Publications by authors named "Mark S Hixon"

50 Publications

Synthetic fluorescent MYC probe: Inhibitor binding site elucidation and development of a high-throughput screening assay.

Bioorg Med Chem 2021 Jul 6;42:116246. Epub 2021 Jun 6.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, United States. Electronic address:

We report the discovery of a fluorescent small molecule probe. This probe exhibits an emission increase in the presence of the oncoprotein MYC that can be attenuated by a competing inhibitor. Hydrogen-deuterium exchange mass spectrometry analysis, rationalized by induced-fit docking, suggests it binds to the "coiled-coil" region of the leucine zipper domain. Point mutations of this site produced functional MYC constructs resistant to inhibition in an oncogenic transformation assay by compounds that displace the probe. Utilizing this probe, we have developed a high-throughput assay to identify MYC inhibitor scaffolds. Screening of a diversity library (N = 1408, 384-well) and a library of pharmacologically active compounds (N = 1280, 1536-well) yielded molecules with greater drug-like properties than the probe. One lead is a potent inhibitor of oncogenic transformation and is specific for MYC relative to resistant mutants and transformation-inducing oncogenes. This method is simple, inexpensive, and does not require protein modification, DNA binding, or the dimer partner MAX. This assay presents an opportunity for MYC inhibition researchers to discover unique scaffolds.
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http://dx.doi.org/10.1016/j.bmc.2021.116246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8279689PMC
July 2021

Salicylanilide Analog Minimizes Relapse of Infection in Mice.

J Med Chem 2020 07 16;63(13):6898-6908. Epub 2020 Jun 16.

Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, Worm Institute of Research and Medicine (WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

infection (CDI) causes serious and sometimes fatal symptoms like diarrhea and pseudomembranous colitis. Although antibiotics for CDI exist, they are either expensive or cause recurrence of the infection due to their altering the colonic microbiota, which is necessary to suppress the infection. Here, we leverage a class of known membrane-targeting compounds that we previously showed to have broad inhibitory activity across multiple strains while preserving the microbiome to develop an efficacious agent. A new series of salicylanilides was synthesized, and the most potent analog was selected through an inhibitory assay to evaluate its pharmacokinetic parameters and potency in a CDI mouse model. The results revealed reduced recurrence of CDI and diminished disturbance of the microbiota in mice compared to standard-of-care vancomycin, thus paving the way for novel therapy that can potentially target the cell membrane of to minimize relapse in the recovering patient.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00123DOI Listing
July 2020

Antipsychotic Benzamides Amisulpride and LB-102 Display Polypharmacy as Racemates, Enantiomers Engage Receptors D and D, while Enantiomers Engage 5-HT.

ACS Omega 2019 Aug 15;4(9):14151-14154. Epub 2019 Aug 15.

Mark S. Hixon Consulting LLC, 11273 Spitfire Road, San Diego, California 92126, United States.

Benzamide antipsychotics such as amisulpride are dosed as racemates though efficacy is assumed to be mediated through enantiomer binding to D receptors. At prescribed doses, the benzamides likely display polypharmacy since brain exposure should be sufficient to engage the 5-HT receptors, as well. Curiously, the studies herein reveal that racemic dosing is required to engage both targets since the D receptor has an almost 40-fold selectivity for the enantiomer, while the 5-HT receptor has greater than 50-fold preference for the enantiomer.
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http://dx.doi.org/10.1021/acsomega.9b02144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714530PMC
August 2019

Monoclonal Antibodies for Combating Synthetic Opioid Intoxication.

J Am Chem Soc 2019 07 25;141(26):10489-10503. Epub 2019 Jun 25.

Departments of Chemistry, Immunology and Microbial Science, Skaggs Institute for Chemical Biology , The Scripps Research Institute , 10550 N Torrey Pines Road , La Jolla , California 92037 , United States.

Opioid abuse in the United States has been declared a national crisis and is exacerbated by an inexpensive, readily available, and illicit supply of synthetic opioids. Specifically, fentanyl and related analogues such as carfentanil pose a significant danger to opioid users due to their high potency and rapid acting depression of respiration. In recent years these synthetic opioids have become the number one cause of drug-related deaths. In our research efforts to combat the public health threat posed by synthetic opioids, we have developed monoclonal antibodies (mAbs) against the fentanyl class of drugs. The mAbs were generated in hybridomas derived from mice vaccinated with a fentanyl conjugate vaccine. Guided by a surface plasmon resonance (SPR) binding assay, we selected six hybridomas that produced mAbs with 10 M binding affinity for fentanyl, yet broad cross-reactivity with related fentanyl analogues. In mouse antinociception models, our lead mAb (6A4) could blunt the effects of both fentanyl and carfentanil in a dose-responsive manner. Additionally, mice pretreated with 6A4 displayed enhanced survival when subjected to fentanyl above LD doses. Pharmacokinetic analysis revealed that the antibody sequesters large amounts of these drugs in the blood, thus reducing drug biodistribution to the brain and other tissue. Lastly, the 6A4 mAb could effectively reverse fentanyl/carfentanil-induced antinociception comparable to the opioid antagonist naloxone, the standard of care drug for treating opioid overdose. While naloxone is known for its short half-life, we found the half-life of 6A4 to be approximately 6 days in mice, thus monoclonal antibodies could theoretically be useful in preventing renarcotization events in which opioid intoxication recurs following quick metabolism of naloxone. Our results as a whole demonstrate that monoclonal antibodies could be a desirable treatment modality for synthetic opioid overdose and possibly opioid use disorder.
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http://dx.doi.org/10.1021/jacs.9b04872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662938PMC
July 2019

Noninvasive Urine Biomarker Lateral Flow Immunoassay for Monitoring Active Onchocerciasis.

ACS Infect Dis 2018 10 21;4(10):1423-1431. Epub 2018 Sep 21.

Department of Chemistry , The Scripps Research Institute , 10550 North Torrey Pines Road , La Jolla , California 92037 , United States.

The parasitic disease onchocerciasis is the second leading cause of preventable blindness, afflicting more than 18 million people worldwide. Despite an available treatment, ivermectin, and control efforts by the World Health Organization, onchocerciasis remains a burden in many regions. With an estimated 120 million people living in areas at risk of infection, efforts are now shifting from prevention to surveillance and elimination. The lack of a robust, point-of-care diagnostic for an active Onchocerca infection has been a limiting factor in these efforts. Previously, we reported the discovery of the biomarker N-acetyl-tyramine- O-glucuronide (NATOG) in human urine samples and its ability to track treatment progression between medicated patients relative to placebo; we also established its capability to monitor disease burden in a jird model. NATOG is a human-produced metabolite of tyramine, which itself is produced as a nematode neurotransmitter. The ability of NATOG to distinguish between active and past infection overcomes the limitations of antibody biomarkers and PCR methodologies. Lateral flow immunoassay (LFIA) diagnostics offer the versatility and simplicity to be employed in the field and are inexpensive enough to be utilized in large-scale screening efforts. Herein, we report the development and assessment of a NATOG-based urine LFIA for onchocerciasis, which accurately identified 85% of analyzed patient samples ( N = 27).
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http://dx.doi.org/10.1021/acsinfecdis.8b00163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189908PMC
October 2018

Synthetic molecules for disruption of the MYC protein-protein interface.

Bioorg Med Chem 2018 08 11;26(14):4234-4239. Epub 2018 Jul 11.

Department of Chemistry, BCC-582, 10550 N Torrey Pines Road, The Scripps Research Institute, La Jolla, CA 92037, United States. Electronic address:

MYC is a key transcriptional regulator involved in cellular proliferation and has established roles in transcriptional elongation and initiation, microRNA regulation, apoptosis, and pluripotency. Despite this prevalence, functional chemical probes of MYC function at the protein level have been limited. Previously, we discovered 5a, that binds to MYC with potency and specificity, downregulates the transcriptional activities of MYC and shows efficacy in vivo. However, this scaffold posed intrinsic pharmacokinetic liabilities, namely, poor solubility that precluded biophysical interrogation. Here, we developed a screening platform based on field-effect transistor analysis (Bio-FET), surface plasmon resonance (SPR), and a microtumor formation assay to analyze a series of new compounds aimed at improving these properties. This blind SAR campaign has produced a new lead compound of significantly increased in vivo stability and solubility for a 40-fold increase in exposure. This probe represents a significant advancement that will not only enable biophysical characterization of this interaction and further SAR, but also contribute to advances in understanding of MYC biology.
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http://dx.doi.org/10.1016/j.bmc.2018.07.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214403PMC
August 2018

Repurposing Suzuki Coupling Reagents as a Directed Fragment Library Targeting Serine Hydrolases and Related Enzymes.

J Med Chem 2017 06 9;60(12):5209-5215. Epub 2017 Jun 9.

Medicinal Chemistry - Gastrointestinal Drug Discovery Unit, ‡Structural Biology & Biophysics, §Modeling & Simulation-Global DMPK, Gastrointestinal Drug Discovery Unit, Takeda California, Inc. , 10410 Science Center Drive, San Diego, California 92121, United States.

Serine hydrolases are susceptible to potent reversible inhibition by boronic acids. Large collections of chemically diverse boronic acid fragments are commercially available because of their utility in coupling chemistry. We repurposed the approximately 650 boronic acid reagents in our collection as a directed fragment library targeting serine hydrolases and related enzymes. Highly efficient hits (LE > 0.6) often result. The utility of the approach is illustrated with the results against autotaxin, a phospholipase implicated in cardiovascular disease.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5483892PMC
June 2017

Design, synthesis and optimization of 7-substituted-pyrazolo[4,3-b]pyridine ALK5 (activin receptor-like kinase 5) inhibitors.

Bioorg Med Chem Lett 2017 05 14;27(9):1955-1961. Epub 2017 Mar 14.

Takeda California, 10410 Science Center Drive, San Diego, CA 92121, United States.

A series of potent ALK5 inhibitors were designed using a SBDD approach and subsequently optimized to improve drug likeness. Starting with a 4-substituted quinoline screening hit, SAR was conducted using a ALK5 binding model to understand the binding site and optimize activity. The resulting inhibitors displayed excellent potency but were limited by high in vitro clearance in rat and human microsomes. Using a scaffold morphing strategy, these analogs were transformed into a related pyrazolo[4,3-b]pyridine series with improved ADME properties.
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http://dx.doi.org/10.1016/j.bmcl.2017.03.026DOI Listing
May 2017

MET Tyrosine Kinase Inhibition Enhances the Antitumor Efficacy of an HGF Antibody.

Mol Cancer Ther 2017 07 24;16(7):1269-1278. Epub 2017 Mar 24.

Department of Biological Sciences, Takeda California, San Diego, California.

Receptor tyrosine kinase therapies have proven to be efficacious in specific cancer patient populations; however, a significant limitation of tyrosine kinase inhibitor (TKI) treatment is the emergence of resistance mechanisms leading to a transient, partial, or complete lack of response. Combination therapies using agents with synergistic activity have potential to improve response and reduce acquired resistance. Chemoreagent or TKI treatment can lead to increased expression of hepatocyte growth factor (HGF) and/or MET, and this effect correlates with increased metastasis and poor prognosis. Despite MET's role in resistance and cancer biology, MET TKI monotherapy has yielded disappointing clinical responses. In this study, we describe the biological activity of a selective, oral MET TKI with slow off-rate and its synergistic antitumor effects when combined with an anti-HGF antibody. We evaluated the combined action of simultaneously neutralizing HGF ligand and inhibiting MET kinase activity in two cancer xenograft models that exhibit autocrine HGF/MET activation. The combination therapy results in additive antitumor activity in KP4 pancreatic tumors and synergistic activity in U-87MG glioblastoma tumors. Pharmacodynamic characterization of biomarkers that correlate with combination synergy reveal that monotherapies induce an increase in the total MET protein, whereas combination therapy significantly reduces total MET protein levels and phosphorylation of 4E-BP1. These results hold promise that dual targeting of HGF and MET by combining extracellular ligand inhibitors with intracellular MET TKIs could be an effective intervention strategy for cancer patients who have acquired resistance that is dependent on total MET protein. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0771DOI Listing
July 2017

T-3364366 Targets the Desaturase Domain of Delta-5 Desaturase with Nanomolar Potency and a Multihour Residence Time.

ACS Med Chem Lett 2016 Sep 10;7(9):868-72. Epub 2016 Aug 10.

Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd. , 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan.

Delta-5 desaturase (D5D) catalyzes the conversion from dihomo-gamma linoleic acid (DGLA) to arachidonic acid (AA). DGLA and AA are common precursors of anti- and pro-inflammatory eicosanoids, respectively, making D5D an attractive drug target for inflammatory-related diseases. Despite several reports on D5D inhibitors, their biochemical mechanisms of action (MOAs) remain poorly understood, primarily due to the difficulty in performing quantitative enzymatic analysis. Herein, we report a radioligand binding assay to overcome this challenge and characterized T-3364366, a thienopyrimidinone D5D inhibitor, by use of the assay. T-3364366 is a reversible, slow-binding inhibitor with a dissociation half-life in excess of 2.0 h. The long residence time was confirmed in cellular washout assays. Domain swapping experiments between D5D and D6D support [(3)H]T-3364366 binding to the desaturase domain of D5D. The present study is the first to demonstrate biochemical MOA of desaturase inhibitors, providing important insight into drug discovery of desaturase enzymes.
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http://dx.doi.org/10.1021/acsmedchemlett.6b00241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5018866PMC
September 2016

Design, synthesis and optimization of novel Alk5 (activin-like kinase 5) inhibitors.

Bioorg Med Chem Lett 2016 09 16;26(17):4334-9. Epub 2016 Jul 16.

Takeda California, 10410 Science Center Drive, San Diego, CA 92121, United States.

Using SBDD, a series of 4-amino-7-azaindoles were discovered as a novel class of Alk5 inhibitors that are potent in both Alk5 enzymatic and cellular assays. Subsequently a ring cyclization strategy was utilized to improve ADME properties leading to the discovery of a series of 1H-imidazo[4,5-c]pyridin-2(3H)-one drug like Alk5 inhibitors.
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http://dx.doi.org/10.1016/j.bmcl.2016.07.030DOI Listing
September 2016

Fragment-based drug discovery of potent and selective MKK3/6 inhibitors.

Bioorg Med Chem Lett 2016 Feb 17;26(3):1086-1089. Epub 2015 Nov 17.

Medicinal Chemistry, Takeda California Inc., 10410 Science Center Drive, San Diego, CA 92121, United States.

The MAPK signaling cascade, comprised of several linear and intersecting pathways, propagates signaling into the nucleus resulting in cytokine and chemokine release. The Map Kinase Kinase isoforms 3 and 6 (MKK3 and MKK6) are responsible for the phosphorylation and activation of p38, and are hypothesized to play a key role in regulating this pathway without the redundancy seen in downstream effectors. Using FBDD, we have discovered efficient and selective inhibitors of MKK3 and MKK6 that can serve as tool molecules to help further understand the role of these kinases in MAPK signaling, and the potential impact of inhibiting kinases upstream of p38.
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http://dx.doi.org/10.1016/j.bmcl.2015.11.054DOI Listing
February 2016

Rapid Determination of the Specificity Constant of Irreversible Inhibitors (kinact/KI) by Means of an Endpoint Competition Assay.

Angew Chem Int Ed Engl 2015 Nov 1;54(47):14099-102. Epub 2015 Oct 1.

Modeling & Simulation, Global DMPK, Takeda California, Inc., 10410 Science Center Drive, San Diego, CA 92121 (USA).

Owing to their covalent target occupancy, irreversible inhibitors require low exposures and offer long duration, and their use thus represents a powerful strategy for achieving pharmacological efficacy. Importantly, the potency metric of irreversible inhibitors is kinact/KI not IC50. A simple approach to measuring kinact/KI was developed that makes use of an irreversible probe for competitive assays run to completion against test compounds. In this system, the kinact/KI value of the test compound is equal to (kinact/KI)probe ×[probe]/IC50. The advantages of this method include simplicity, high throughput, and application to all target classes, and it only requires an in-depth kinetic evaluation of the probe.
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http://dx.doi.org/10.1002/anie.201505800DOI Listing
November 2015

Toward the discovery of dual inhibitors for botulinum neurotoxin A: concomitant targeting of endocytosis and light chain protease activity.

Chem Commun (Camb) 2015 Apr;51(28):6226-9

Departments of Chemistry and Immunology and Microbial Sciences, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Dyngo-4a™ has been found to be an endocytic inhibitor of BoNT/A neurotoxicity through dynamin inhibition. Herein, we demonstrate this molecule to have a previously unrecognized dual activity against BoNT/A, dynamin-protease inhibition. To establish the importance of this dual activity, detailed kinetic analysis of Dyngo-4a's inhibition of BoNT/A metalloprotease as well as cellular and animal toxicity studies have been described. The research presented is the first polypharmacological approach to counteract BoNT/A intoxication.
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http://dx.doi.org/10.1039/c5cc00677eDOI Listing
April 2015

A simple and widely applicable hit validation strategy for protein-protein interaction inhibitors based on a quantitative ligand displacement assay.

Bioorg Med Chem Lett 2014 Dec 2;24(24):5836-5839. Epub 2014 Oct 2.

Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.

Identification of inhibitors for protein-protein interactions (PPIs) from high-throughput screening (HTS) is challenging due to the weak affinity of primary hits. We present a hit validation strategy of PPI inhibitors using quantitative ligand displacement assay. From an HTS for Bcl-xL/Mcl-1 inhibitors, we obtained a hit candidate, I1, which potentially forms a reactive Michael acceptor, I2, inhibiting Bcl-xL/Mcl-1 through covalent modification. We confirmed rapid reversible and competitive binding of I1 with a probe peptide, suggesting non-covalent binding. The advantages of our approach over biophysical assays include; simplicity, higher throughput, low protein consumption and universal application to PPIs including insoluble membrane proteins.
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http://dx.doi.org/10.1016/j.bmcl.2014.09.073DOI Listing
December 2014

Probing BoNT/A protease exosites: implications for inhibitor design and light chain longevity.

Biochemistry 2014 Nov 21;53(43):6820-4. Epub 2014 Oct 21.

Departments of Chemistry and Immunology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

Botulinum neurotoxin serotype A (BoNT/A) is one of the most lethal toxins known. Its extreme toxicity is due to its light chain (LC), a zinc protease that cleaves SNAP-25, a synaptosome-associated protein, leading to the inhibition of neuronal activity. Studies on BoNT/A LC have revealed that two regions, termed exosites, can play an important role in BoNT catalytic activity. A clear understanding of how these exosites influence neurotoxin catalytic activity would provide a critical framework for deciphering the mechanism of SNAP-25 cleavage and the design of inhibitors. Herein, based on the crystallographic structure of BoNT/A LC complexed with its substrate, we designed an α-exosite binding probe. Experiments with this unique probe demonstrated that α-exosite binding enhanced both catalytic activity and stability of the LC. These data help delineate why α-exosite binding is needed for SNAP-25 cleavage and also provide new insights into the extended lifetime observed for BoNT/A LC in vivo.
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http://dx.doi.org/10.1021/bi500950xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4222541PMC
November 2014

A platform stratifying a sequestering agent and a pharmacological antagonist as a means to negate botulinum neurotoxicity.

ACS Chem Neurosci 2014 Aug 11;5(8):632-6. Epub 2014 Jul 11.

Departments of Chemistry, Immunology and Microbial Sciences, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

Botulinum neurotoxicity is characterized by peripheral neuromuscular blockade/flaccid paralysis that can lead to respiratory failure and ultimately death. Current therapeutic options provide relief in a pre-exposure scenario, but there are no clinically approved postexposure medical countermeasures. Here, we introduce a platform that utilizes a combination of a toxin sequestering agent and a pharmacological antagonist to ablate botulinum neurotoxicity in a well-defined mouse lethality assay. The platform was constructed to allow for ready exchange of sequestering agent and/or pharmacological antagonist for therapeutic optimization. As such, we attempted to improve upon the pharmacological antagonist, a potassium channel blocker, 3,4-diaminopyridine, through a prodrug approach; thus, a complete kinetic decomposition pathway is described. These experiments provide the first proof-of-principle that a synergistic combination strategy can be used to reduce toxin burden in the peripheral using a sequestering antibody, while restoring muscle action via a pharmacological small molecule antagonist.
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http://dx.doi.org/10.1021/cn500135hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140587PMC
August 2014

Benzoquinones as inhibitors of botulinum neurotoxin serotype A.

Bioorg Med Chem 2014 Aug 16;22(15):3971-81. Epub 2014 Jun 16.

Departments of Chemistry and Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

Although botulinum neurotoxin serotype A (BoNT/A) is known for its use in cosmetics, it causes a potentially fatal illness, botulism, and can be used as a bioterror weapon. Many compounds have been developed that inhibit the BoNTA zinc-metalloprotease light chain (LC), however, none of these inhibitors have advanced to clinical trials. In this study, a fragment-based approach was implemented to develop novel covalent inhibitors of BoNT/A LC. First, electrophilic fragments were screened against BoNT/A LC, and benzoquinone (BQ) derivatives were found to be active. In kinetic studies, BQ compounds acted as irreversible inhibitors that presumably covalently modify cysteine 165 of BoNT/A LC. Although most BQ derivatives were highly reactive toward glutathione in vitro, a few compounds such as natural product naphthazarin displayed low thiol reactivity and good BoNT/A inhibition. In order to increase the potency of the BQ fragment, computational docking studies were employed to elucidate a scaffold that could bind to sites adjacent to Cys165 while positioning a BQ fragment at Cys165 for covalent modification; 2-amino-N-arylacetamides met these criteria and when linked to BQ displayed at least a 20-fold increase in activity to low μM IC₅₀ values. Unlike BQ alone, the linked-BQ compounds demonstrated only weak irreversible inhibition and therefore acted mainly as non-covalent inhibitors. Further kinetic studies revealed a mutual exclusivity of BQ covalent inactivation and competitive inhibitor binding to sites adjacent to Cys165, refuting the viability of the current strategy for developing more potent irreversible BoNT/A inhibitors. The highlights of this study include the discovery of BQ compounds as irreversible BoNT/A inhibitors and the rational design of low μM IC50 competitive inhibitors that depend on the BQ moiety for activity.
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http://dx.doi.org/10.1016/j.bmc.2014.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4138410PMC
August 2014

ATP allosterically activates the human 5-lipoxygenase molecular mechanism of arachidonic acid and 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid.

Biochemistry 2014 Jul 2;53(27):4407-19. Epub 2014 Jul 2.

Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.

5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid. This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE (dehydration of the hydroperoxide) has a rate of substrate capture (Vmax/Km) significantly lower than that of AA hydroperoxidation (oxidation of AA to form the hydroperoxide); however, hyperbolic kinetic parameters for ATP activation indicate a similar activation for AA and 5(S)-HpETE. Solvent isotope effect results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly because of a lowering of the dependence of the rate-limiting step on hydrogen atom abstraction and an increase in the dependency on hydrogen bond rearrangement. Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation.
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http://dx.doi.org/10.1021/bi401621dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215895PMC
July 2014

A fragment-based approach to identifying S-adenosyl-l-methionine -competitive inhibitors of catechol O-methyl transferase (COMT).

J Med Chem 2014 Jun 4;57(12):5459-63. Epub 2014 Jun 4.

Medicinal Chemistry, ‡Structural Biology, §Discovery Biology, ∥Analytical Chemistry, Takeda California Inc. , 10410 Science Center Drive San Diego California 92121, United States.

Catechol O-methyl transferase belongs to the diverse family of S-adenosyl-l-methionine transferases. It is a target involved in the treatment of Parkinson's disease. Here we present a fragment-based screening approach to discover noncatechol derived COMT inhibitors which bind at the SAM binding pocket. We describe the identification and characterization of a series of highly ligand efficient SAM competitive bisaryl fragments (LE = 0.33-0.58). We also present the first SAM-competitive small-molecule COMT co-complex crystal structure.
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http://dx.doi.org/10.1021/jm500475kDOI Listing
June 2014

Probing the effects of hapten stability on cocaine vaccine immunogenicity.

Mol Pharm 2013 Nov 4;10(11):4176-84. Epub 2013 Oct 4.

Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, The Worm Institute of Research and Medicine, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

Judicious hapten design has been shown to be of importance when trying to generate a viable vaccine against a drug of abuse. Hapten design has typically been predicated upon faithfully emulating the unique chemical architecture that each drug presents. However, the need for drug-hapten congruency may also compromise vaccine immunogenicity if the drug-hapten conjugate possesses chemical epitope instability. There has been no systematic study on the impact of hapten stability as it relates to vaccine immunogenicity. As a starting point, we have probed the stability of a series of cocaine haptens through varying several of its structural elements, including functionality at the C2-position, the nature of the linker, and its site of attachment. Accordingly, a hydrolytic stability profile of four cocaine haptens (GNNA, GNNS, GNE, and GNC) was produced, and these results were compared through each hapten's immunological properties, which were generated via active vaccination. From this group of four, three of the haptens, GNE, GNNA, and GNC, were further examined in an animal behavioral model, and findings here were again measured in relationship to hapten stability. We demonstrate a corresponding relationship between the half-life of the hapten and its immunogenicity, wherein haptens presenting a fully representative cocaine framework elicited higher concentrations of cocaine-specific IgG in sera and also conferred better protection against cocaine-induced locomotor activity. Our results indicate that hapten half-life plays an important role in vaccine immunogenicity and this in turn can impact animal behavioral effects when challenged with a drug of abuse.
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http://dx.doi.org/10.1021/mp400214wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3946501PMC
November 2013

Mechanistic insights into the LsrK kinase required for autoinducer-2 quorum sensing activation.

J Am Chem Soc 2013 May 16;135(21):7827-30. Epub 2013 May 16.

The Skaggs Institute for Chemical Biology and Department of Chemistry and the Worm Institute for Research and Medicine (WIRM), the Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California, 92037, USA.

In enteric bacteria, the kinase LsrK catalyzes the phosphorylation of the C5-hydroxyl group in the linear form of 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). This phosphorylation is required for AI-2 sequestration in the cytoplasm and subsequent derepression of AI-2-related genes necessary for quorum development. While LsrK is a critical enzyme within the DPD quorum sensing relay system, kinetic details of this kinase have yet to be reported. A continuous UV-vis spectrophotometric assay was developed that allowed steady-state kinetic analysis of LsrK to be undertaken with the substrates ATP and DPD. The data was most consistent with a rapid equilibrium ordered mechanism with ATP binding first: kcat (7.4 ± 0.6 s(-1)), Km,ATP (150 ± 30 μM) and Km(app),DPD (1.0 ± 0.2 mM). The assay also allowed a DPD substrate profile to be conducted, which provided an unexpected biochemical disconnect between the previous agonist/antagonist cell-based reporter assay and the LsrK assay presented herein. Together these findings raise the importance of LsrK and lay the foundation not only for further understanding of this enzyme and its critical biological role but also for the rational design of regulatory molecules targeting AI-2 quorum sensing in pathogenic bacteria.
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http://dx.doi.org/10.1021/ja4024989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3736694PMC
May 2013

Probing active cocaine vaccination performance through catalytic and noncatalytic hapten design.

J Med Chem 2013 May 29;56(9):3701-9. Epub 2013 Apr 29.

Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, The Worm Institute of Research and Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

Presently, there are no FDA-approved medications to treat cocaine addiction. Active vaccination has emerged as one approach to intervene through the rapid sequestering of the circulating drug, thus terminating both psychoactive effects and drug toxicity. Herein, we report our efforts examining two complementary, but mechanistically distinct active vaccines, i.e., noncatalytic and catalytic, for cocaine treatment. A cocaine-like hapten GNE and a cocaine transition-state analogue GNT were used to generate the active vaccines, respectively. GNE-KLH (keyhole limpet hemocyannin) was found to elicit persistent high-titer, cocaine-specific antibodies and blunt cocaine-induced locomotor behaviors. Catalytic antibodies induced by GNT-KLH were also shown to produce potent titers and suppress locomotor response in mice; however, upon repeated cocaine challenges, the vaccine's protecting effects waned. In depth kinetic analysis suggested that loss of catalytic activity was due to antibody modification by cocaine. The work provides new insights for the development of active vaccines for the treatment of cocaine abuse.
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http://dx.doi.org/10.1021/jm400228wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3691275PMC
May 2013

The C-terminus of Botulinum A Protease Has Profound and Unanticipated Kinetic Consequences Upon the Catalytic Cleft.

ACS Med Chem Lett 2013 Feb 23;4(2):283-287. Epub 2012 Dec 23.

Departments of Chemistry and Immunology, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA, 92037, USA.

Botulinum neurotoxins (BoNTs) are among the most deadly poisons known though ironically, they also are of great therapeutic utility. A number of research programs have been initiated to discover small molecule inhibitors of BoNTs metalloprotease activity. Many, though not all of these programs have screened against a truncated and more stable form of the enzyme, that possess comparable catalytic properties to the full length enzyme. Interestingly, several classes of inhibitors notably the hydroxamates, display a large shift in potency between the two enzyme forms. In this report we compare the kinetics of active-site, alpha-exosite and beta-exosite inhibitors versus truncated and full length enzyme. Molecular dynamics simulations conducted with the truncated and homology models of the fully length BoNT LC/A indicate the flexibility of the C-terminus of the full length enzyme is responsible for the potency shifts of active-site proximally binding inhibitors while distal binding (alpha-exosite) inhibitors remain equipotent.
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http://dx.doi.org/10.1021/ml300428sDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3615567PMC
February 2013

Onchocerca volvulus-neurotransmitter tyramine is a biomarker for river blindness.

Proc Natl Acad Sci U S A 2013 Mar 25;110(11):4218-23. Epub 2013 Feb 25.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.

Onchocerciasis, also known as "river blindness", is a neglected tropical disease infecting millions of people mainly in Africa and the Middle East but also in South America and Central America. Disease infectivity initiates from the filarial parasitic nematode Onchocerca volvulus, which is transmitted by the blackfly vector Simulium sp. carrying infectious third-stage larvae. Ivermectin has controlled transmission of microfilariae, with an African Program elimination target date of 2025. However, there is currently no point-of-care diagnostic that can distinguish the burden of infection--including active and/or past infection--and enable the elimination program to be effectively monitored. Here, we describe how liquid chromatography-MS-based urine metabolome analysis can be exploited for the identification of a unique biomarker, N-acetyltyramine-O,β-glucuronide (NATOG), a neurotransmitter-derived secretion metabolite from O. volvulus. The regulation of this tyramine neurotransmitter was found to be linked to patient disease infection, including the controversial antibiotic doxycycline treatment that has been shown to both sterilize and kill adult female worms. Further clues to its regulation have been elucidated through biosynthetic pathway determination within the nematode and its human host. Our results demonstrate that NATOG tracks O. volvulus metabolism in both worms and humans, and thus can be considered a host-specific biomarker for onchocerciasis progression. Liquid chromatography-MS-based urine metabolome analysis discovery of NATOG not only has broad implications for a noninvasive host-specific onchocerciasis diagnostic but provides a basis for the metabolome mining of other neglected tropical diseases for the discovery of distinct biomarkers and monitoring of disease progression.
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http://dx.doi.org/10.1073/pnas.1221969110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3600455PMC
March 2013

A Back-to-Front Fragment-Based Drug Design Search Strategy Targeting the DFG-Out Pocket of Protein Tyrosine Kinases.

ACS Med Chem Lett 2012 Apr 28;3(4):342-6. Epub 2012 Feb 28.

Discovery Research Laboratories, Oncology Drug Discovery Unit, Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd. , 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.

We present a straightforward process for the discovery of novel back pocket-binding fragment molecules against protein tyrosine kinases. The approach begins by screening against the nonphosphorylated target kinase with subsequent counterscreening of hits against the phosphorylated enzyme. Back pocket-binding fragments are inactive against the phosphorylated kinase. Fragment molecules are of insufficient size to span both regions of the ATP binding pocket; thus, the outcome is binary (back pocket-binding or hinge-binding). Next, fragments with the appropriate binding profile are assayed in combination with a known hinge-binding fragment and subsequently with a known back pocket-binding fragment. Confirmation of back pocket-binding by Yonetani-Theorell plot analysis progresses candidate fragments to crystallization trials. The method is exemplified by a fragment screening campaign against vascular endothelial growth factor receptor 2, and a novel back pocket-binding fragment is presented.
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http://dx.doi.org/10.1021/ml3000403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4025632PMC
April 2012

Formulating a new basis for the treatment against botulinum neurotoxin intoxication: 3,4-Diaminopyridine prodrug design and characterization.

Bioorg Med Chem 2011 Nov 14;19(21):6203-9. Epub 2011 Sep 14.

Department of Chemistry, Immunology and Microbial Sciences, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Botulism is a disease characterized by neuromuscular paralysis and is produced from botulinum neurotoxins (BoNTs) found within the Gram positive bacterium Clostridium botulinum. This bacteria produces the most deadliest toxin known, with lethal doses as low as 1 ng/kg. Due to the relative ease of production and transport, the use of these agents as potential bioterrorist weapons has become of utmost concern. No small molecule therapies against BoNT intoxication have been approved to date. However, 3,4-diaminopyridine (3,4-DAP), a potent reversible inhibitor of voltage-gated potassium channels, is an effective cholinergic agonist used in the treatment of neuromuscular degenerative disorders that require cholinergic enhancement. 3,4-DAP has also been shown to facilitate recovery of neuromuscular action potential post botulinum intoxication by blocking K(+) channels. Unfortunately, 3,4-DAP displays toxicity largely due to blood-brain-barrier (BBB) penetration. As a dual-action prodrug approach to cholinergic enhancement we have designed carbamate and amide conjugates of 3,4-DAP. The carbamate prodrug is intended to be a slowly reversible inhibitor of acetylcholinesterase (AChE) along the lines of the stigmines thereby allowing increased persistence of released acetylcholine within the synaptic cleft. As a secondary activity, cleavage of the carbamate prodrug by AChE will afford the localized release of 3,4-DAP, which in turn, will enhance the pre-synaptic release of additional acetylcholine. Being a competitive inhibitor with respect to acetylcholine, the activity of the prodrug will be greatest at the synaptic junctions most depleted of acetylcholine. Here we report upon the synthesis and biochemical characterization of three new classes of prodrugs intended to limit previously reported stability and toxicity issues. Of the prodrugs examined, compound 32, demonstrated the most clinically relevant half-life of 2.76 h, while selectively inhibiting AChE over butyrylcholinesterase--a plasma-based high activity esterase. Future in vivo studies could provide validation of prodrug 32 as a potential treatment against BoNT intoxication as well as other neuromuscular disorders.
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http://dx.doi.org/10.1016/j.bmc.2011.09.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196298PMC
November 2011

Identification of α2 macroglobulin as a major serum ghrelin esterase.

Angew Chem Int Ed Engl 2011 Nov 16;50(45):10699-702. Epub 2011 Sep 16.

Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

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http://dx.doi.org/10.1002/anie.201104512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515674PMC
November 2011

Biochemical characterization of a novel type-II VEGFR2 kinase inhibitor: comparison of binding to non-phosphorylated and phosphorylated VEGFR2.

Bioorg Med Chem 2011 Sep 6;19(18):5342-51. Epub 2011 Aug 6.

Discovery Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 26-1, Muraoka-Higashi, 2-chome, Fujisawa, Kanagawa 251-8555, Japan.

A pyrrolo[3,2-d]pyrimidine-based type-II vascular endothelial growth factor receptor 2 (VEGFR2) kinase inhibitor, compound 20d, displayed time-dependent inhibition of the non-phosphorylated catalytic domain of VEGFR2. In contrast, 20d did not show time-dependent inhibition of the phosphorylated enzyme. Dissociation of 20d from non-phosphorylated VEGFR2 was slow and the half-life of the complex was longer than 4h. In contrast, dissociation of 20d from the phosphorylated enzyme was very fast (half-life <5min). A fluorescent tracer based displacement assay and surface plasmon resonance (SPR) analysis confirmed the slow dissociation of 20d from only non-phosphorylated VEGFR2. Thus, activity based and binding kinetic analyses both supported slow dissociation of 20d from only non-phosphorylated VEGFR2. Additionally SPR analysis revealed that association rates were rapid and nearly identical for these two phosphorylation forms of VEGFR2. From these results, the preferential effect of 20d on non-phosphorylated VEGFR2 is dominated by its slow dissociation from the enzyme and this characteristically long residence time may increase its potency in vivo. The present findings may assist in the design of novel type-II kinase inhibitors.
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http://dx.doi.org/10.1016/j.bmc.2011.08.002DOI Listing
September 2011

A cross-over inhibitor of the botulinum neurotoxin light chain B: a natural product implicating an exosite mechanism of action.

Chem Commun (Camb) 2011 Feb 4;47(6):1713-5. Epub 2011 Jan 4.

Department of Chemistry, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Clostridium botulinum produces the most lethal toxins known to man, as such they are high risk terrorist threats, and alarmingly there is no approved therapeutic. We report the first cross-over small molecule inhibitor of these neurotoxins and propose a mechanism by which it may impart its inhibitory activity.
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http://dx.doi.org/10.1039/c0cc04078aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3065946PMC
February 2011
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