Publications by authors named "Patrick J McDevitt"

14 Publications

  • Page 1 of 1

A Scalable Platform for Producing Recombinant Nucleosomes with Codified Histone Methyltransferase Substrate Preferences.

Protein Expr Purif 2019 12 12;164:105455. Epub 2019 Jul 12.

Janssen Pharmaceutical Companies of Johnson and Johnson, Philadelphia, PA, USA.

Wolf-Hirschhorn Syndrome Candidate 1 (WHSC1; also known as NSD2) is a SET domain-containing histone lysine methyltransferase. A chromosomal translocation occurs in 15-20% of multiple myeloma patients and is associated with increased production of WHSC1 and poor clinical prognosis. To define the substrate requirements of NSD2, we established a platform for the large-scale production of recombinant polynucleosomes, based on authentic human histone proteins, expressed in E. coli, and complexed with linearized DNA. A brief survey of methyltransferases whose substrate requirements are recorded in the literature yielded expected results, lending credence to the fitness of our approach. This platform was readily 'codified' with respect to both position and extent of methylation at histone 3 lysines 18 and 36 and led to the conclusion that the most readily discernible activity of NSD2 in contact with a nucleosome substrate is dimethylation of histone 3 lysine 36. We further explored reaction mechanism, and conclude a processive, rather than distributive mechanism best describes the interaction of NSD2 with intact nucleosome substrates. The methods developed feature scale and flexibility and are suited to thorough pharmaceutical-scale drug discovery campaigns.
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http://dx.doi.org/10.1016/j.pep.2019.105455DOI Listing
December 2019

Nucleosome Binding Alters the Substrate Bonding Environment of Histone H3 Lysine 36 Methyltransferase NSD2.

J Am Chem Soc 2016 06 23;138(21):6699-702. Epub 2016 May 23.

Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.

Nuclear receptor-binding SET domain protein 2 (NSD2) is a histone H3 lysine 36 (H3K36)-specific methyltransferase enzyme that is overexpressed in a number of cancers, including multiple myeloma. NSD2 binds to S-adenosyl-l-methionine (SAM) and nucleosome substrates to catalyze the transfer of a methyl group from SAM to the ε-amino group of histone H3K36. Equilibrium binding isotope effects and density functional theory calculations indicate that the SAM methyl group is sterically constrained in complex with NSD2, and that this steric constraint is released upon nucleosome binding. Together, these results show that nucleosome binding to NSD2 induces a significant change in the chemical environment of enzyme-bound SAM.
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http://dx.doi.org/10.1021/jacs.6b01612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702673PMC
June 2016

Structure-Based Design of a Novel SMYD3 Inhibitor that Bridges the SAM-and MEKK2-Binding Pockets.

Structure 2016 05 7;24(5):774-781. Epub 2016 Apr 7.

Cancer Epigenetics Discovery Performance Unit, GlaxoSmithKline, Collegeville, PA 19426, USA. Electronic address:

SMYD3 is a lysine methyltransferase overexpressed in colorectal, breast, prostate, and hepatocellular tumors, and has been implicated as an oncogene in human malignancies. Methylation of MEKK2 by SMYD3 is important for regulation of the MEK/ERK pathway, suggesting the possibility of selectively targeting SMYD3 in RAS-driven cancers. Structural and kinetic characterization of SMYD3 was undertaken leading to a co-crystal structure of SMYD3 with a MEKK2-peptide substrate bound, and the observation that SMYD3 follows a partially processive mechanism. These insights allowed for the design of GSK2807, a potent and selective, SAM-competitive inhibitor of SMYD3 (Ki = 14 nM). A high-resolution crystal structure reveals that GSK2807 bridges the gap between the SAM-binding pocket and the substrate lysine tunnel of SMYD3. Taken together, our data demonstrate that small-molecule inhibitors of SMYD3 can be designed to prevent methylation of MEKK2 and these could have potential use as anticancer therapeutics.
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http://dx.doi.org/10.1016/j.str.2016.03.010DOI Listing
May 2016

Discovery of a Novel 2,6-Disubstituted Glucosamine Series of Potent and Selective Hexokinase 2 Inhibitors.

ACS Med Chem Lett 2016 Mar 28;7(3):217-22. Epub 2015 Dec 28.

Cancer Metabolism Chemistry; Cancer Metabolism Biology; and Platform Technology & Sciences, GlaxoSmithKline , 1250 South Collegeville Road, Collegeville, Pennsylvania 19426-0989, United States.

A novel series of potent and selective hexokinase 2 (HK2) inhibitors, 2,6-disubstituted glucosamines, has been identified based on HTS hits, exemplified by compound 1. Inhibitor-bound crystal structures revealed that the HK2 enzyme could adopt an "induced-fit" conformation. The SAR study led to the identification of potent HK2 inhibitors, such as compound 34 with greater than 100-fold selectivity over HK1. Compound 25 inhibits in situ glycolysis in a UM-UC-3 bladder tumor cell line via (13)CNMR measurement of [3-(13)C]lactate produced from [1,6-(13)C2]glucose added to the cell culture.
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http://dx.doi.org/10.1021/acsmedchemlett.5b00214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4789681PMC
March 2016

Transition state for the NSD2-catalyzed methylation of histone H3 lysine 36.

Proc Natl Acad Sci U S A 2016 Feb 19;113(5):1197-201. Epub 2016 Jan 19.

Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461;

Nuclear receptor SET domain containing protein 2 (NSD2) catalyzes the methylation of histone H3 lysine 36 (H3K36). It is a determinant in Wolf-Hirschhorn syndrome and is overexpressed in human multiple myeloma. Despite the relevance of NSD2 to cancer, there are no potent, selective inhibitors of this enzyme reported. Here, a combination of kinetic isotope effect measurements and quantum chemical modeling was used to provide subangstrom details of the transition state structure for NSD2 enzymatic activity. Kinetic isotope effects were measured for the methylation of isolated HeLa cell nucleosomes by NSD2. NSD2 preferentially catalyzes the dimethylation of H3K36 along with a reduced preference for H3K36 monomethylation. Primary Me-(14)C and (36)S and secondary Me-(3)H3, Me-(2)H3, 5'-(14)C, and 5'-(3)H2 kinetic isotope effects were measured for the methylation of H3K36 using specifically labeled S-adenosyl-l-methionine. The intrinsic kinetic isotope effects were used as boundary constraints for quantum mechanical calculations for the NSD2 transition state. The experimental and calculated kinetic isotope effects are consistent with an SN2 chemical mechanism with methyl transfer as the first irreversible chemical step in the reaction mechanism. The transition state is a late, asymmetric nucleophilic displacement with bond separation from the leaving group at (2.53 Å) and bond making to the attacking nucleophile (2.10 Å) advanced at the transition state. The transition state structure can be represented in a molecular electrostatic potential map to guide the design of inhibitors that mimic the transition state geometry and charge.
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http://dx.doi.org/10.1073/pnas.1521036113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747696PMC
February 2016

Structures of human DPP7 reveal the molecular basis of specific inhibition and the architectural diversity of proline-specific peptidases.

PLoS One 2012 29;7(8):e43019. Epub 2012 Aug 29.

Institute of Molecular Biosciences, University of Graz, Graz, Austria.

Proline-specific dipeptidyl peptidases (DPPs) are emerging targets for drug development. DPP4 inhibitors are approved in many countries, and other dipeptidyl peptidases are often referred to as DPP4 activity- and/or structure-homologues (DASH). Members of the DASH family have overlapping substrate specificities, and, even though they share low sequence identity, therapeutic or clinical cross-reactivity is a concern. Here, we report the structure of human DPP7 and its complex with a selective inhibitor Dab-Pip (L-2,4-diaminobutyryl-piperidinamide) and compare it with that of DPP4. Both enzymes share a common catalytic domain (α/β-hydrolase). The catalytic pocket is located in the interior of DPP7, deep inside the cleft between the two domains. Substrates might access the active site via a narrow tunnel. The DPP7 catalytic triad is completely conserved and comprises Ser162, Asp418 and His443 (corresponding to Ser630, Asp708 and His740 in DPP4), while other residues lining the catalytic pockets differ considerably. The "specificity domains" are structurally also completely different exhibiting a β-propeller fold in DPP4 compared to a rare, completely helical fold in DPP7. Comparing the structures of DPP7 and DPP4 allows the design of specific inhibitors and thus the development of less cross-reactive drugs. Furthermore, the reported DPP7 structures shed some light onto the evolutionary relationship of prolyl-specific peptidases through the analysis of the architectural organization of their domains.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043019PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3430648PMC
February 2013

Structures of human DPP7 reveal the molecular basis of specific inhibition and the architectural diversity of proline-specific peptidases.

PLoS One 2012 29;7(8):e43019. Epub 2012 Aug 29.

Institute of Molecular Biosciences, University of Graz, Graz, Austria.

Proline-specific dipeptidyl peptidases (DPPs) are emerging targets for drug development. DPP4 inhibitors are approved in many countries, and other dipeptidyl peptidases are often referred to as DPP4 activity- and/or structure-homologues (DASH). Members of the DASH family have overlapping substrate specificities, and, even though they share low sequence identity, therapeutic or clinical cross-reactivity is a concern. Here, we report the structure of human DPP7 and its complex with a selective inhibitor Dab-Pip (L-2,4-diaminobutyryl-piperidinamide) and compare it with that of DPP4. Both enzymes share a common catalytic domain (α/β-hydrolase). The catalytic pocket is located in the interior of DPP7, deep inside the cleft between the two domains. Substrates might access the active site via a narrow tunnel. The DPP7 catalytic triad is completely conserved and comprises Ser162, Asp418 and His443 (corresponding to Ser630, Asp708 and His740 in DPP4), while other residues lining the catalytic pockets differ considerably. The "specificity domains" are structurally also completely different exhibiting a β-propeller fold in DPP4 compared to a rare, completely helical fold in DPP7. Comparing the structures of DPP7 and DPP4 allows the design of specific inhibitors and thus the development of less cross-reactive drugs. Furthermore, the reported DPP7 structures shed some light onto the evolutionary relationship of prolyl-specific peptidases through the analysis of the architectural organization of their domains.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043019PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3430648PMC
February 2013

An overview of the history and current status of clergy health.

J Prev Interv Community 2012 ;40(3):177-9

Duke Global Health Institute, Duke Center for Health Policy and Inequalities Research, Durham, North Carolina 27705, USA.

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http://dx.doi.org/10.1080/10852352.2012.680407DOI Listing
November 2012

Ministerial burnout: motivation and renewal for mission.

J Pastoral Care Counsel 2010 ;64(4):6.1-10

Department of Counseling and Special Education, School of Education, DePaul University, Chicago, Illinois, USA.

Roman Catholic priests are at high risk for stressors, burnout, and other emotional problems due to aging, role confusion, lack of support, changes in occupational focus, and ideological questions (Fruehle, Gautier, Bendyna, 2000; Hamel, 2000; Sammon, Reznikoff, & Gersinger, 1985; USCCB, 1982; USCCB, 2000). The business theories of Organizational Citizen Behavior and Survivor's Syndrome provide organizational explanations for factors contributing to the lack of motivation among priests. The techniques in Motivational Interviewing provide tools for religious leaders to employ when addressing the lack of motivation with individual priests. The article provides recommendations for seminaries and priests' programs of ongoing formation in addressing the issues of burnout, low-morale, and the lack of motivation.
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http://dx.doi.org/10.1177/154230501006400406DOI Listing
April 2011

Massive abdominal venous cavernous transformation diagnosed by use of EUS.

Gastrointest Endosc 2010 Apr 8;71(4):878-9. Epub 2009 Dec 8.

Division of Gastroenterology and Hepatology, Penn State Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA.

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http://dx.doi.org/10.1016/j.gie.2009.09.022DOI Listing
April 2010

Optimized procedures for producing biologically active chemokines.

Protein Expr Purif 2009 Jun;65(2):251-60

GlaxoSmithKline, Biological Reagents & Assay Development, Mail Code: UE0548, 709 Swedeland Road, King of Prussia, PA 19406, USA.

We describe here two strategies to produce biologically active chemokines with authentic N-terminal amino acid residues. The first involves producing the target chemokine with an N-terminal 6xHis-SUMO tag in Escherichia coli as inclusion bodies. The fusion protein is solubilized and purified with Ni-NTA-agarose in denaturing reagents. This is further followed by tag removal and refolding in a redox refolding buffer. The second approach involves expressing the target chemokine with an N-terminal 6xHis-Trx-SUMO tag in an engineered E. coli strain that facilitates formation of disulfide bonds in the cytoplasm. Following purification of the fusion protein via Ni-NTA and tag removal, the target chemokine is refolded without redox buffer and purified by reverse phase chromatography. Using the procedures, we have produced more than 15 biologically active chemokines, with a yield of up to 15 mg/L.
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http://dx.doi.org/10.1016/j.pep.2009.01.017DOI Listing
June 2009

A bicistronic expression system for bacterial production of authentic human interleukin-18.

Protein Expr Purif 2003 Feb;27(2):279-92

Department of Gene Expression, Protein Biochemistry, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Rd, King of Prussia, PA 19406, USA.

Interleukin-18 (IL-18) is activated and released from immune effector cells to stimulate acquired and innate immune responses involving T and natural killer (NK) cells. The release of IL-18 from mammalian cells is linked to its proteolytic activation by caspases including interleukin 1 converting enzyme (ICE). The absence of a signal peptide sequence and the requirement for coupled activation and cellular release have presented challenges for the large-scale recombinant production of IL-18. In this study, we have explored methods for the direct production of authentic human IL-18 toward the development of a large-scale production system. Expression of mature IL-18 directly in Escherichia coli with a methionine initiating codon leads to the production of MetIL-18 that is dramatically less potent in bioassays than IL-18 produced as a pro-peptide and activated in vitro. To produce an authentic IL-18, we have devised a bicistronic expression system for the coupled transcription and translation of ProIL-18 with caspase-1 (ICE) or caspase-4 (ICE-rel II, TX, ICH-2). Mature IL-18 with an authentic N-terminus was produced and has a biological activity and potency comparable to that of in vitro processed mature IL-18. Optimization of this system for the maximal production yields can be accomplished by modulating the temperature, to affect the rate of caspase activation and to favor the accumulation of ProIL-18, prior to its proteolytic processing by activated caspase. The effect of temperature is particularly profound for the caspase-4 co-expression process, enabling optimized production levels of over 150 mg/L in shake flasks at 25 degrees C. An alternative bicistronic expression design utilizing a precise ubiquitin IL-18 fusion, processed by co-expressed ubiquitinase, was also successfully used to generate fully active IL-18, thereby demonstrating that the pro-sequence of IL-18 is not required for recombinant IL-18 production.
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http://dx.doi.org/10.1016/s1046-5928(02)00606-xDOI Listing
February 2003

Structural basis for Chk1 inhibition by UCN-01.

J Biol Chem 2002 Nov 19;277(48):46609-15. Epub 2002 Sep 19.

Department of Structural Biology, GlaxoSmithKline, King of Prussia, Pennsylvania 19406, USA.

Chk1 is a serine-threonine kinase that plays an important role in the DNA damage response, including G(2)/M cell cycle control. UCN-01 (7-hydroxystaurosporine), currently in clinical trials, has recently been shown to be a potent Chk1 inhibitor that abrogates the G(2)/M checkpoint induced by DNA-damaging agents. To understand the structural basis of Chk1 inhibition by UCN-01, we determined the crystal structure of the Chk1 kinase domain in complex with UCN-01. Chk1 structures with staurosporine and its analog SB-218078 were also determined. All three compounds bind in the ATP-binding pocket of Chk1, producing only slight changes in the protein conformation. Selectivity of UCN-01 toward Chk1 over cyclin-dependent kinases can be explained by the presence of a hydroxyl group in the lactam moiety interacting with the ATP-binding pocket. Hydrophobic interactions and hydrogen-bonding interactions were observed in the structures between UCN-01 and the Chk1 kinase domain. The high structural complementarity of these interactions is consistent with the potency and selectivity of UCN-01.
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http://dx.doi.org/10.1074/jbc.M201233200DOI Listing
November 2002