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Activin receptor-like kinases 1-7 (ALK1-7) regulate a complex network of SMAD-independent as well as SMAD-dependent signaling pathways. One of the widely used inhibitors for functional investigations of these processes, in particular for bone morphogenetic protein (BMP) signaling, is LDN-193189 . However, LDN-193189 has insufficient kinome-wide selectivity complicating its use in cellular target validation assays. Herein, we report the identification and comprehensive characterization of two chemically distinct highly selective inhibitors of ALK1 and ALK2, M4K2234 and MU1700 , along with their negative controls. We show that both MU1700 and M4K2234 efficiently block the BMP pathway via selective in cellulo inhibition of ALK1/2 kinases and exhibit favorable in vivo profiles in mice. MU1700 is highly brain penetrant and shows remarkably high accumulation in the brain. These high-quality orthogonal chemical probes offer the selectivity required to become widely used tools for in vitro and in vivo investigation of BMP signaling.

Despite decades of research on new diffuse intrinsic pontine glioma (DIPG) treatments, little or no progress has been made on improving patient outcomes. In this work, we explored novel scaffold modifications of M4K2009 , a 3,5-diphenylpyridine ALK2 inhibitor previously reported by our group. Here we disclose the design, synthesis, and evaluation of a first-in-class set of 5- to 7-membered ether-linked and 7-membered amine-linked constrained inhibitors of ALK2. This rigidification strategy led us to the discovery of the ether-linked inhibitors M4K2308 and M4K2281 and the amine-linked inhibitors M4K2304 and M4K2306 , each with superior potency against ALK2. Notably, M4K2304 and M4K2306 exhibit exceptional selectivity for ALK2 over ALK5, surpassing the reference compound. Preliminary studies on their in vivo pharmacokinetics, including blood-brain barrier penetration, revealed that these constrained scaffolds have favorable exposure and do open a novel chemical space for further optimization and future evaluation in orthotopic models of DIPG.

Drug resistance to front-line malarial treatments represents an ongoing threat to control malaria, a vector borne infectious disease. The malarial parasite, Plasmodium falciparum has developed genetic variants, conferring resistance to the current standard therapeutic artemisinin and its derivatives commonly referred to as artemisinin-combination therapies (ACTs). Emergence of multi-drug resistance parasite genotypes is a warning of potential treatment failure, reaffirming the urgent and critical need to find and validate alternate drug targets to prevent the spread of disease. An attractive and novel drug target includes glucose-regulated protein 78 kDa (GRP78, or BiP), an essential molecular chaperone protein involved in the unfolded protein response that is upregulated in ACT treated P. falciparum parasites. We have shown that both sequence and structure are closely related to human GRP78 (hGRP78), a chaperone belonging to the HSP70 class of ATPase proteins, which is often upregulated in cellular stress responses and cancer. By screening a library of nucleoside analogues, we identified eight 'hit' compounds binding at the active site of the ATP binding domain of P. falciparum GRP78 using a high-throughput ligand soaking screen using x-ray crystallography. These compounds were further evaluated using protein thermal shift assays to assess target binding activity. The nucleoside analogues identified from our screen provide a starting point for the development of more potent and selective antimalarial inhibitors. In addition, we have established a well-defined, high-throughput crystal-based screening approach that can be applied to many crystallizable P. falciparum proteins for generating anti- Plasmodium specific compounds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mrozek, Antoshchenko, Chen, Zepeda-Velázquez, Smil, Kumar, Lu and Park.)

The COVID-19 pandemic has clearly brought the healthcare systems worldwide to a breaking point, along with devastating socioeconomic consequences. The SARS-CoV-2 virus, which causes the disease, uses RNA capping to evade the human immune system. Nonstructural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small-molecule inhibitors of nsp14 methyltransferase (MTase) activity, we developed and employed a radiometric MTase assay to screen a library of 161 in-house synthesized S -adenosylmethionine (SAM) competitive MTase inhibitors and SAM analogs. Among six identified screening hits, SS148 inhibited nsp14 MTase activity with an IC 50 value of 70 ± 6 nM and was selective against 20 human protein lysine MTases, indicating significant differences in SAM binding sites. Interestingly, DS0464 with an IC 50 value of 1.1 ± 0.2 µM showed a bisubstrate competitive inhibitor mechanism of action. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein MTases. The structure-activity relationship provided by these compounds should guide the optimization of selective bisubstrate nsp14 inhibitors and may provide a path toward a novel class of antivirals against COVID-19, and possibly other coronaviruses.

Mutations in the gene encoding activin receptor-like kinase 2 (ALK2) are implicated in the pathophysiology of a pediatric brainstem cancer, diffuse intrinsic pontine glioma (DIPG). Inhibitors of ALK2 that cross the blood-brain barrier have been proposed as a method of treatment for DIPG. As part of an open science approach to radiopharmaceutical and drug discovery, we developed 11 C-labeled radiotracers from potent and selective lead ALK2 inhibitors to investigate their brain permeability through positron emission tomography (PET) neuroimaging. Four radiotracers were synthesized by 11 C-methylation and assessed by dynamic PET imaging in healthy Sprague-Dawley rats. One of the compounds, [ 11 C]M4K2127 , showed high initial brain uptake (SUV ∼ 2), including in the region of interest (pons). This data supports the use of this chemotype as a brain penetrant ALK2 inhibitor that permeates evenly into the pons with potential application for the treatment of DIPG., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

Protein arginine N-methyl transferase 4 (PRMT4) asymmetrically dimethylates the arginine residues of histone H3 and nonhistone proteins. The overexpression of PRMT4 in several cancers has stimulated interest in the discovery of inhibitors as biological tools and, potentially, therapeutics. Although several PRMT4 inhibitors have been reported, most display poor selectivity against other members of the PRMT family of methyl transferases. Herein, we report the structure-based design of a new class of alanine-containing 3-arylindoles as potent and selective PRMT4 inhibitors, and describe key structure-activity relationships for this class of compounds., (© 2021 Wiley-VCH GmbH.)

There are currently no effective chemotherapeutic drugs approved for the treatment of diffuse intrinsic pontine glioma (DIPG), an aggressive pediatric cancer resident in the pons region of the brainstem. Radiation therapy is beneficial but not curative, with the condition being uniformly fatal. Analysis of the genomic landscape surrounding DIPG has revealed that activin receptor-like kinase-2 (ALK2) constitutes a potential target for therapeutic intervention given its dysregulation in the disease. We adopted an open science approach to develop a series of potent, selective, orally bioavailable, and brain-penetrant ALK2 inhibitors based on the lead compound LDN-214117 . Modest structural changes to the C-3, C-4, and C-5 position substituents of the core pyridine ring afforded compounds M4K2009 , M4K2117 , and M4K2163 , each with a superior potency, selectivity, and/or blood-brain barrier (BBB) penetration profile. Robust in vivo pharmacokinetic (PK) properties and tolerability mark these inhibitors as advanced preclinical compounds suitable for further development and evaluation in orthotopic models of DIPG.

Diffuse intrinsic pontine glioma is an aggressive pediatric cancer for which no effective chemotherapeutic drugs exist. Analysis of the genomic landscape of this disease has led to the identification of the serine/threonine kinase ALK2 as a potential target for therapeutic intervention. In this work, we adopted an open science approach to develop a series of potent type I inhibitors of ALK2 which are orally bio-available and brain-penetrant. Initial efforts resulted in the discovery of M4K2009 , an analogue of the previously reported ALK2 inhibitor LDN-214117 . Although highly selective for ALK2 over the TGF-βR1 receptor ALK5, M4K2009 is also moderately active against the hERG potassium channel. Varying the substituents of the trimethoxyphenyl moiety gave rise to an equipotent benzamide analogue M4K2149 with reduced off-target affinity for the ion channel. Additional modifications yielded 2-fluoro-6-methoxybenzamide derivatives ( 26a-c ), which possess high inhibitory activity against ALK2, excellent selectivity, and superior pharmacokinetic profiles.

SET domain bifurcated protein 1 (SETDB1) is a human histone-lysine methyltransferase which is amplified in human cancers and was shown to be crucial in the growth of non-small and small cell lung carcinoma. In addition to its catalytic domain, SETDB1 harbors a unique tandem tudor domain which recognizes histone sequences containing both methylated and acetylated lysines, and likely contributes to its localization on chromatin. Using X-ray crystallography and NMR spectroscopy fragment screening approaches, we have identified the first small molecule fragment hits that bind to histone peptide binding groove of the Tandem Tudor Domain (TTD) of SETDB1. Herein, we describe the binding modes of these fragments and analogues and the biophysical characterization of key compounds. These confirmed small molecule fragments will inform the development of potent antagonists of SETDB1 interaction with histones., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Protein methyltransferases (PMTs) comprise a major class of epigenetic regulatory enzymes with therapeutic relevance. Here we present a collection of chemical probes and associated reagents and data to elucidate the function of human and murine PMTs in cellular studies. Our collection provides inhibitors and antagonists that together modulate most of the key regulatory methylation marks on histones H3 and H4, providing an important resource for modulating cellular epigenomes. We describe a comprehensive and comparative characterization of the probe collection with respect to their potency, selectivity, and mode of inhibition. We demonstrate the utility of this collection in CD4 + T cell differentiation assays revealing the potential of individual probes to alter multiple T cell subpopulations which may have implications for T cell-mediated processes such as inflammation and immuno-oncology. In particular, we demonstrate a role for DOT1L in limiting Th1 cell differentiation and maintaining lineage integrity. This chemical probe collection and associated data form a resource for the study of methylation-mediated signaling in epigenetics, inflammation and beyond.

PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is crucial for maturation of ribosomes and has been implicated in several diseases. We recently disclosed a highly potent, selective, and cell-active allosteric inhibitor of PRMT3, compound 4. Here, we report comprehensive structure-activity relationship studies that target the allosteric binding site of PRMT3. We conducted design, synthesis, and evaluation of novel compounds in biochemical, selectivity, and cellular assays that culminated in the discovery of 4 and other highly potent (IC 50 values: ∼10-36 nM), selective, and cell-active allosteric inhibitors of PRMT3 (compounds 29, 30, 36, and 37). In addition, we generated compounds that are very close analogs of these potent inhibitors but displayed drastically reduced potency as negative controls (compounds 49-51). These inhibitors and negative controls are valuable chemical tools for the biomedical community to further investigate biological functions and disease associations of PRMT3.

Well-characterized selective inhibitors of protein arginine methyltransferases (PRMTs) are invaluable chemical tools for testing biological and therapeutic hypotheses. Based on 4, a fragment-like inhibitor of type I PRMTs, we conducted structure-activity relationship (SAR) studies and explored three regions of this scaffold. The studies led to the discovery of a potent, selective, and cell-active dual inhibitor of PRMT4 and PRMT6, 17 (MS049). As compared to 4, 17 displayed much improved potency for PRMT4 and PRMT6 in both biochemical and cellular assays. It was selective for PRMT4 and PRMT6 over other PRMTs and a broad range of other epigenetic modifiers and nonepigenetic targets. We also developed 46 (MS049N), which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. Considering possible overlapping substrate specificity of PRMTs, 17 and 46 are valuable chemical tools for dissecting specific biological functions and dysregulation of PRMT4 and PRMT6 in health and disease.

The potent antistaphylococcal activity of N-substituted pantothenamides (PanAms) has been shown to at least partially be due to the inhibition of Staphylococcus aureus's atypical type II pantothenate kinase (SaPanK II ), the first enzyme of coenzyme A biosynthesis. This mechanism of action follows from SaPanK II having a binding mode for PanAms that is distinct from those of other PanKs. To dissect the molecular interactions responsible for PanAm inhibitory activity, we conducted a mini SAR study in tandem with the cocrystallization of SaPanK II with two classic PanAms (N5-Pan and N7-Pan), culminating in the synthesis and characterization of two new PanAms, N-Pip-PanAm and MeO-N5-PanAm. The cocrystal structures showed that all of the PanAms are phosphorylated by SaPanK II but remain bound at the active site; this occurs primarily through interactions with Tyr240' and Thr172'. Kinetic analysis showed a strong correlation between k cat (slow PanAm turnover) and IC 50 (inhibition of pantothenate phosphorylation) values, suggesting that SaPanK II inhibition occurs via a delay in product release. In-depth analysis of the PanAm-bound structures showed that the capacity for accepting a hydrogen bond from the amide of Thr172' was a stronger determinant for PanAm potency than the capacity to π-stack with Tyr240'. The two new PanAms, N-Pip-PanAm and MeO-N5-PanAm, effectively combine both hydrogen bonding and hydrophobic interactions, resulting in the most potent SaPanK II inhibition described to date. Taken together, our results are consistent with an inhibition mechanism wherein PanAms act as SaPanK II substrates that remain bound upon phosphorylation. The phospho-PanAm-SaPanK II interactions described herein may help future antistaphylococcal drug development.

Protein arginine methyltransferases (PRMTs) represent an emerging target class in oncology and other disease areas. So far, the most successful strategy to identify PRMT inhibitors has been to screen large to medium-size chemical libraries. Attempts to develop PRMT inhibitors using receptor-based computational methods have met limited success. Here, using virtual screening approaches, we identify 11 CARM1 (PRMT4) inhibitors with ligand efficiencies ranging from 0.28 to 0.84. CARM1 selective hits were further validated by orthogonal methods. Two structure-based rounds of optimization produced 27 (SGC2085), a CARM1 inhibitor with an IC50 of 50 nM and more than hundred-fold selectivity over other PRMTs. These results indicate that virtual screening strategies can be successfully applied to Rossmann-fold protein methyltransferases.

Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis.

WD repeat-containing protein 5 (WDR5) is an important component of the multiprotein complex essential for activating mixed-lineage leukemia 1 (MLL1). Rearrangement of the MLL1 gene is associated with onset and progression of acute myeloid and lymphoblastic leukemias, and targeting the WDR5-MLL1 interaction may result in new cancer therapeutics. Our previous work showed that binding of small molecule ligands to WDR5 can modulate its interaction with MLL1, suppressing MLL1 methyltransferase activity. Initial structure-activity relationship studies identified N-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides as potent and selective antagonists of this protein-protein interaction. Guided by crystal structure data and supported by in silico library design, we optimized the scaffold by varying the C-1 benzamide and C-5 substituents. This allowed us to develop the first highly potent (Kdisp < 100 nM) small molecule antagonists of the WDR5-MLL1 interaction and demonstrate that N-(4-(4-methylpiperazin-1-yl)-3'-(morpholinomethyl)-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide 16d (OICR-9429) is a potent and selective chemical probe suitable to help dissect the biological role of WDR5.

Protein methyltransferases (PMTs) are a promising target class in oncology and other disease areas. They are composed of SET domain methyltransferases and structurally unrelated Rossman-fold enzymes that include protein arginine methyltransferases (PRMTs). In the absence of a well-defined medicinal chemistry tool-kit focused on PMTs, most current inhibitors were identified by screening large and diverse libraries of leadlike molecules. So far, no successful fragment-based approach was reported against this target class. Here, by deconstructing potent PRMT inhibitors, we find that chemical moieties occupying the substrate arginine-binding site can act as efficient fragment inhibitors. Screening a fragment library against PRMT6 produced numerous hits, including a 300 nM inhibitor (ligand efficiency of 0.56) that decreased global histone 3 arginine 2 methylation in cells, and can serve as a warhead for the development of PRMT chemical probes.

Mutations in the histone 3 variant H3.3 have been identified in one-third of pediatric glioblastomas (GBMs), but not in adult tumors. Here we show that H3.3 is a dynamic determinant of functional properties in adult GBM. H3.3 is repressed by mixed lineage leukemia 5 (MLL5) in self-renewing GBM cells. MLL5 is a global epigenetic repressor that orchestrates reorganization of chromatin structure by punctuating chromosomes with foci of compacted chromatin, favoring tumorigenic and self-renewing properties. Conversely, H3.3 antagonizes self-renewal and promotes differentiation. We exploited these epigenetic states to rationally identify two small molecules that effectively curb cancer stem cell properties in a preclinical model. Our work uncovers a role for MLL5 and H3.3 in maintaining self-renewal hierarchies in adult GBM., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cytochrome P450scc (CYP 11A1) catalyzes the conversion of cholesterol (Ch) to pregnenolone, the precursor to steroid hormones. This process proceeds via three sequential monooxygenation reactions: two hydroxylations of Ch first form 22(R)-hydroxycholesterol (HC) and then 20α,22(R)-dihydroxycholesterol (DHC); a lyase reaction then cleaves the C20-C22 bond to form pregnenolone. Recent cryoreduction/annealing studies that employed electron paramagnetic resonance (EPR)/electron nuclear double resonance (ENDOR) spectroscopy [Davydov, R., et al. (2012) J. Am. Chem. Soc. 134, 17149] showed that compound I (Cpd I) is the active intermediate in the first step, hydroxylation of Ch. Herein, we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the second and third steps of the enzymatic process, as conducted by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc complexes with HC and DHC. This procedure is validated by showing that the cryoreduced ternary complexes of oxy-cytochrome P450scc with HC and DHC are catalytically competent and during annealing generate DHC and pregnenolone, respectively. Cryoreduction of the oxy-P450scc-HC ternary complex trapped at 77K produces the superoxo-ferrous P450scc intermediate along with a minor fraction of ferric hydroperoxo intermediates. The superoxo-ferrous intermediate converts into a ferric-hydroperoxo species after annealing at 145 K. During subsequent annealing at 170-180 K, the ferric-hydroperoxo intermediate converts to the primary product complex with the large solvent kinetic isotope effect that indicates Cpd I is being formed, and (1)H ENDOR measurements of the primary product formed in D2O demonstrate that Cpd I is the active species. They show that the primary product contains Fe(III) coordinated to the 20-O(1)H of DHC with the (1)H derived from substrate, the signature of the Cpd I reaction. Hydroperoxo ferric intermediates are the primary species formed during cryoreduction of the oxy-P450scc-DHC ternary complex, and they decay at 185 K with a strong solvent kinetic isotope effect to form low-spin ferric P450scc. Together, these observations indicated that Cpd I also is the active intermediate in the C20,22 lyase final step. In combination with our previous results, this study thus indicates that Cpd I is the active species in each of the three sequential monooxygenation reactions by which P450scc catalytically converts Ch to pregnenolone.

The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short (30-kDa) CCAAT-enhancer binding protein-α (C/EBPα) translational isoform, termed p30, represents the most common type of CEBPA mutation in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL (SET-domain/mixed-lineage leukemia) histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as downregulation of the latter inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a new small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30 cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML.

PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is essential for maturation of ribosomes, may have a role in lipogenesis, and is implicated in several diseases. A potent, selective, and cell-active PRMT3 inhibitor would be a valuable tool for further investigating PRMT3 biology. Here we report the discovery of the first PRMT3 chemical probe, SGC707, by structure-based optimization of the allosteric PRMT3 inhibitors we reported previously, and thorough characterization of this probe in biochemical, biophysical, and cellular assays. SGC707 is a potent PRMT3 inhibitor (IC50 =31±2 nM, KD =53±2 nM) with outstanding selectivity (selective against 31 other methyltransferases and more than 250 non-epigenetic targets). The mechanism of action studies and crystal structure of the PRMT3-SGC707 complex confirm the allosteric inhibition mode. Importantly, SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies. This well-characterized chemical probe is an excellent tool to further study the role of PRMT3 in health and disease., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The protein arginine methyltransferases PRMT7 and PRMT5, respectively, monomethylate and symmetrically dimethylate arginine side-chains of proteins involved in diverse cellular mechanisms, including chromatin-mediated control of gene transcription, splicing, and the RAS to ERK transduction cascade. It is believed that PRMT5 and PRMT7 act in conjunction to methylate their substrates, and genetic deletions support the notion that these enzymes derepress cell proliferation and migration in cancer. Using available structures of PRMT5, we designed DS-437, a PRMT5 inhibitor with an IC50 value of 6 μM against both PRMT5 and PRMT7 that is inactive against 29 other human protein-, DNA-, and RNA-methyltransferases and inhibits symmetrical dimethylation of PRMT5 substrates in cells. This compound behaves as a cofactor competitor and represents a valid scaffold to interrogate the potential of the PRMT5-PRMT7 axis as a target for therapy.

Pantothenate kinase (PanK) is the rate-limiting enzyme in Coenzyme A biosynthesis, catalyzing the ATP-dependent phosphorylation of pantothenate. We solved the co-crystal structures of PanKs from Staphylococcus aureus (SaPanK) and Klebsiella pneumonia (KpPanK) with N-[2-(1,3-benzodioxol-5-yl)ethyl] pantothenamide (N354-Pan). Two different N354-Pan conformers interact with polar/nonpolar mixed residues in SaPanK and aromatic residues in KpPanK. Additionally, phosphorylated N354-Pan is found at the closed active site of SaPanK but not at the open active site of KpPanK, suggesting an exchange of the phosphorylated product with a new N354-Pan only in KpPanK. Together, pantothenamides conformational flexibility and binding pocket are two key considerations for selective compound design., (© 2014 Wiley Periodicals, Inc.)

Unlabelled: Coronaviruses express a deubiquitinating protein, the papain-like protease-2 (PLP2), that removes both ubiquitin and the ubiquitin-like interferon (IFN)-stimulated gene 15 (ISG15) protein from target proteins. ISG15 has antiviral activity against a number of viruses; therefore, we examined the effect of ISG15 conjugation (ISGylation) in a model of acute viral hepatitis induced by the murine hepatitis virus strain 3 (MHV-3) coronavirus. Mice deficient in the ISG15 deconjugating enzyme, ubiquitin-specific peptidase-18 (USP18), accumulate high levels of ISG15-conjugated proteins and are hypersensitive to type I IFN. Infecting USP18(-/-) mice with MHV-3 resulted in extended survival (8 ± 1.2 versus 4 days) and in improved liver histology, a decreased inflammatory response, and viral titers 1 to 2 logs lower than in USP18(+/+) mice. The suppression of viral replication was not due to increased IFN since infected USP18(-/-) mice had neither increased hepatic IFN-α, -β, or -γ mRNA nor circulating protein. Instead, delayed MHV-3 replication coincided with high levels of cellular ISGylation. Decreasing ISGylation by knockdown of the ISG15 E1 enzyme, Ube1L, in primary USP18(+/+) and USP18(-/-) hepatocytes led to increased MHV-3 replication. Both in vitro and in vivo, increasing MHV-3 titers were coincident with increased PLP2 mRNA and decreased ISGylation over the course of infection. The pharmacologic inhibition of the PLP2 enzyme in vitro led to decreased MHV-3 replication. Overall, these results demonstrate the antiviral effect of ISGylation in an in vivo model of coronavirus-induced mouse hepatitis and illustrate that PLP2 manipulates the host innate immune response through the ISG15/USP18 pathway., Importance: There have been a number of serious worldwide pandemics due to widespread infections by coronavirus. This virus (in its many forms) is difficult to treat, in part because it is very good at finding "holes" in the way that the host (the infected individual) tries to control and eliminate the virus. In this study, we demonstrate that an important host viral defense-the ISG15 pathway-is only partially effective in controlling severe coronavirus infection. Activation of the pathway is very good at suppressing viral production, but over time the virus overwhelms the host response and the effects of the ISG15 pathway. These data provide insight into host-virus interactions during coronavirus infection and suggest that the ISG15 pathway is a reasonable target for controlling severe coronavirus infection although the best treatment will likely involve multiple pathways and targets.

N-Substituted pantothenamides are derivatives of pantothenate, the precursor in the biosynthesis of the essential metabolic cofactor coenzyme A (CoA). These compounds are substrates of pantothenate kinase (PanK) in the first step of CoA biosynthesis and possess antimicrobial activity against various pathogenic bacteria. Here we solved the crystal structure of the Klebsiella pneumoniae PanK (KpPanK) in complex with N-pentylpantothenamide (N5-Pan) to understand the molecular basis of its antimicrobial activity. The structure reveals a polar pocket interacting with the pantothenate moiety of N5-Pan and an aromatic pocket loosely protecting the pentyl tail, suggesting that the introduction of an aromatic ring to a new pantothenamide may enhance the compound's affinity to KpPanK. To test this idea, we synthesized N-pyridin-3-ylmethylpantothenamide (Np-Pan) and solved its co-crystal structure with KpPanK. The structure reveals two alternat conformations of the aromatic ring of Np-Pan bound at the aromatic pocket, providing the basis for further improvement of pantothenamide binding to KpPanK., (Copyright © 2013 Wiley Periodicals, Inc.)

Chemical inhibition of proteins involved in chromatin-mediated signaling is an emerging strategy to control chromatin compaction with the aim to reprogram expression networks to alter disease states. Protein methyltransferases constitute one of the protein families that participate in epigenetic control of gene expression, and represent a novel therapeutic target class. Recruitment of the protein lysine methyltransferase DOT1L at aberrant loci is a frequent mechanism driving acute lymphoid and myeloid leukemias, particularly in infants, and pharmacological inhibition of DOT1L extends survival in a mouse model of mixed lineage leukemia. A better understanding of the structural chemistry of DOT1L inhibition would accelerate the development of improved compounds. Here, we report that the addition of a single halogen atom at a critical position in the cofactor product S-adenosylhomocysteine (SAH, an inhibitor of SAM-dependent methyltransferases) results in an 8-fold increase in potency against DOT1L, and reduced activities against other protein and non-protein methyltransferases. We solved the crystal structure of DOT1L in complex with Bromo-deaza-SAH and rationalized the observed effects. This discovery reveals a simple strategy to engineer selectivity and potency towards DOT1L into the adenosine scaffold of the cofactor shared by all methyltransferases, and can be exploited towards the development of clinical candidates against mixed lineage leukemia., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Protein methyltransferases (PMTs) are a novel gene family of therapeutic relevance involved in chromatin-mediated signaling and other biological mechanisms. Most PMTs are organized around the structurally conserved SET domain that catalyzes the methylation of a substrate lysine. A few potent chemical inhibitors compete with the protein substrate, and all are anchored in the channel recruiting the methyl-accepting lysine. We propose a novel strategy to design focused chemical libraries targeting the substrate binding site, where a limited number of warheads each occupying the lysine-channel of multiple enzymes would be decorated by different substituents. A variety of sequence and structure-based approaches used to analyze the diversity of the lysine channel of SET domain PMTs support the relevance of this strategy. We show that chemical fragments derived from published inhibitors are valid warheads that can be used in the design of novel focused libraries targeting other PMTs.

Protein arginine methyltransferases (PRMTs) play an important role in diverse biological processes. Among the nine known human PRMTs, PRMT3 has been implicated in ribosomal biosynthesis via asymmetric dimethylation of the 40S ribosomal protein S2 and in cancer via interaction with the DAL-1 tumor suppressor protein. However, few selective inhibitors of PRMTs have been discovered. We recently disclosed the first selective PRMT3 inhibitor, which occupies a novel allosteric binding site and is noncompetitive with both the peptide substrate and cofactor. Here we report comprehensive structure-activity relationship studies of this series, which resulted in the discovery of multiple PRMT3 inhibitors with submicromolar potencies. An X-ray crystal structure of compound 14u in complex with PRMT3 confirmed that this inhibitor occupied the same allosteric binding site as our initial lead compound. These studies provide the first experimental evidence that potent and selective inhibitors can be created by exploiting the allosteric binding site of PRMT3.