Publications by authors named "Ruben Abagyan"

246 Publications

Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis.

Commun Biol 2021 Mar 16;4(1):340. Epub 2021 Mar 16.

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA.

Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This technique describes and compares the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways.
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http://dx.doi.org/10.1038/s42003-021-01838-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966745PMC
March 2021

Concomitant drugs associated with increased mortality for MDMA users reported in a drug safety surveillance database.

Sci Rep 2021 Mar 16;11(1):5997. Epub 2021 Mar 16.

College of Pharmacy, Touro University California, Vallejo, CA, USA.

3,4-Methylenedioxymethamphetamine (MDMA) is currently being evaluated by the Food and Drug Administration (FDA) for the treatment of post-traumatic stress disorder (PTSD). If MDMA is FDA-approved it will be important to understand what medications may pose a risk of drug-drug interactions. The goal of this study was to evaluate the risks due to MDMA ingestion alone or in combination with other common medications and drugs of abuse using the FDA drug safety surveillance data. To date, nearly one thousand reports of MDMA use have been reported to the FDA. The majority of these reports include covariates such as co-ingested substances and demographic parameters. Univariate and multivariate logistic regression was employed to uncover the contributing factors to the reported risk of death among MDMA users. Several drug classes (MDMA metabolites or analogs, anesthetics, muscle relaxants, amphetamines and stimulants, benzodiazepines, ethanol, opioids), four antidepressants (bupropion, sertraline, venlafaxine and citalopram) and olanzapine demonstrated increased odds ratios for the reported risk of death. Future drug-drug interaction clinical trials should evaluate if any of the other drug-drug interactions described in our results actually pose a risk of morbidity or mortality in controlled medical settings.
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http://dx.doi.org/10.1038/s41598-021-85389-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966744PMC
March 2021

Structure based design and synthesis of novel Toll-like Receptor 2 (TLR 2) lipid antagonists.

Bioorg Med Chem Lett 2021 Feb 23;40:127861. Epub 2021 Feb 23.

Neuropore Therapies Inc., 10835 Road to the Cure, Suite 230, San Diego, CA 92121, USA. Electronic address:

Toll-like receptors (TLRs) play key role in innate immune response to Damage Associated Molecular Patterns (DAMPs) and Pathogen Associated Molecular Patterns (PAMPs). DAMP/PAMP-mediated activation of TLRs triggers NFκB signaling resulting in pro-inflammatory cytokine release. Using TLR2-Pam2CSK4 agonist co-crystal structure information, we designed and synthesized a novel series of Toll-like Receptor 2 (TLR2) lipid antagonists and identified compounds 14, 15 and 17 with sub-micromolar potency. TLR2 antagonists that we identified are stable for > 1.0 h in both gastric juice and PBS buffer and could be used as research tools.
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http://dx.doi.org/10.1016/j.bmcl.2021.127861DOI Listing
February 2021

Antineoplastic kinase inhibitors: A new class of potent anti-amoebic compounds.

PLoS Negl Trop Dis 2021 Feb 8;15(2):e0008425. Epub 2021 Feb 8.

Center for Discovery and Innovation in Parasitic Diseases, Skaggs School for Pharmacy and Pharmaceutical Sciences, University of California-San Diego, La Jolla, California, United States of America.

Entamoeba histolytica is a protozoan parasite which infects approximately 50 million people worldwide, resulting in an estimated 70,000 deaths every year. Since the 1960s E. histolytica infection has been successfully treated with metronidazole. However, drawbacks to metronidazole therapy exist, including adverse effects, a long treatment course, and the need for an additional drug to prevent cyst-mediated transmission. E. histolytica possesses a kinome with approximately 300-400 members, some of which have been previously studied as potential targets for the development of amoebicidal drug candidates. However, while these efforts have uncovered novel potent inhibitors of E. histolytica kinases, none have resulted in approved drugs. In this study we took the alternative approach of testing a set of twelve previously FDA-approved antineoplastic kinase inhibitors against E. histolytica trophozoites in vitro. This resulted in the identification of dasatinib, bosutinib, and ibrutinib as amoebicidal agents at low-micromolar concentrations. Next, we utilized a recently developed computational tool to identify twelve additional drugs with human protein target profiles similar to the three initial hits. Testing of these additional twelve drugs led to the identification of ponatinib, neratinib, and olmutinib were identified as highly potent, with EC50 values in the sub-micromolar range. All of these six drugs were found to kill E. histolytica trophozoites as rapidly as metronidazole. Furthermore, ibrutinib was found to kill the transmissible cyst stage of the model organism E. invadens. Ibrutinib thus possesses both amoebicidal and cysticidal properties, in contrast to all drugs used in the current therapeutic strategy. These findings together reveal antineoplastic kinase inhibitors as a highly promising class of potent drugs against this widespread and devastating disease.
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http://dx.doi.org/10.1371/journal.pntd.0008425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895358PMC
February 2021

Mtor inhibitors associated with higher cardiovascular adverse events-A large population database analysis.

Clin Transplant 2021 Apr 2;35(4):e14228. Epub 2021 Feb 2.

Skaggs School of Pharmacy and Pharmaceutical Sciences, La Jolla, CA, USA.

There are limited real-world data available regarding adverse events (AEs) of immunosuppressants. We utilized the FDA Adverse Event Reporting System (FAERS) database from 2004 to 2018 to perform a retrospective database analysis. We analyzed AE reports due to the individual agents tacrolimus, sirolimus, or everolimus and compared reporting odds ratios of the mTOR inhibitors to tacrolimus. The mTOR inhibitors arm had 1282 reports with 4176 AEs, while the tacrolimus arm had a total of 7587 reports with 20 940 individual AEs. mTOR inhibitors had significantly higher incidences of cardiovascular (ROR 1.95, 95% CI 1.70, 2.23), dermatologic (ROR 1.34, 95% CI 1.04, 1.73), endocrine (ROR 1.52, 95% CI 1.26, 1.82), gastrointestinal (ROR 1.15, 95% CI 1.01, 1.30), infectious disease (ROR 1.35, 95% 1.20, 1.52), musculoskeletal (ROR 1.39, 95% CI 1.13, 1.70), pulmonary (ROR 3.46, 95% 2.97, 4.03), renal (ROR 1.27, 95% CI 1.10, 1.46), and vascular AEs (ROR 3.10, 95% CI 2.14, 4.49). Across every organ type, mTOR inhibitors had greater cardiovascular AEs compared to tacrolimus, specifically in arteriosclerosis, heart failure, hypotension, tachycardia, chest pain, edema, and pericardial disorders. mTOR inhibitors may be associated with higher cardiovascular AEs. Further investigation is required to determine the potential mechanism of this effect.
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http://dx.doi.org/10.1111/ctr.14228DOI Listing
April 2021

A receptor-like protein mediates plant immune responses to herbivore-associated molecular patterns.

Proc Natl Acad Sci U S A 2020 12 23;117(49):31510-31518. Epub 2020 Nov 23.

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093;

Herbivory is fundamental to the regulation of both global food webs and the extent of agricultural crop losses. Induced plant responses to herbivores promote resistance and often involve the perception of specific herbivore-associated molecular patterns (HAMPs); however, precisely defined receptors and elicitors associated with herbivore recognition remain elusive. Here, we show that a receptor confers signaling and defense outputs in response to a defined HAMP common in caterpillar oral secretions (OS). Staple food crops, including cowpea () and common bean (), specifically respond to OS via recognition of proteolytic fragments of chloroplastic ATP synthase, termed inceptins. Using forward-genetic mapping of inceptin-induced plant responses, we identified a corresponding leucine-rich repeat receptor, termed INR, specific to select legume species and sufficient to confer inceptin-induced responses and enhanced defense against armyworms () in tobacco. Our results support the role of plant immune receptors in the perception of chewing herbivores and defense.
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http://dx.doi.org/10.1073/pnas.2018415117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733821PMC
December 2020

Cardiac adverse events associated with chloroquine and hydroxychloroquine exposure in 20 years of drug safety surveillance reports.

Sci Rep 2020 11 5;10(1):19199. Epub 2020 Nov 5.

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA.

Chloroquine (CQ) and hydroxychloroquine (HCQ) are on the World Health Organization's List of Essential Medications for treating non-resistant malaria, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). In addition, both drugs are currently used off-label in hospitals worldwide and in numerous clinical trials for the treatment of SARS-CoV-2 infection. However, CQ and HCQ use has been associated with cardiac side effects, which is of concern due to the higher risk of COVID-19 complications in patients with heart related disorders, and increased mortality associated with COVID-19 cardiac complications. In this study we analyzed over thirteen million adverse event reports form the United States Food and Drug Administration Adverse Event Reporting System to confirm and quantify the association of cardiac side effects of CQ and HCQ. Additionally, we identified several confounding factors, including male sex, NSAID coadministration, advanced age, and prior diagnoses contributing to drug related cardiotoxicity. These findings may help guide therapeutic decision making and ethical trial design for COVID-19 treatment.
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http://dx.doi.org/10.1038/s41598-020-76258-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644696PMC
November 2020

Differential activities of maize plant elicitor peptides as mediators of immune signaling and herbivore resistance.

Plant J 2020 12 2;104(6):1582-1602. Epub 2020 Dec 2.

Division of Biology, University of California San Diego, La Jolla, CA, USA.

Plant elicitor peptides (Peps) are conserved regulators of defense responses and models for the study of damage-associated molecular pattern-induced immunity. Although present as multigene families in most species, the functional relevance of these multigene families remains largely undefined. While Arabidopsis Peps appear largely redundant in function, previous work examining Pep-induced responses in maize (Zm) implied specificity of function. To better define the function of individual ZmPeps and their cognate receptors (ZmPEPRs), activities were examined by assessing changes in defense-associated phytohormones, specialized metabolites and global gene expression patterns, in combination with heterologous expression assays and analyses of CRISPR/Cas9-generated knockout plants. Beyond simply delineating individual ZmPep and ZmPEPR activities, these experiments led to a number of new insights into Pep signaling mechanisms. ZmPROPEP and other poaceous precursors were found to contain multiple active Peps, a phenomenon not previously observed for this family. In all, seven new ZmPeps were identified and the peptides were found to have specific activities defined by the relative magnitude of their response output rather than by uniqueness. A striking correlation was observed between individual ZmPep-elicited changes in levels of jasmonic acid and ethylene and the magnitude of induced defense responses, indicating that ZmPeps may collectively regulate immune output through rheostat-like tuning of phytohormone levels. Peptide structure-function studies and ligand-receptor modeling revealed structural features critical to the function of ZmPeps and led to the identification of ZmPep5a as a potential antagonist peptide able to competitively inhibit the activity of other ZmPeps, a regulatory mechanism not previously observed for this family.
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http://dx.doi.org/10.1111/tpj.15022DOI Listing
December 2020

Biomimetic microbioreactor-supramolecular nanovesicles improve enzyme therapy of hepatic cancer.

Nanomedicine 2021 01 1;31:102311. Epub 2020 Oct 1.

Chongqing Research Center for Pharmaceutical Engineering, Chongqing Medical University, Chongqing, China. Electronic address:

A novel biomimetic nanovesicle-loaded supramolecular enzyme-based therapeutics has been developed. Here, using a biomimetic lipid-D-α-tocopherol polyethylene glycol succinate (TPGS) hybrid semi-permeable membrane, cyclodextrin supramolecular docking, metal-ion-aided coordination complexing, we combined multiple functional motifs into a single biomimetic microbioreactor-supramolecular nanovesicle (MiSuNv) that allowed effective transport of arginine deiminase (ADI) to hepatic tumor cells to enhance arginine depletion. We compared two intercalated enzyme-carrying supermolecular motifs mainly comprising of 2-hydroxypropyl-β-cyclodextrin and sulfobutyl-ether-β-cyclodextrin, the only two cyclodextrin derivatives approved for injection by the United States Food and Drug Administration. The ADI-specific antitumor effects were enhanced by TPGS (one constituent of MiSuNv, having synergistic antitumor effects), as ADI was separated from adverse external environment by a semi-permeable membrane and sequestered in a favorable internal microenvironment with an optimal pH and metal-ion combination. ADI@MiSuNv contributed to cell cycle arrest, apoptosis and autophagy through the enhanced efficacy of enzyme treatment against Hep3B xenograft tumors in rats.
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http://dx.doi.org/10.1016/j.nano.2020.102311DOI Listing
January 2021

HMG-CoA Reductase Inhibitors as Drug Leads against .

ACS Chem Neurosci 2020 10 19;11(19):3089-3096. Epub 2020 Sep 19.

Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093-0756, United States.

Primary amebic meningoencephalitis (PAM), caused by the free-living ameba , has a fatality rate of over 97%. Treatment of PAM relies on amphotericin B in combination with other drugs, but few patients have survived with the existing drug treatment regimens. Therefore, development of effective drugs is a critical unmet need to avert deaths from PAM. Since ergosterol is one of the major sterols in the membrane of , disruption of isoprenoid and sterol biosynthesis by small-molecule inhibitors may be an effective intervention strategy against . The genome of contains a gene encoding HMG-CoA reductase (HMGR); the catalytic domains of human and HMGR share <60% sequence identity with only two amino acid substitutions in the active site of the enzyme. Considering the similarity of human and HMGR, we tested well-tolerated and widely used HMGR inhibitors, known as cholesterol-lowering statins, against We identified blood-brain-barrier-permeable pitavastatin as a potent amebicidal agent against the U.S., Australian, and European strains of . Pitavastatin was equipotent to amphotericin B against the European strain of ; it killed about 80% of trophozoites within 16 h of drug exposure. Pretreatment of trophozoites with mevalonate, the product of HMGR, rescued from inhibitory effects of statins, demonstrating that HMGR of is the target of statins. Because of the good safety profile and availability for both adult and pediatric uses, consideration should be given to repurposing the fast-acting pitavastatin for the treatment of PAM.
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http://dx.doi.org/10.1021/acschemneuro.0c00428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544672PMC
October 2020

Synthesis, Optimization, Antifungal Activity, Selectivity, and CYP51 Binding of New 2-Aryl-3-azolyl-1-indolyl-propan-2-ols.

Pharmaceuticals (Basel) 2020 Aug 8;13(8). Epub 2020 Aug 8.

EA1155-IICiMed, Institut de Recherche en Santé 2, Département de Pharmacochimie, Nantes Atlantique Universités, Université de Nantes, F-44200 Nantes, France.

A series of 2-aryl-3-azolyl-1-indolyl-propan-2-ols was designed as new analogs of fluconazole (FLC) by replacing one of its two triazole moieties by an indole scaffold. Two different chemical approaches were then developed. The first one, in seven steps, involved the synthesis of the key intermediate 1-(1-benzotriazol-1-yl)methyl-1-indole and the final opening of oxiranes by imidazole or 1-1,2,4-triazole. The second route allowed access to the target compounds in only three steps, this time with the ring opening by indole and analogs. Twenty azole derivatives were tested against and other species. The enantiomers of the best anti- compound, 2-(2,4-dichlorophenyl)-3-(1-indol-1-yl)-1-(1-1,2,4-triazol-1-yl)-propan-2-ol (), were analyzed by X-ray diffraction to determine their absolute configuration. The (-)- enantiomer (Minimum inhibitory concentration (MIC) = IC = 0.000256 µg/mL on CA98001) was found with the -absolute configuration. In contrast the ()- enantiomer was found with the -absolute configuration (MIC = 0.023 µg/mL on CA98001). By comparison, the MIC value for FLC was determined as 0.020 µg/mL for the same clinical isolate. Additionally, molecular docking calculations and molecular dynamics simulations were carried out using a crystal structure of lanosterol 14α-demethylase (CaCYP51). The (-)-()- enantiomer aligned with the positioning of posaconazole within both the heme and access channel binding sites, which was consistent with its biological results. All target compounds have been also studied against human fetal lung fibroblast (MRC-5) cells. Finally, the selectivity of four compounds on a panel of human P450-dependent enzymes (CYP19, CYP17, CYP26A1, CYP11B1, and CYP11B2) was investigated.
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http://dx.doi.org/10.3390/ph13080186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464559PMC
August 2020

Postmarketing safety surveillance data reveals antidepressant effects of botulinum toxin across various indications and injection sites.

Sci Rep 2020 07 30;10(1):12851. Epub 2020 Jul 30.

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA.

The World Health Organization estimates the number of people suffering from depression to be over 264 million. Current monoamine transmission modulating therapeutics, even with proper adherence and acceptable tolerability, are not effective for nearly one third of the patients, leading clinicians to explore other therapeutic options such as electroconvulsive therapy, transcranial magnetic stimulation, ketamine infusions, and, more recently, glabellar botulinum toxin, BoNT, injections. The scale and mechanism of antidepressant action of BoNT is unclear and maybe hypothetically attributed to the disruption of proprioceptive facial feedback reinforcing negative emotions. Here we verify the antidepressant effect of botulinum toxin by analysis of over 40 thousand BoNT treatment reports out of thirteen million postmarketing safety reports in the FDA Adverse Event Reporting System, FAERS. The results of the analysis indicate that patients who received BoNT injections to treat hyperhidrosis, facial wrinkles, migraine prophylaxis, spasticity, and spasms, had a significantly lower number of depression reports when compared to patients undergoing different treatments for the same conditions. These findings suggest that the antidepressant effect of BoNT is significant, and, surprisingly, is observed for a broad range of injection sites.
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http://dx.doi.org/10.1038/s41598-020-69773-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393507PMC
July 2020

Biomimetic polysaccharide-cloaked lipidic nanovesicles/microassemblies for improving the enzymatic activity and prolonging the action time for hyperuricemia treatment.

Nanoscale 2020 Jul;12(28):15222-15235

Chongqing Research Center for Pharmaceutical Engineering, Chongqing Medical University, Chongqing 400016, China.

The improvement and maintenance of enzymatic activities represent major challenges. However, to address these we developed novel biomimetic polysaccharide hyaluronan (Hn)-cloaked lipidic nanovesicles (BHLN) and microassemblies (BHLNM) as enzyme carriers that function by entrapping enzymes in the core or by tethering them to the inner/outer surfaces via covalent interactions. The effectiveness of these enzyme carriers was demonstrated through an evaluation of the enzymatic activity and anti-hyperuricemia bioactivity of urate oxidase (also called uricase, Uase). We showed that Uase was effectively loaded within the BHLN/BHLNM (UHLN/UHLNM) and maintained good enzymatic bioactivity through a range of effects, including isolation from the external environment due to the vesicle-carrying (shielding effect), avoidance of recognition by the reticuloendothelial system due to Hn-cloaking (long-term effect), production of beneficial conformational changes (allosteric effect) due to a favorable internal microenvironment of construction and vesicle loading, and stabilization due to the reversible conjugation of Uase or vesicle and serum albumin (deposit effect). UHLN/UHLNM had significantly increased bioavailability (∼533% and ∼331% compared to Uase) and demonstrated greatly improved efficacy, whereby the time required for UHLN/UHLNM to lower the plasma uric acid concentration to a normal level was much shorter than that for free Uase. The interactions of the therapeutic enzyme (Uase), biomimetic membrane components (Hn and phospholipid), and serum albumin were investigated with a fluorescent probe and computational simulations to help understand the superior properties of UHLN/UHLNM.
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http://dx.doi.org/10.1039/d0nr02651dDOI Listing
July 2020

Biomimetic Membrane-Structured Nanovesicles Carrying a Supramolecular Enzyme to Cure Lung Cancer.

ACS Appl Mater Interfaces 2020 Jul 29;12(28):31112-31123. Epub 2020 Jun 29.

Department of Thoracic Surgery, Daping Hospital of Army Medical University, PLA, Chongqing 400042, China.

Platforms for enzyme delivery must simultaneously have plasma stability, high catalytic activity, and low/no immunogenicity of the enzyme. Here, we designed a novel biomimetic membrane-structured nanovesicle (BNV) to efficiently carry supramolecular enzymes to meet the above requirements. We complexed l-asparaginase (Aase) with hydroxypropyl-β-cyclodextrin (HPCD) to form a supramolecular amphiphile (AS) by self-assembly via noncovalent reversible interactions. We then used the first synthesized polyethylene glycol (PEG 2 kDa)-decorated hyaluronan (12 kDa) and HPCD to self-assemble a semipermeable biomimetic membrane-structured nanovesicle (BNV) together with AS loading. As compared to native Aase, AS@BNV exhibited superior catalytic activity preservation, improved catalytic activity, better pharmacokinetics in rats, enhanced cytotoxic effects, increased antitumor efficacy, and decreased side effects. The underlying mechanisms, such as the autophagy inhibition action against tumor cells, protein-protein docking of the interaction between Aase-serum albumin, and decreased hepatic enzymatic activity, were investigated. This approach paves the way for new types of powerful biomimetic-, supramolecular-, and nanocarrier-based enzymatic therapies.
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http://dx.doi.org/10.1021/acsami.0c06207DOI Listing
July 2020

Cytomembrane-mimicking nanocarriers with a scaffold consisting of a CD44-targeted endogenous component for effective asparaginase supramolecule delivery.

Nanoscale 2020 Jun;12(22):12083-12097

Chongqing Research Center for Pharmaceutical Engineering, Chongqing Medical University, Chongqing 400016, China.

Highly effective and safe delivery of therapeutic enzymes is pivotal to the success of antitumor therapy. Herein, we report on a targeted enzyme delivery system based on cytomembrane-mimicking nanocarriers (CmN) and a supramolecular technique (SmT). Specifically, each CmN had a scaffold that mainly consisted of a CD44-targeted endogenous component conjugated with polyethylene glycol 2000 (HA-g-PEG) that self-assembled with α-cyclodextrin (ACD). The CmN acted as a microbioreactor with an inner hollow space with the capacity to confine the large molecule asparaginase (Asp) in an Asp/ACD-supramolecular complex conjugated to the inner region. The supramolecular Asp loaded into the CmN (A-S-CmN) exhibited superior stability, kinetic properties, catalytic activity and antitumor effects compared to free Asp due to the dual protection of the supramolecular complex and the nanovesicle, the CD44 targeting-homing ability, the prolonged effects of HA-g-PEG, and the favorable inner microenvironment of the constructed supramolecular CmN. The A-S-CmN also showed a decrease in in vivo toxicity and immunogenicity. CmN combined with SmT therapeutics are easy to implement and extend for use in the delivery of various enzymes and for many types of cancer treatment.
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http://dx.doi.org/10.1039/d0nr02588gDOI Listing
June 2020

How can proton pump inhibitors damage central and peripheral nervous systems?

Neural Regen Res 2020 Nov;15(11):2041-2042

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA.

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http://dx.doi.org/10.4103/1673-5374.282252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716020PMC
November 2020

Crosslinking-guided geometry of a complete CXC receptor-chemokine complex and the basis of chemokine subfamily selectivity.

PLoS Biol 2020 04 9;18(4):e3000656. Epub 2020 Apr 9.

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.

Chemokines and their receptors are orchestrators of cell migration in humans. Because dysregulation of the receptor-chemokine system leads to inflammation and cancer, both chemokines and receptors are highly sought therapeutic targets. Yet one of the barriers for their therapeutic targeting is the limited understanding of the structural principles behind receptor-chemokine recognition and selectivity. The existing structures do not include CXC subfamily complexes and lack information about the receptor distal N-termini, despite the importance of the latter in signaling, regulation, and bias. Here, we report the discovery of the geometry of the complex between full-length CXCR4, a prototypical CXC receptor and driver of cancer metastasis, and its endogenous ligand CXCL12. By comprehensive disulfide cross-linking, we establish the existence and the structure of a novel interface between the CXCR4 distal N-terminus and CXCL12 β1-strand, while also recapitulating earlier findings from nuclear magnetic resonance, modeling and crystallography of homologous receptors. A cross-linking-informed high-resolution model of the CXCR4-CXCL12 complex pinpoints the interaction determinants and reveals the occupancy of the receptor major subpocket by the CXCL12 proximal N terminus. This newly found positioning of the chemokine proximal N-terminus provides a structural explanation of CXC receptor-chemokine selectivity against other subfamilies. Our findings challenge the traditional two-site understanding of receptor-chemokine recognition, suggest the possibility of new affinity and signaling determinants, and fill a critical void on the structural map of an important class of therapeutic targets. These results will aid the rational design of selective chemokine-receptor targeting small molecules and biologics with novel pharmacology.
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http://dx.doi.org/10.1371/journal.pbio.3000656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173943PMC
April 2020

Systems Biology Analysis Reveals Eight SLC22 Transporter Subgroups, Including OATs, OCTs, and OCTNs.

Int J Mol Sci 2020 Mar 5;21(5). Epub 2020 Mar 5.

Department of Medicine, University of California San Diego, San Diego, CA 92093, USA.

The SLC22 family of OATs, OCTs, and OCTNs is emerging as a central hub of endogenous physiology. Despite often being referred to as "drug" transporters, they facilitate the movement of metabolites and key signaling molecules. An in-depth reanalysis supports a reassignment of these proteins into eight functional subgroups, with four new subgroups arising from the previously defined OAT subclade: OATS1 (SLC22A6, SLC22A8, and SLC22A20), OATS2 (SLC22A7), OATS3 (SLC22A11, SLC22A12, and Slc22a22), and OATS4 (SLC22A9, SLC22A10, SLC22A24, and SLC22A25). We propose merging the OCTN (SLC22A4, SLC22A5, and Slc22a21) and OCT-related (SLC22A15 and SLC22A16) subclades into the OCTN/OCTN-related subgroup. Using data from GWAS, in vivo models, and in vitro assays, we developed an SLC22 transporter-metabolite network and similar subgroup networks, which suggest how multiple SLC22 transporters with mono-, oligo-, and multi-specific substrate specificity interact to regulate metabolites. Subgroup associations include: OATS1 with signaling molecules, uremic toxins, and odorants, OATS2 with cyclic nucleotides, OATS3 with uric acid, OATS4 with conjugated sex hormones, particularly etiocholanolone glucuronide, OCT with neurotransmitters, and OCTN/OCTN-related with ergothioneine and carnitine derivatives. Our data suggest that the SLC22 family can work among itself, as well as with other ADME genes, to optimize levels of numerous metabolites and signaling molecules, involved in organ crosstalk and inter-organismal communication, as proposed by the remote sensing and signaling theory.
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http://dx.doi.org/10.3390/ijms21051791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084758PMC
March 2020

Druggable exosites of the human kino-pocketome.

J Comput Aided Mol Des 2020 03 10;34(3):219-230. Epub 2020 Jan 10.

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, USA.

Small molecules binding at any of the multiple regulatory sites on the molecular surface of a protein kinase may stabilize or disrupt the corresponding interaction, leading to consequent modulation of the kinase cellular activity. As such, each of these sites represents a potential drug target. Even targeting sites outside the immediate ATP site, the so-called exosites, may cause desirable biological effects through an allosteric mechanism. Targeting exosites can alleviate adverse effects and toxicity that is common when ATP-site compounds bind promiscuously to many other types of kinases. In this study we have identified, catalogued, and annotated all potentially druggable exosites on the protein kinase domains within the existing structural human kinome. We then priority-ranked these exosites by those most amenable to drug design. In order to identify pockets that are either consistent across the kinome, or unique and specific to a particular structure, we have also implemented a normalized representation of all pockets, and displayed these graphically. Finally, we have built a database and designed a web-based interface for users interested in accessing the 3-dimensional representations of these pockets. We envision this information will assist drug discovery efforts searching for untargeted binding pockets in the human kinome.
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http://dx.doi.org/10.1007/s10822-019-00276-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082431PMC
March 2020

Unique metabolite preferences of the drug transporters OAT1 and OAT3 analyzed by machine learning.

J Biol Chem 2020 02 2;295(7):1829-1842. Epub 2020 Jan 2.

Department of Pediatrics, University of California San Diego, La Jolla, California 92093-0693; Department of Medicine, University of California San Diego, La Jolla, California 92093-0693. Electronic address:

The multispecific organic anion transporters, OAT1 (SLC22A6) and OAT3 (SLC22A8), the main kidney elimination pathways for many common drugs, are often considered to have largely-redundant roles. However, whereas examination of metabolomics data from -knockout mice ( and ) revealed considerable overlap, over a hundred metabolites were increased in the plasma of one or the other of these knockout mice. Many of these relatively unique metabolites are components of distinct biochemical and signaling pathways, including those involving amino acids, lipids, bile acids, and uremic toxins. Cheminformatics, together with a "logical" statistical and machine learning-based approach, identified a number of molecular features distinguishing these unique endogenous substrates. Compared with OAT1, OAT3 tends to interact with more complex substrates possessing more rings and chiral centers. An independent "brute force" approach, analyzing all possible combinations of molecular features, supported the logical approach. Together, the results suggest the potential molecular basis by which OAT1 and OAT3 modulate distinct metabolic and signaling pathways As suggested by the Remote Sensing and Signaling Theory, the analysis provides a potential mechanism by which "multispecific" kidney proximal tubule transporters exert distinct physiological effects. Furthermore, a strong metabolite-based machine-learning classifier was able to successfully predict unique OAT1 OAT3 drugs; this suggests the feasibility of drug design based on knockout metabolomics of drug transporters. The approach can be applied to other SLC and ATP-binding cassette drug transporters to define their nonredundant physiological roles and for analyzing the potential impact of drug-metabolite interactions.
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http://dx.doi.org/10.1074/jbc.RA119.010729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029110PMC
February 2020

Transcriptome and binding data indicate that citral inhibits single strand DNA-binding proteins.

Physiol Plant 2020 May 24;169(1):99-109. Epub 2020 Feb 24.

USDA, ARS, Natural Products Utilization Research Unit, Oxford, MS, 38677, USA.

The mechanism of phytotoxicity of citral was probed in Arabidopsis thaliana using RNA-Seq and in silico binding analyses. Inhibition of growth by 50% by citral downregulated transcription of 9156 and 5541 genes in roots and shoots, respectively, after 1 h. Only 56 and 62 genes in roots and shoots, respectively, were upregulated. In the shoots, the downregulation increased at 3 h (6239 genes downregulated, vs 66 upregulated). Of all genes affected in roots at 1 h (time of greatest effect), 7.69% of affected genes were for nucleic acid binding functions. Genes for single strand DNA binding proteins (SSBP) WHY1, WHY 2 and WHY3 were strongly downregulated in the shoot up until 12 h after citral exposure. Effects were strong in the root at just 1 h after the treatment and then at 12 and 24 h. Similar effects occurred with the transcription factors MYC-2, ANAC and SCR-SHR, which were also significantly downregulated for the first hour of treatment, and downregulation occurred again after 12 and 24 h treatment. Downregulation of ANAC in the first hour of treatment was significantly (P < 0.0001) decreased more than eight times compared to the control. In silico molecular docking analysis suggests binding of citral isomers to the SSBPs WHY1, WHY2, and WHY3, as well as with other transcription factors such as MYC-2, ANAC and SCR-SHR. Such effects could account for the profound and unusual effects of citral on downregulation of gene transcription.
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http://dx.doi.org/10.1111/ppl.13055DOI Listing
May 2020

Proton-pump inhibitor use is associated with a broad spectrum of neurological adverse events including impaired hearing, vision, and memory.

Sci Rep 2019 11 21;9(1):17280. Epub 2019 Nov 21.

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA.

Proton-pump inhibitors, PPIs, are considered effective therapy for stomach acid suppression due to their irreversible inhibition of the hydrogen/potassium pump in the gastric parietal cells. They are widely prescribed and are considered safe for over-the-counter use. Recent studies have shown an association between PPI use and Alzheimer dementia, while others have disputed that connection. We analyzed over ten million United States Food and Drug Administration Adverse Event Reporting System reports, including over forty thousand reports containing PPIs, and provided evidence of increased propensity for memory impairment among PPI reports when compared to histamine-2 receptor antagonist control group. Furthermore, we found significant associations of PPI use with a wide range of neurological adverse reactions including, migraine, several peripheral neuropathies, and visual and auditory neurosensory abnormalities.
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http://dx.doi.org/10.1038/s41598-019-53622-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6872761PMC
November 2019

Discovery of holoenzyme-disrupting chemicals as substrate-selective CK2 inhibitors.

Sci Rep 2019 11 4;9(1):15893. Epub 2019 Nov 4.

University of California, San Diego, Skaggs School of Pharmacy and Pharmaceutical Sciences, La Jolla, CA, 92093, USA.

CK2 is a constitutively active protein kinase overexpressed in numerous malignancies. Interaction between CK2α and CK2β subunits is essential for substrate selectivity. The CK2α/CK2β interface has been previously targeted by peptides to achieve functional effects; however, no small molecules modulators were identified due to pocket flexibility and open shape. Here we generated numerous plausible conformations of the interface using the fumigation modeling protocol, and virtually screened a compound library to discover compound 1 that suppressed CK2α/CK2β interaction in vitro and inhibited CK2 in a substrate-selective manner. Orthogonal SPR, crystallography, and NMR experiments demonstrated that 4 and 6, improved analogs of 1, bind to CK2α as predicted. Both inhibitors alter CK2 activity in cells through inhibition of CK2 holoenzyme formation. Treatment with 6 suppressed MDA-MB231 triple negative breast cancer cell growth and induced apoptosis. Altogether, our findings exemplify an innovative computational-experimental approach and identify novel non-peptidic inhibitors of CK2 subunit interface disclosing substrate-selective functional effects.
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http://dx.doi.org/10.1038/s41598-019-52141-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828666PMC
November 2019

Discovery of New Inhibitors of Hepatitis C Virus NS3/4A Protease and Its D168A Mutant.

ACS Omega 2019 Oct 2;4(16):16999-17008. Epub 2019 Oct 2.

Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Chemistry and Biochemistry, and School of Medicine, University of California San Diego, La Jolla, California 92093, United States.

Hepatitis C virus (HCV) is a human pathogen with high morbidity. The HCV NS3/4A protease is essential for viral replication and is one of the top three drug targets. Several drugs targeting the protease have been developed, but drug-resistant mutant strains emerged. Here, we screened a library and synthesized a novel class of small molecules based on a tryptophan derivative scaffold identified as HCV NS3/4A protease inhibitors that are active against both wild type and mutant form of the protease. Only the compounds with predicted binding poses not affected by the most frequent mutations in the active site were selected for experimental validation. The antiviral activities were evaluated by replicon and enzymatic assays. Twenty-two compounds were found to inhibit HCV with EC values ranging between 0.64 and 63 μM with compound being the most active. In protease assays, had a comparable inhibition profile for the common mutant HCV GT1b D168A and the wild-type enzyme. However, in the same assay, the potency of the approved drug, simeprevir, decreased 5.7-fold for the mutant enzyme relative to the wild type. The top three inhibitors were also tested against four human serine proteases and were shown to be specific to the viral protease. The fluorescence-based cell viability assay demonstrated a sufficient therapeutic range for the top three candidates.
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http://dx.doi.org/10.1021/acsomega.9b02491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796237PMC
October 2019

Multi-center screening of the Pathogen Box collection for schistosomiasis drug discovery.

Parasit Vectors 2019 Oct 22;12(1):493. Epub 2019 Oct 22.

Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA.

Background: Over the past five years, as a public service to encourage and accelerate drug discovery for diseases of poverty, the Medicines for Malaria Venture (MMV) has released box sets of 400 compounds named the Malaria, Pathogen and Stasis Boxes. Here, we screened the Pathogen Box against the post-infective larvae (schistosomula) of Schistosoma mansoni using assays particular to the three contributing institutions, namely, the University of California San Diego (UCSD) in the USA, the Swiss Tropical and Public Health Institute (Swiss TPH) in Switzerland, and the Fundação Oswaldo Cruz (FIOCRUZ) in Brazil. With the same set of compounds, the goal was to determine the degree of inter-assay variability and identify a core set of active compounds common to all three assays. New drugs for schistosomiasis would be welcome given that current treatment and control strategies rely on chemotherapy with just one drug, praziquantel.

Methods: Both the UCSD and Swiss TPH assays utilize daily observational scoring methodologies over 72 h, whereas the FIOCRUZ assay employs XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) at 72 h to measure viability as a function of NAD/NADH redox state. Raw and transformed data arising from each assay were assembled for comparative analysis.

Results: For the UCSD and Swiss TPH assays, there was strong concordance of at least 87% in identifying active and inactive compounds on one or more of the three days. When all three assays were compared at 72 h, concordance remained a robust 74%. Further, robust Pearson's correlations (0.48-0.68) were measured between the assays. Of those actives at 72 h, the UCSD, Swiss TPH and FIOCRUZ assays identified 86, 103 and 66 compounds, respectively, of which 35 were common. Assay idiosyncrasies included the identification of unique compounds, the differential ability to identify known antischistosomal compounds and the concept that compounds of interest might include those that increase metabolic activity above baseline.

Conclusions: The inter-assay data generated were in good agreement, including with previously reported data. A common set of antischistosomal molecules for further exploration has been identified .
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http://dx.doi.org/10.1186/s13071-019-3747-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805474PMC
October 2019

Macrocycle modeling in ICM: benchmarking and evaluation in D3R Grand Challenge 4.

J Comput Aided Mol Des 2019 12 9;33(12):1057-1069. Epub 2019 Oct 9.

Molsoft L.L.C., 11199 Sorrento Valley Road, S209, San Diego, CA, 92121, USA.

Macrocycles represent a potentially vast extension of drug chemical space still largely untapped by synthetic compounds. Sampling of flexible rings is incorporated in the ICM-dock protocol. We tested the ability of ICM-dock to reproduce macrocyclic ligand-protein receptor complexes, first in a large retrospective benchmark (246 complexes), and next, in context of the D3R Grand Challenge 4 (GC4), where we modeled bound complexes and predicted activities for a series of macrocyclic BACE inhibitors. Sub-angstrom accuracy was achieved in ligand pose prediction both in cross-docking (D3R Challenge Stage 1A) and cognate (Stage 1B) setup. Stage 1B submission was top ranked by mean and average RMSDs, even though no ligand knowledge was used in our simulations on this Stage. Furthermore, we demonstrate successful receptor conformational selection in Stage 1A, aided by the enhanced '4D' multiple receptor conformation docking protocol with optimized scoring offsets. In the activity 3D QSAR modeling, predictivity of the BACE pKd model was modest, while for the second target (Cathepsin-S), leading performance was achieved. Difference in activity prediction performance between the targets is likely explained by the amount of available and relevant training data.
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http://dx.doi.org/10.1007/s10822-019-00225-9DOI Listing
December 2019

Identification of Four Amoebicidal Nontoxic Compounds by a Molecular Docking Screen of Sterol Δ8-Δ7-Isomerase and Phenotypic Assays.

ACS Infect Dis 2019 12 17;5(12):2029-2038. Epub 2019 Oct 17.

Skaggs School of Pharmacy and Pharmaceutical Science , University of California, San Diego , 9500 Gilman Drive , La Jolla , California 92093 , United States.

is a free-living amoeba causing primary amoebic meningoencephalitis, a rapid-onset brain infection in humans with over 97% mortality rate. Despite some progress in the treatment of the disease, there is no single, proven, evidence-based treatment with a high probability of cure. Here we report the chemical library screening and experimental identification of four new compounds with amoebicidal effects against . The chemical library was screened by molecular docking against a homology model of sterol Δ8-Δ7 isomerase (NfERG2). Thirty top-ranking hits were then tested in a cell-based assay for antiproliferative/amoebicidal activities. Eight chemicals exhibited nearly 100% inhibition of at 50 μM, with the EC values ranging from 6 to 25 μM. A cell toxicity assay using human HEK-293 cells was also performed. Four of the compounds preferentially kill amoeba cells with no apparent human cell toxicities. These compounds fall into two distinct chemical scaffolds with druglike properties.
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http://dx.doi.org/10.1021/acsinfecdis.9b00227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7085920PMC
December 2019

Recommendations for Improving Methods and Models for Aquatic Hazard Assessment of Ionizable Organic Chemicals.

Environ Toxicol Chem 2020 02;39(2):269-286

ExxonMobil Biomedical Sciences, Spring, Texas, USA.

Ionizable organic chemicals (IOCs) such as organic acids and bases are an important substance class requiring aquatic hazard evaluation. Although the aquatic toxicity of IOCs is highly dependent on the water pH, many toxicity studies in the literature cannot be interpreted because pH was not reported or not kept constant during the experiment, calling for an adaptation and improvement of testing guidelines. The modulating influence of pH on toxicity is mainly caused by pH-dependent uptake and bioaccumulation of IOCs, which can be described by ion-trapping and toxicokinetic models. The internal effect concentrations of IOCs were found to be independent of the external pH because of organisms' and cells' ability to maintain a stable internal pH milieu. If the external pH is close to the internal pH, existing quantitative structure-activity relationships (QSARs) for neutral organics can be adapted by substituting the octanol-water partition coefficient by the ionization-corrected liposome-water distribution ratio as the hydrophobicity descriptor, demonstrated by modification of the target lipid model. Charged, zwitterionic and neutral species of an IOC can all contribute to observed toxicity, either through concentration-additive mixture effects or by interaction of different species, as is the case for uncoupling of mitochondrial respiration. For specifically acting IOCs, we recommend a 2-step screening procedure with ion-trapping/QSAR models used to predict the baseline toxicity, followed by adjustment using the toxic ratio derived from in vitro systems. Receptor- or plasma-binding models also show promise for elucidating IOC toxicity. The present review is intended to help demystify the ecotoxicity of IOCs and provide recommendations for their hazard and risk assessment. Environ Toxicol Chem 2020;39:269-286. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.
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http://dx.doi.org/10.1002/etc.4602DOI Listing
February 2020

Nilotinib, an approved leukemia drug, inhibits smoothened signaling in Hedgehog-dependent medulloblastoma.

PLoS One 2019 20;14(9):e0214901. Epub 2019 Sep 20.

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego (UCSD), La Jolla, California, United States of America.

Dysregulation of the seven-transmembrane (7TM) receptor Smoothened (SMO) and other components of the Hedgehog (Hh) signaling pathway contributes to the development of cancers including basal cell carcinoma (BCC) and medulloblastoma (MB). However, SMO-specific antagonists produced mixed results in clinical trials, marked by limited efficacy and high rate of acquired resistance in tumors. Here we discovered that Nilotinib, an approved inhibitor of several kinases, possesses an anti-Hh activity, at clinically achievable concentrations, due to direct binding to SMO and inhibition of SMO signaling. Nilotinib was more efficacious than the SMO-specific antagonist Vismodegib in inhibiting growth of two Hh-dependent MB cell lines. It also reduced tumor growth in subcutaneous MB mouse xenograft model. These results indicate that in addition to its known activity against several tyrosine-kinase-mediated proliferative pathways, Nilotinib is a direct inhibitor of the Hh pathway. The newly discovered extension of Nilotinib's target profile holds promise for the treatment of Hh-dependent cancers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214901PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754133PMC
March 2020