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1.  RecO and RecR Are Necessary for RecA Loading in Response to DNA Damage and Replication Fork Stress 
Journal of Bacteriology  2014;196(15):2851-2860.
RecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examined, including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify single-stranded DNA binding protein (SSB) as an additional component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest.
PMCID: PMC4135682  PMID: 24891441
2.  Degrasyn-like Symmetrical Compounds: Possible Therapeutic Agents for Multiple Myeloma (MM-I) 
Bioorganic & medicinal chemistry  2014;22(4):1450-1458.
A series of degrasyn-like symmetrical compounds have been designed, synthesized, and screened against B cell malignancy (multiple myeloma, mantle cell lymphoma) cell lines. The lead compounds T5165804 and CP2005 showed higher nanomolar potency against these tumor cells in comparison to degrasyn and inhibited Usp9x activity in vitro and in intact cells. These observations suggest that this new class of compounds holds promise as cancer therapeutic agents
PMCID: PMC4158738  PMID: 24457091
3.  Small Molecule Deubiquitinase Inhibitors Promote Macrophage Anti-Infective Capacity 
PLoS ONE  2014;9(8):e104096.
The global spread of anti-microbial resistance requires urgent attention, and diverse alternative strategies have been suggested to address this public health concern. Host-directed immunomodulatory therapies represent one approach that could reduce selection for resistant bacterial strains. Recently, the small molecule deubiquitinase inhibitor WP1130 was reported as a potential anti-infective drug against important human food-borne pathogens, notably Listeria monocytogenes and noroviruses. Utilization of WP1130 itself is limited due to poor solubility, but given the potential of this new compound, we initiated an iterative rational design approach to synthesize new derivatives with increased solubility that retained anti-infective activity. Here, we test a small library of novel synthetic molecules based on the structure of the parent compound, WP1130, for anti-infective activity in vitro. Our studies identify a promising candidate, compound 9, which reduced intracellular growth of L. monocytogenes at concentrations that caused minimal cellular toxicity. Compound 9 itself had no bactericidal activity and only modestly slowed Listeria growth rate in liquid broth culture, suggesting that this drug acts as an anti-infective compound by modulating host-cell function. Moreover, this new compound also showed anti-infective activity against murine norovirus (MNV-1) and human norovirus, using the Norwalk virus replicon system. This small molecule inhibitor may provide a chemical platform for further development of therapeutic deubiquitinase inhibitors with broad-spectrum anti-infective activity.
PMCID: PMC4122495  PMID: 25093325
4.  Chemical Derivatives of a Small Molecule Deubiquitinase Inhibitor Have Antiviral Activity against Several RNA Viruses 
PLoS ONE  2014;9(4):e94491.
Most antiviral treatment options target the invading pathogen and unavoidably encounter loss of efficacy as the pathogen mutates to overcome replication restrictions. A good strategy for circumventing drug resistance, or for pathogens without treatment options, is to target host cell proteins that are utilized by viruses during infection. The small molecule WP1130 is a selective deubiquitinase inhibitor shown previously to successfully reduce replication of noroviruses and some other RNA viruses. In this study, we screened a library of 31 small molecule derivatives of WP1130 to identify compounds that retained the broad-spectrum antiviral activity of the parent compound in vitro but exhibited improved drug-like properties, particularly increased aqueous solubility. Seventeen compounds significantly reduced murine norovirus infection in murine macrophage RAW 264.7 cells, with four causing decreases in viral titers that were similar or slightly better than WP1130 (1.9 to 2.6 log scale). Antiviral activity was observed following pre-treatment and up to 1 hour postinfection in RAW 264.7 cells as well as in primary bone marrow-derived macrophages. Treatment of the human norovirus replicon system cell line with the same four compounds also decreased levels of Norwalk virus RNA. No significant cytotoxicity was observed at the working concentration of 5 µM for all compounds tested. In addition, the WP1130 derivatives maintained their broad-spectrum antiviral activity against other RNA viruses, Sindbis virus, LaCrosse virus, encephalomyocarditis virus, and Tulane virus. Thus, altering structural characteristics of WP1130 can maintain effective broad-spectrum antiviral activity while increasing aqueous solubility.
PMCID: PMC3983190  PMID: 24722666
5.  Lipid Storage Disorders Block Lysosomal Trafficking By Inhibiting TRP Channel and Calcium Release 
Nature Communications  2012;3:731.
Lysosomal lipid accumulation, defects in membrane trafficking, and altered Ca2+ homeostasis are common features in many lysosomal storage diseases. Mucolipin TRP channel 1 (TRPML1) is the principle Ca2+ channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca2+ release, measured using a genetically-encoded Ca2+ indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann-Pick (NP) disease cells. Sphingomyelins (SMs) are plasma membrane lipids that undergo Sphingomyelinase (SMase)-mediated hydrolysis in the lysosomes of normal cells, but accumulate distinctively in NP cell lysosomes. Patch-clamp analyses revealed that TRPML1 channel activity is inhibited by SMs, but potentiated by SMases. In NP type C (NPC) cells, increasing TRPML1’s expression/activity was sufficient to correct the trafficking defects and reduce lysosome storage and cholesterol accumulation. We propose that abnormal accumulation of luminal lipids causes secondary lysosome storage by blocking TRPML1- and Ca2+-dependent lysosomal trafficking.
PMCID: PMC3347486  PMID: 22415822
Sphingomyelin; TRP channel; Mucolipin; Ca2+ release; Membrane trafficking; Lysosome; Lysosome Storage Disease; Niemann-Pick disease
6.  Use of Enterally Delivered Angiotensin II Type Ia Receptor Antagonists to Reduce the Severity of Colitis 
Digestive diseases and sciences  2011;56(9):2553-2565.
Renin-angiotensin system blockade reduces inflammation in several organ systems. Having found a fourfold increase in angiotensin II type Ia receptor expression in a dextran sodium sulfate colitis model, we targeted blockade with angiotensin II type Ia receptor antagonists to prevent colitis development. Because hypotension is a major complication of angiotensin II type Ia receptor antagonists use, we hypothesized that use of angiotensin II type Ia receptor antagonists compounds which lack cell membrane permeability, and thus enteric absorption, would allow for direct enteral delivery at far higher concentrations than would be tolerated systemically, yet retain efficacy.
Based on the structure of the angiotensin II type Ia receptor antagonist losartan, deschloro-losartan was synthesized, which has extremely poor cell membrane permeability. Angiotensin II type Ia receptor antagonist efficacy was evaluated by determining the ability to block NF-κB activation in vitro. Dextran sodium sulfate colitis was induced in mice and angiotensin II type Ia receptor antagonist efficacy delivered transanally was assessed.
In vitro, deschloro-losartan demonstrated near equal angiotensin II type Ia receptor blockade compared to losartan as well as another angiotensin II type Ia receptor antagonist, candesartan. In the dextran sodium sulfate model, each compound significantly improved clinical and histologic scores and epithelial cell apoptosis. Abundance of TNF-α, IL-1β, and IL6 mRNA were significantly decreased with each compound. In vitro and in vivo intestinal drug absorption, as well as measures of blood pressure and mucosal and colonic blood flow, showed significantly lower uptake of deschloro-losartan compared to losartan and candesartan.
This study demonstrated efficacy of high-dose angiotensin II type Ia receptor antagonists in this colitis model. We postulate that a specially designed angiotensin II type Ia receptor antagonist with poor oral absorption may have great potential as a new therapeutic agent for inflammatory bowel disease in the future.
PMCID: PMC3163034  PMID: 21399927
Angiotensin II type Ia receptor; Dextran sodium sulfate; Colitis; Angiotensin II type Ia receptor antagonist; Nuclear factor κB
7.  Menin-MLL Inhibitors Reverse Oncogenic Activity of MLL Fusion Proteins in Leukemia 
Nature Chemical Biology  2012;8(3):277-284.
Translocations involving the Mixed Lineage Leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the MEN1 gene. Here, we present the first small molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind to menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.
PMCID: PMC3401603  PMID: 22286128
8.  Antiviral Activity of a Small Molecule Deubiquitinase Inhibitor Occurs via Induction of the Unfolded Protein Response 
PLoS Pathogens  2012;8(7):e1002783.
Ubiquitin (Ub) is a vital regulatory component in various cellular processes, including cellular responses to viral infection. As obligate intracellular pathogens, viruses have the capacity to manipulate the ubiquitin (Ub) cycle to their advantage by encoding Ub-modifying proteins including deubiquitinases (DUBs). However, how cellular DUBs modulate specific viral infections, such as norovirus, is poorly understood. To examine the role of DUBs during norovirus infection, we used WP1130, a small molecule inhibitor of a subset of cellular DUBs. Replication of murine norovirus in murine macrophages and the human norovirus Norwalk virus in a replicon system were significantly inhibited by WP1130. Chemical proteomics identified the cellular DUB USP14 as a target of WP1130 in murine macrophages, and pharmacologic inhibition or siRNA-mediated knockdown of USP14 inhibited murine norovirus infection. USP14 is a proteasome-associated DUB that also binds to inositol-requiring enzyme 1 (IRE1), a critical mediator of the unfolded protein response (UPR). WP1130 treatment of murine macrophages did not alter proteasome activity but activated the X-box binding protein-1 (XBP-1) through an IRE1-dependent mechanism. In addition, WP1130 treatment or induction of the UPR also reduced infection of other RNA viruses including encephalomyocarditis virus, Sindbis virus, and La Crosse virus but not vesicular stomatitis virus. Pharmacologic inhibition of the IRE1 endonuclease activity partially rescued the antiviral effect of WP1130. Taken together, our studies support a model whereby induction of the UPR through cellular DUB inhibition blocks specific viral infections, and suggest that cellular DUBs and the UPR represent novel targets for future development of broad spectrum antiviral therapies.
Author Summary
Deubiquitinases (DUBs) are enzymes, which are implicated in many cellular processes but their functions during virus infection are not well understood. We used WP1130, a small molecule inhibitor of a subset of DUBs, as a probe to unravel the functions of DUBs during norovirus infections. We identified USP14 as a cellular DUB target of WP1130 that is required for optimal norovirus infection. Furthermore, we demonstrated that chemical induction of the unfolded protein response can significantly inhibit viral progeny production of several RNA viruses, including noroviruses. These results suggest that chemical inhibition of cellular DUBs and/or modulation of the unfolded protein response could represent novel targets for therapy against a variety of viral pathogens.
PMCID: PMC3390402  PMID: 22792064
9.  Complementary Cell-Based High Throughput Screens Identify Novel Modulators of the Unfolded Protein Response 
Journal of Biomolecular Screening  2011;16(8):825-835.
Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than two decades indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, we hypothesized that high throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small molecule activators of the apoptotic arm of the UPR to control or kill OSCC. We have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR sub-pathways. A ~66K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of pre-fractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80μM. A series of citrinin derivatives were isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds we examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, we found that patulin at 2.5 – 10μM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34 and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.
PMCID: PMC3374590  PMID: 21844328
unfolded protein response; endoplasmic reticulum stress; cell-based assay; luciferase reporter; natural products
10.  A Small Molecule Deubiquitinase Inhibitor Increases Localization of Inducible Nitric Oxide Synthase to the Macrophage Phagosome and Enhances Bacterial Killing▿†  
Infection and Immunity  2011;79(12):4850-4857.
Macrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogen Listeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with the Listeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking.
PMCID: PMC3232648  PMID: 21911458
11.  Evolution of eukaryal tRNA-guanine transglycosylase: insight gained from the heterocyclic substrate recognition by the wild-type and mutant human and Escherichia coli tRNA-guanine transglycosylases 
Nucleic Acids Research  2010;39(7):2834-2844.
The enzyme tRNA-guanine transglycosylase (TGT) is involved in the queuosine modification of tRNAs in eukarya and eubacteria and in the archaeosine modification of tRNAs in archaea. However, the different classes of TGTs utilize different heterocyclic substrates (and tRNA in the case of archaea). Based on the X-ray structural analyses, an earlier study [Stengl et al. (2005) Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism. Chembiochem, 6, 1926–1939] has made a compelling case for the divergent evolution of the eubacterial and archaeal TGTs. The X-ray structure of the eukaryal class of TGTs is not known. We performed sequence homology and phylogenetic analyses, and carried out enzyme kinetics studies with the wild-type and mutant TGTs from Escherichia coli and human using various heterocyclic substrates that we synthesized. Observations with the Cys145Val (E. coli) and the corresponding Val161Cys (human) TGTs are consistent with the idea that the Cys145 evolved in eubacterial TGTs to recognize preQ1 but not queuine, whereas the eukaryal equivalent, Val161, evolved for increased recognition of queuine and a concomitantly decreased recognition of preQ1. Both the phylogenetic and kinetic analyses support the conclusion that all TGTs have divergently evolved to specifically recognize their cognate heterocyclic substrates.
PMCID: PMC3074131  PMID: 21131277

Results 1-11 (11)