|Lab P.I./Group Leader:|
|Focus:||Biochemistry, Biology, Chemical Biology, Chemistry|
|Research Experience:||Trying to make science better!|
|Lab P.I./Group Leader:||Elly Nedivi|
|Department:||Picower Institute for Learning and Memory|
|Research Experience:||I focused on elucidating the mechanism of action of a CPG15, a novel activity-regulated growth factor expressed in the brain.|
|Collaborators:||Tad Fujino, Jeremie Boucher|
|Lab P.I./Group Leader:||Charles Craik and Paul Ortiz de Montellano|
|Employer/University:||University of California, San Francisco|
|Location:||San Francisco CA|
|Focus:||Biochemistry, Chemistry, Chemistry and Chemical Biology, Structural Biology, Virology|
|Research Experience:||The project focused on designing, synthesizing and testing inhibitors targeting the viral protease encoded by Kaposi's Sarcoma-associated Herpesvirus (KSHV). In addition to validating the viral protease as a potential therapeutic target, we utilized the inhibitors to define the mechanism of activation of the dimeric protease.|
|Collaborators:||Anson Nomura, Nobuhisa Shimba, Sami Mahrus|
|Degree Earned:||Ph.D. Chemistry and Chemical Biology|
|Lab P.I./Group Leader:||Palmer W. Taylor|
|Employer/University:||Unviersity of California, San Diego|
|Location:||San Diego CA|
|Research Experience:||Cloned, purified and assayed thiol-labeled mutant acetylcholinesterases for investigation into the molecular structure and mechanism of the enzyme.|
|Collaborators:||Aileen Boyd, Lilly Wong|
|Lab P.I./Group Leader:||Lawrence J. Marnett|
|Focus:||Chemistry, Medicinal Chemistry, Organic Chemistry|
|Research Experience:||Derivatives of the NSAID indomethacin were synthesized to address cyclooxygenase-2 (COX-2) selectivity.|
|Position:||Undergraduate summer researcher|
|Lab P.I./Group Leader:||James V. Staros|
|Focus:||Chemistry, Organic Chemistry|
|Research Experience:||Spin-labeled ATP derivatives were synthesized as biophysical probes for the binding of ATP to EGFR.|
|Collaborators:||Cheryl Guyer, Jonathan Ewald|
|Lab P.I./Group Leader:||Michael P. Doyle|
|Location:||San Antonio TX|
|Focus:||Chemistry, Organic Chemistry|
|Research Experience:||Use of Cu(I) and dirhodium(II) catalysts for selective decomposition of diazoacetates leading to intramolecular cyclopropanation.|
|Degree Earned:||B.A. Chemistry|
|When I'm not at the Bench, I'm:||Playing golf, shooting videos or waiting for the summer finally to show up in Boston.|
|Who/What inspired me to become a Scientist:||My father and the belief that through science, I had an opportunity to make a difference in the world.|
|My scientific idol is:||Linus Pauling|
|In 10 years I hope to be:||Overwhelmed by the efficiency and popularity of science!|
|Websites:|| • http://www.alanmarnett.com
1: Substrate modulation of enzyme activity in the herpesvirus protease family.
Journal of Molecular Biology 373(4) 913-923 2007
Lazic A, Goetz DH, Nomura AM, Marnett AB, Craik CS.
The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 A resolution structure of Kaposi's sarcoma-associated herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme. Extended substrate binding sites are induced upon peptide binding. In particular, 104 A2 of surface are buried in the newly formed S4 pocket when tyrosine binds at this site. The peptide inhibitor also induces a rearrangement of residues that stabilizes the oxyanion hole and the dimer interface. Concomitant with the structural changes, an increase in catalytic efficiency of the enzyme results upon extended substrate binding. A nearly 20-fold increase in kcat/KM results upon extending the peptide substrate from a tetrapeptide to a hexapeptide exclusively due to a KM effect. This suggests that the mechanism by which herpesvirus proteases achieve their high specificity is by using extended substrates to modulate both the structure and activity of the enzyme.
2: One Functional Switch Mediates Reversible and Irreversible Inactivation of a Herpesvirus Protease.
Biochemistry 45(11) 3572-3579 2006
Nomura AM, Marnett AB, Shimba N, Dotsch V and Craik CS.
Distinct mechanisms have evolved to regulate the function of proteolytic enzymes. Viral proteases in particular have developed novel regulatory mechanisms, presumably due to their comparatively rapid life cycles and responses to constant evolutionary pressure. Herpesviruses are a family of human pathogens that require a viral protease with a concentration-dependent zymogen activation involving folding of two alpha-helices and activation of the catalytic machinery, which results in formation of infectious virions. Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) is unique among the herpesvirus proteases in possessing an autolysis site in the dimer interface, which removes the carboxyl-terminal 27 amino acids comprising an alpha-helix adjacent to the active site. Truncation results in the irreversible loss of dimerization and concomitant inactivation. We characterized the conformational and functional differences between the active dimer, inactive monomer, and inactive truncated protease to determine the different protease regulatory mechanisms that control the KSHV lytic cycle. Circular dichroism revealed a loss of 31% alpha-helicity upon dimer dissociation. Comparison of the full-length and truncated monomers by NMR showed differences in 21% of the protein structure, mainly located adjacent to the dimer interface, with little perturbation of the overall protein upon truncation. Fluorescence polarization and active site labeling, with a transition state mimetic, characterized the functional effects of these conformational changes. Substrate turnover is abolished in both the full-length and truncated monomers; however, substrate binding remained intact. Disruption of the helix 6 interaction with the active site oxyanion loop is therefore used in two independent regulatory mechanisms of proteolytic activity.
3: Papa's got a brand new tag: Recent advances in identification of proteases and their natural substrates.
Trends in Biotechnology 23(2) 59-64 2005
Marnett AB and Craik CS.
Characterization of proteolytic enzymes and their substrates presents a formidable challenge in the context of biological systems. Despite the fact that an estimated 2% of the human genome codes for proteases, only a small fraction of these enzymes have well-characterized functions. Much of the difficulty in understanding protease biology is a direct result of the complexity of regulation, localization and activation exhibited by this class of enzymes. Here, we focus on several recently developed techniques representing crucial advances toward identification of proteases and their natural substrates.
4: Induced structure: A transitional helix switch that regulates enzyme activity.
Nature Structural & Molecular Biology 12 1019-1020 2005
Nomura AM*, Marnett AB*, Shimba N, Dotsch V and Craik CS.
Herpesviruses encode a protease that is activated by homodimerization at high enzyme concentrations during lytic replication. The homodimer contains two active sites, which are distal from the dimer interface. Assignment of backbone NMR resonances and engineering of a redox switch show that two helices position a loop containing catalytic residues within each active site.
5: Communication between the active sites and dimer interface of a herpesvirus protease revealed by a transition-state inhibitor.
Proceedings of the National Academy of Sciences 101(18) 6870-6875 2004
Marnett AB, Nomura AM, Shimba N, Ortiz de Montellano PR, and Craik CS.
Structurally diverse organophosphonate inhibitors targeting the active site of the enzyme were used to investigate the relationship of the active site and the dimer interface of wild-type protease in solution. Positional scanning synthetic combinatorial libraries revealed Kaposi's sarcoma-associated herpesvirus protease to be highly specific, even at sites distal to the peptide bond undergoing hydrolysis. Specificity results were used to synthesize a hexapeptide diphenylphosphonate inhibitor of Kaposi's sarcoma-associated herpesvirus protease. The transition state analog inhibitors covalently phosphonylate the active site serine, freezing the enzyme structure during catalysis. An NMR-based assay was developed to monitor the native monomer-dimer equilibrium in solution and was used to demonstrate the effect of protease inhibition on the quaternary structure of the enzyme. NMR, circular dichroism, and size exclusion chromatography analysis showed that active site inhibition strongly regulates the binding affinity of the monomer-dimer equilibrium at the spatially separate dimer interface of the protease, shifting the equilibrium to the dimeric form of the enzyme. Furthermore, inhibitor studies revealed that the catalytic cycles of the spatially separate active sites are independent. These results (i) provide direct evidence that peptide bond hydrolysis is integrally linked to the quaternary structure of the enzyme, (ii) establish a molecular mechanism of protease activation and stabilization during catalysis, and (iii) highlight potential implications of substoichiometric inhibition of the viral protease in developing herpesviral therapeutics.
6: Herpesvirus protease inhibition by dimer disruption.
Journal of Virology 78(12) 6657-6665 2004
Shimba N, Nomura AM, Marnett AB and Craik CS.
Kaposi's sarcoma-associated herpesvirus (KSHV), like all herpesviruses, encodes a protease (KSHV Pr), which is necessary for the viral lytic cycle. Herpesvirus proteases function as obligate dimers; however, each monomer has an intact, complete active site which does not interact directly with the other monomer across the dimer interface. Protein grafting of an interfacial KSHV Pr alpha-helix onto a small stable protein, avian pancreatic polypeptide, generated a helical 30-amino-acid peptide designed to disrupt the dimerization of KSHV Pr. The chimeric peptide was optimized through protein modeling of the KSHV Pr-peptide complex. Circular dichroism analysis and gel filtration chromatography revealed that the rationally designed peptide adopts a helical conformation and is capable of disrupting KSHV Pr dimerization, respectively. Additionally, the optimized peptide inhibits KSHV Pr activity by 50% at a approximately 200-fold molar excess of peptide to KSHV Pr, and the dissociation constant was estimated to be 300 microM. Mutagenesis of the interfacial residue M197 to a leucine resulted in an inhibitory concentration which was twofold higher for KSHV Pr M197L than for KSHV Pr, in agreement with the model that the dimer interface is involved in peptide binding. These results indicate that the dimer interface, as well as the active sites, of herpesvirus proteases is a viable target for inhibiting enzyme activity.
7: Biochemically based design of cyclooxygenase-2 (COX-2) inhibitors: Facile conversion of nonsteroidal antiinflammatory drugs to potent and highly selective COX-2 inhibitors.
Proceedings of the National Academy of Sciences 97(2) 925-930 2000
Kalgutkar AS, Crews BC, Rowlinson SW, Marnett AB, Kozak KR, Remmel RP, and Marnett LJ.
All nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the cyclooxygenase (COX) isozymes to different extents, which accounts for their anti-inflammatory and analgesic activities and their gastrointestinal side effects. We have exploited biochemical differences between the two COX enzymes to identify a strategy for converting carboxylate-containing NSAIDs into selective COX-2 inhibitors. Derivatization of the carboxylate moiety in moderately selective COX-1 inhibitors, such as 5,8,11,14-eicosatetraynoic acid (ETYA) and arylacetic and fenamic acid NSAIDs, exemplified by indomethacin and meclofenamic acid, respectively, generated potent and selective COX-2 inhibitors. In the indomethacin series, esters and primary and secondary amides are superior to tertiary amides as selective inhibitors. Only the amide derivatives of ETYA and meclofenamic acid inhibit COX-2; the esters are either inactive or nonselective. Inhibition kinetics reveal that indomethacin amides behave as slow, tight-binding inhibitors of COX-2 and that selectivity is a function of the time-dependent step. Site-directed mutagenesis of murine COX-2 indicates that the molecular basis for selectivity differs from the parent NSAIDs and from diarylheterocycles. Selectivity arises from novel interactions at the opening and at the apex of the substrate-binding site. Lead compounds in the present study are potent inhibitors of COX-2 activity in cultured inflammatory cells. Furthermore, indomethacin amides are orally active, nonulcerogenic, anti-inflammatory agents in an in vivo model of acute inflammation. Expansion of this approach can be envisioned for the modification of all carboxylic acid-containing NSAIDs into selective COX-2 inhibitors.
8: Probing the active center gorge of acetylcholinesterase by fluorophores linked to substituted cysteines.
Journal of Biological Chemistry 275(29) 22401-22408 2000
Boyd AE, Marnett AB, Wong L, and Taylor P.
To examine the influence of individual side chains in governing rates of ligand entry into the active center gorge of acetylcholinesterase and to characterize the dynamics and immediate environment of these residues, we have conjugated reactive groups with selected charge and fluorescence characteristics to cysteines substituted by mutagenesis at specific positions on the enzyme. Insertion of side chains larger than in the native tyrosine at position 124 near the constriction point of the active site gorge confers steric hindrance to affect maximum catalytic throughput (k(cat)/K(m)) and rates of diffusional entry of trifluoroketones to the active center. Smaller groups appear not to present steric constraints to entry; however, cationic side chains selectively and markedly reduce cation ligand entry through electrostatic repulsion in the gorge. The influence of side chain modification on ligand kinetics has been correlated with spectroscopic characteristics of fluorescent side chains and their capacity to influence the binding of a peptide, fasciculin, which inhibits catalysis peripherally by sealing the mouth of the gorge. Acrylodan conjugated to cysteine was substituted for tyrosine at position 124 within the gorge, for histidine 287 on the surface adjacent to the gorge and for alanine 262 on a mobile loop distal to the gorge. The 124 position reveals the most hydrophobic environment and the largest hypsochromic shift of the emission maximum with fasciculin binding. This finding likely reflects a sandwiching of the acrylodan in the complex with the tip of fasciculin loop II. An intermediate spectral shift is found for the 287 position, consistent with partial occlusion by loops II and III of fasciculin in the complex. Spectroscopic properties of the acrylodan at the 262 position are unaltered by fasciculin addition. Hence, combined spectroscopic and kinetic analyses reveal distinguishing characteristics in various regions of acetylcholinesterase that influence ligand association.
9: Enantiocontrolled macrocycle formation by catalytic intramolecular cyclopropanation,
Journal of the American Chemical Society 122(24) 5718-5728 2000
Doyle MP, Hu W, Chapman B, Marnett AB, Peterson CS, Vitale JP, and Stanley SA.
Stereoselectivity in intramolecular cyclopropanation reactions resulting in cyclopropane fusion with ten- and larger-membered rings has been examined using chiral copper(I) and dirhodium(II) catalysts. The influence of alkene structure and catalyst has been obtained using the 1,2-benzenedimethanol linker between the allylic double bond and diazoacetate. Control features in the addition reaction, especially those for diastereoselectivity and enantioselectivity, have been elucidated, and they are associated with the metal itself or its attendant ligands that influence the trajectory of the alkene to the carbene center. The influence of ring size, from five- to twenty-membered rings, on stereoselectivity has been determined with selected copper(I) and dirhodium(II) catalysts, and the changes in stereocontrol as a function of ring size can be understood as being due to a change in the olefin trajectory to the carbene center. Hydride abstraction from a benzylic position accompanies addition when dirhodium catalysts are employed, and intramolecular Câˆ’H insertion into an allylic site to form a nine-membered ring has been observed as a major competing reaction but with negligible enantiocontrol. The use of 1,8-naphthalenedimethanol as a linker results in lower enantioselectivity than does use of 1,2-benzenedimethanol.
10: Ester and amide derivatives of the nonsteroidal antiinflammatory drug, indomethacin, as selective cyclooxygenase-2 inhibitors.
Journal of Medicinal Chemistry 43(15) 2860-2870 2000
Kalgutkar AS, Marnett AB, Crews BC, Remmel RP, and Marnett LJ.
Recent studies from our laboratory have shown that derivatization of the carboxylate moiety in substrate analogue inhibitors, such as 5,8,11,14-eicosatetraynoic acid, and in nonsteroidal antiinflammatory drugs (NSAIDs), such as indomethacin and meclofenamic acid, results in the generation of potent and selective cyclooxygenase-2 (COX-2) inhibitors (Kalgutkar et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 925-930). This paper summarizes details of the structure-activity studies involved in the transformation of the arylacetic acid NSAID, indomethacin, into a COX-2-selective inhibitor. Many of the structurally diverse indomethacin esters and amides inhibited purified human COX-2 with ICo5 values in the low-nanomolar range but did not inhibit ovine COX-1 activity at concentrations as high as 66 microM. Primary and secondary amide analogues of indomethacin were more potent as COX-2 inhibitors than the corresponding tertiary amides. Replacement of the 4-chlorobenzoyl group in indomethacin esters or amides with the 4-bromobenzyl functionality or hydrogen afforded inactive compounds. Likewise, exchanging the 2-methyl group on the indole ring in the ester and amide series with a hydrogen also generated inactive compounds. Inhibition kinetics revealed that indomethacin amides behave as slow, tight-binding inhibitors of COX-2 and that selectivity is a function of the time-dependent step. Conversion of indomethacin into ester and amide derivatives provides a facile strategy for generating highly selective COX-2 inhibitors and eliminating the gastrointestinal side effects of the parent compound.
11: Macrocycle formation by catalytic intramolecular cyclopropanation. A new general methodology for the synthesis of macrolides
Journal of the American Chemical Society 119(38) 8826-8837 1997
Doyle MP, Peterson CS, Protopopova MN, Marnett AB, Parker DL, Ene DG, and Lynch V.
Catalytic intramolecular cyclopropanation by diazoacetates onto a remote carbonâˆ’carbon double bond resulting in the formation of 9- to 20-membered ring lactones is reported. When competition exists between proximal allylic and remote olefinic cyclopropanation, macrocyclization is favored by catalysts of increasing electrophilicity: Rh2(pfb)4 > Rh2(OAc)4, Cu(MeCN)4PF6 > Rh(cap)4, and Cu(acac)2. Terpene systems, cis-nerolidyl diazoacetate and related structures, malonic ester derivatives, and those with 1,2-benzenedimethanol, pentaerythritol, and cis-2-buten-1,4-diol linkers all undergo cyclopropanation onto the most remote carbonâˆ’carbon double bond in good yield. Generally, only one cyclopropane diastereoisomer is observed, but increasing ring size allows stereochemistries in macrocyclization reactions that resemble those of their intermolecular cyclopropanation counterparts. In one system (25) macrocyclic addition is accompanied by ylide formation/[2,3]-sigmatropic rearrangement resulting in the formation of a 10-membered ring lactone. Overall, few limits to macrocycle formation are evident, and the methodology appears to have general applicability.