in vitro and a human AML xenograft mouse model. Methods: We designed, synthesized, and performed structure-activity relationships of several rationally designed BiQ analogues with amino alcohol functional groups on the naphthoquinone core rings. The compounds were screened for anti-leukemic activity and the mechanism as well as in vivo tolerability and efficacy of our lead compound was investigated. Results: We report that a dimeric naphthoquinone (designated BaltBiQ) demonstrated potent nanomolar anti-leukemic activity in AML cell lines. BaltBiQ treatment resulted in the generation of reactive oxygen species, induction of DNA damage, and inhibition of indoleamine dioxygenase 1. Although BaltBiQ was tolerated well in vivo, it did not significantly improve survival as a single agent, but in combination with the specific Bcl-2 inhibitor, Venetoclax, tumor growth was significantly inhibited compared to untreated mice. Conclusion: We synthesized a novel amino alcohol dimeric naphthoquinone, investigated its main mechanisms of action, reported its in vitro anti-AML cytotoxic activity, and showed its in vivo promising activity combined with a clinically available Bcl-2 inhibitor in a patient-derived xenograft model of AML.

INTRODUCTION:Discovery of small molecules that impede the activity of single-strand DNA repair enzyme, PARP1, has led to four marketed drugs for the treatment of advanced-stage cancers. Hence, there is a renewed enthusiasm in the PARP inhibitor discovery arena. To reduce nonspecific interactions or potential toxicities, and to understand the role of other minimally explored PARP enzymes, exciting new findings have emerged toward the development of selective inhibitors and targeted chemical biology probes. Importantly, the conventional PARP inhibitor design has evolved in a way that could potentially lead to multienzyme-targeting - a polypharmacological approach against aggressive cancers.AREAS COVERED:This review comprises recent progress made in the development of PARP inhibitors, primarily focused on human cancers. Discovery of novel PARP inhibitors with pan, selective, and multi-target inhibition using in vitro and in vivo cancer models is summarized and critically evaluated. Emphasis is given to patents published during 2016-2020, excluding TNKS 1/2 inhibitors.EXPERT OPINION:The outstanding success demonstrated by the FDA approved PARP inhibitors has fueled further clinical evaluations for expansion of their clinical utilities. The current clinical investigations include new candidates as well as marketed PARP-targeted drugs, both as single agents and in combination with other chemotherapeutics. Recent advances have also unveiled critical roles of other PARPs in oncogenic signal transduction, in addition to those of the well-documented PARP1/2 and TNKS1/2 enzymes. Further studies on lesser-known PARP members are urgently needed for functional annotations and for understanding their roles in cancer progression and other human diseases.

Two critical steps in drug development are 1) the discovery of molecules that have the desired effects on a target, and 2) the optimization of such molecules into lead compounds with the required potency and pharmacokinetic properties for translation. DNA-encoded chemical libraries (DECLs) can nowadays yield hits with unprecedented ease, and lead-optimization is becoming the limiting step. Here we integrate DECL screening with structure-based computational methods to streamline the development of lead compounds. The presented workflow consists of enumerating a virtual combinatorial library (VCL) derived from a DECL screening hit and using computational binding prediction to identify molecules with enhanced properties relative to the original DECL hit. As proof-of-concept demonstration, we applied this approach to identify an inhibitor of PARP10 that is more potent and druglike than the original DECL screening hit.

Acute myeloid leukemia (AML) is a neoplastic disorder resulting from clonal proliferation of poorly differentiated immature myeloid cells. Distinct genetic and epigenetic aberrations are key features of AML that account for its variable response to standard therapy. Irrespective of their oncogenic mutations, AML cells produce elevated levels of reactive oxygen species (ROS). They also alter expression and activity of antioxidant enzymes to promote cell proliferation and survival. Subsequently, selective targeting of redox homeostasis in a molecularly heterogeneous disease, such as AML, has been an appealing approach in the development of novel anti-leukemic chemotherapeutics. Naphthoquinones are able to undergo redox cycling and generate ROS in cancer cells, which have made them excellent candidates for testing against AML cells. In addition to inducing oxidative imbalance in AML cells, depending on their structure, naphthoquinones negatively affect other cellular apparatus causing neoplastic cell death. Here we provide an overview of the anti-AML activities of naphthoquinone derivatives, as well as analysis of their mechanism of action, including induction of reduction-oxidation imbalance, alteration in mitochondrial transmembrane potential, Bcl-2 modulation, initiation of DNA damage, and modulation of MAPK and STAT3 activity, alterations in the unfolded protein response and translocation of FOX-related transcription factors to the nucleus.

ADP-ribosylation is a ubiquitous post-translational addition of either monomers or polymers of ADP-ribose to target proteins by ADP-ribosyltransferases, usually by interferon-inducible diphtheria toxin-like enzymes known as PARPs. While several PARPs have known antiviral activities, these activities are mostly independent of ADP-ribosylation. Consequently, less is known about the antiviral effects of ADP-ribosylation. Several viral families, including Coronaviridae, Togaviridae, and Hepeviridae, encode for macrodomain proteins that bind to and hydrolyze ADP-ribose from proteins and are critical for optimal replication and virulence. These results suggest that macrodomains counter cellular ADP-ribosylation, but whether PARPs or, alternatively, other ADP-ribosyltransferases cause this modification is not clear. Here we show that pan-PARP inhibition enhanced replication and inhibited interferon production in primary macrophages infected with macrodomain-mutant but not wild-type coronavirus. Specifically, knockdown of two abundantly expressed PARPs, PARP12 and PARP14, led to increased replication of mutant but did not significantly affect wild-type virus. PARP14 was also important for the induction of interferon in mouse and human cells, indicating a critical role for this PARP in the regulation of innate immunity. In summary, these data demonstrate that the macrodomain is required to prevent PARP-mediated inhibition of coronavirus replication and enhancement of interferon production.

A series of carbamate-based inhibitors of glutamate carboxypeptidase II (GCPII) were designed and synthesized using ZJ-43, N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-l-glutamic acid, as a molecular template in order to better understand the impact of replacing one of the two nitrogen atoms in the urea-based GCPII inhibitor with an oxygen atom. Compound 7 containing a C-terminal 2-oxypentanedioic acid was more potent than compound 5 containing a C-terminal glutamic acid (2-aminopentanedioic acid) despite GCPII's preference for peptides containing an N-terminal glutamate as substrates. Subsequent crystallographic analysis revealed that ZJ-43 and its two carbamate analogs 5 and 7 with the same (S,S)-stereochemical configuration adopt a nearly identical binding mode while (R,S)-carbamate analog 8 containing a d-leucine forms a less extensive hydrogen bonding network. QM and QM/MM calculations have identified no specific interactions in the GCPII active site that would distinguish ZJ-43 from compounds 5 and 7 and attributed the higher potency of ZJ-43 and compound 7 to the free energy changes associated with the transfer of the ligand from bulk solvent to the protein active site as a result of the lower ligand strain energy and solvation/desolvation energy. Our findings underscore a broader range of factors that need to be taken into account in predicting ligand-protein binding affinity. These insights should be of particular importance in future efforts to design and develop GCPII inhibitors for optimal inhibitory potency.

Introduction: Acute myeloid leukemia (AML) is a genetically heterogeneous neoplasm with poor clinical outcome after treatment with current conventional chemotherapy. New treatments are therefore needed to combat this aggressive cancer. One of the hallmarks of AML blasts is disrupted cellular oxidative state resulting from elevated level of reactive oxygen species (ROS) and overexpressed antioxidant proteins. Another characteristic of AML blasts is escaping host immune surveillance mediated in part by overexpression of the tryptophan-metabolizing enzyme, indoleamine 2,3-dioxygenase (IDO). We have designed and synthesized a novel hydroxylamine dimeric naphthoquinone (MCD-66), which can kill leukemia cells by dual mechanisms: 1) negatively perturbing cellular oxidative balance, and 2) effectively inhibiting IDO-1. Results: Two AML cell lines (MV4-11 and MOLM14) were treated with increasing doses of the clinically used naphthoquinones, atovaquone, and vitamin K, as well as with MCD-66, and IC 50 curves were generated. With nanomolar range IC 50 s, MCD-66 was 25-30 fold more potent than other naphthoquinones against AML cells. MCD-66 effectively inhibited clonogenicity of AML cells in a dose-dependent manner. When MV4-11 cells were treated with MCD-66 at IC 50 concentration, ROS levels significantly peaked after 2 hours and returned to baseline within 24 hours. The augmented production of ROS led to concomitant increase in DNA damage, evidenced by γH 2 AX foci in treated AML blasts. Additionally, MCD-66 inhibited IDO-1 activity at 1 µM (37%) and 10 µM (79%), but not its isozymes IDO-2 or tryptophan-2,3-dioxygenase (TDO). Conclusion: The novel amino-alcohol dimeric naphthoquinone MCD-66 has shown promising activity against AML blast in vitro with dual mechanisms of action. The in vivo studies focusing on tolerability and efficacy, including modulating immune surveillance in xenograft models of AML, are ongoing. Citation Format: Elizabeth Tsuying Chang, Brandon A. Carter-Cooper, Smaraki Dash, Dana Ferraris, Ashkan Emadi, Rena G. Lapidus. 3,3′-bis(2-aminoethanol)-2,2′-binaphthoquinone promotes killing of acute myeloid leukemia cells by augmenting oxidative stress and inhibiting indoleamine dioxygenase [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B122.

A series of diaryl ethers were designed and synthesized to discern the structure activity relationships against the two closely related mono-(ADP-ribosyl)transferases PARP10 and PARP14. Structure activity studies identified 8b as a sub-micromolar inhibitor of PARP10 with similar to 15-fold selectivity over PARP14. In addition, 8k and 8m were discovered to have sub-micromolar potency against PARP14 and demonstrated moderate selectivity over PARP10. A crystal structure of the complex of PARP14 and 8b shows binding of the compound in a novel hydrophobic pocket and explains both potency and selectivity over other PARP family members. In addition, 8b, 8k and 8m also demonstrate selectivity over PARP1. Together, this study identified novel, potent and metabolically stable derivatives to use as chemical probes for these biologically interesting therapeutic targets. (C) 2018 Elsevier Ltd. All rights reserved.