Human Immunodeficiency Virus 1 Integrase (HIV-1 IN) is the enzyme responsible for integrating the viral DNA into the host genome, and is essential to the life cycle of the virus . L-chicoric acid (L-CA) is a bidentate catechol that has been identified as a potent inhibitor of HIV-1 IN. By combining high level ab initio calculations, a flexible docking scheme, and the new Autodock 4.0 free-energy function we have obtained a L-CA binding mode that explains its observed potency and is consistent with available experimental data. Because of the a,b-unsaturated ester functionality of the side arms of L-CA we first performed an extensive conformational analysis of L-CA using molecular mechanics, semiempirical calculations, and high level ab initio calculations. As a result we have identified two distinct L-CA binding modes, one for the s-cis /s-cis and another for the s-cis /s-trans isomers. The most stable conformer was found to be the structure with the a,b-unsaturated ester in the s-cis conformation for both arms of L-CA. This conformer also gave the top ranked docking solution. Analysis of the interactions with key IN residues, combined with results using a L-CA tetramethoxy derivative and a Q148A IN mutant, correlate well with the experimental data.
Cite: Eamonn F. Healy, Jonathan Sanders†, Peter J. King and W. Edward Robinson, Jr “A Docking Study of L-Chicoric Acid with HIV-1 Integrase” J Mol. Graph. Model. 2009, 27 , 14.
† student author
The conformational flexibility exhibited by protein kinases poses an enormous challenge to the design of cancer therapeutics. Additionally the high degree of structural conservation within the kinase superfamiliy often leads to inhibitors that exhibit little selectivity and substantial cross reactivity. This work investigates the conformational changes that accompany the binding of Gleevec, or imatinib mesylate, to the tyrosine kinases c-Kit and c-Abl. Our analysis is that this fit is driven, at least in part, by the need to exclude water from solvent-exposed backbone hydrogen bonds. Both experimental and molecular modeling studies of the active state inhibitor of the tyrosine kinase c-Abl indicate that solvent exclusion also plays a role in this system.
Cite: Eamonn F. Healy , Skylar Johnson† , Charles Hauser, and Peter King “Tyrosine kinase inhibition: Ligand binding and conformational change in c-Kit and c-Abl.” FEBS Lett. 2009, 583, 2899-2906.
† student author
ADAM10 and ADAM17
The matrix metalloproteinase family has been a pharmaceutical target for most of the last three decades, but success has been hampered by unwanted side-effects caused by lack of selectivity, poor oral bioavailability and decreased potency in vivo. The surface-expressed metalloproteinases ADAM10 and ADAM17, the latter also also referred to as TACE, play important roles in various physiological processes, especially involving tissue repair and development. Because of its role in the release of the cytokine TNF-alpha TACE has been a key target for pharmaceutical intervention in the treatment of rheumatoid arthritis. An extensive body of structural activity data has been developed for a series of small molecule inhibitors of TACE based on a sulfonamide scaffold containing key acetylenic substituents. We have undertaken an extensive molecular modeling study of select members of this ligand group to better understand the structural nuances involved in the development of ever more potent TACE inhibitors, and identify those elements of structure-based design that would enhance the selectivity of such inhibitors for TACE over ADAM10. Results include the identification of a flexible loop, comparable to that found in other MMPs, that plays a subtle, yet significant, role in determining inhibitor potency.
Cite: Eamonn F. Healy, Pablo Romano†, Moises Mejia† and Gunnar Lindfors III†, Acetylenic Inhibitors of ADAM10 and ADAM17: In silico analysis of potency and selectivity J. Mol. Graph. Model., 2010, 29, 436-442.
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The Nucleophilic Halogenases
The nucleophilic halogenases 5’-fluoro-5’-deoxyadenosine synthase (FDAS) and salinosporamide synthase (SalL) share 35% sequence identity, and both have been shown to catalyze chlorination, though only SalL does so under native conditions. High-resolution crystal structures of both enzymes support SN2 as the substitution mechanism, and thus desolvation of the halide through the exchange of protein residues for water must be a key component for both catalytic processes. QM/MM calculations and Molecular Dynamics (MD) simulations indicate that microsolvation of the nucleophile within the active site can serve to dramatically weaken the interaction between the chloride and a critical backbone amide. The resulting exposure of an otherwise shielded backbone amide is shown to induce structural disorder in the protein, thereby reducing the enzyme’s catalytic efficiency. This model highlights the importance of excluding water from the vicinity of a backbone amide through inclusion of a tightly bound halide ion, and raises the possibility that desolvation is driven, in part, by a need serve to preserve the integrity of the protein structure of the nucleophilic halogenases,
Cite: E.F. Healy, “The effect of desolvation on nucleophilic halogenase activity”, Computational and Theoretical Chemistry, 2011, 964, 91-93.
Small Heat Shock Proteins
Molecular Dynamics simulations of a fitted multimeric structure of Mycobacterium tuberculosis a-crystallin (Mtb Acr) identify solvent exclusion from the b4- b8 hydrophobic groove as a critical factor driving subunit assembly. Dehydration is also implicated as a determinant factor governing the chaperone activity of the dimer upon its dissociation from the oligomer. Two exposed hydrogen bonds, responsible for stabilizing the b8- b9 fold are identified as key mechanistic elements in this process. Based on the overproduction of the chemokine CXCL16, observed after macrophage exposure to Mtb Acr, the proteases ADAM10 and ADAM17 are mooted as possible targets of this chaperone activity.
Cite: Eamonn F. Healy, and Peter J. King "A Mechanism of Action for Small Heat Shock Proteins", Biochem. Biophys. Res. Commun., 2012, 417, 268–273.
Small heat shock proteins (sHsp) are widely distributed molecular chaperones that bind to misfolded proteins to prevent irreversible aggregation and aid in refolding to a competent state. The sHsps chatacterized thus far all contain a conserved alpha-crystallin, and variable N- and C- termini critical for chaperone activity and oligomerization. The E. coli sHsps IbpA and IbpB share 48% sequence homology , are induced by heat shock and oxidative stress, and each requires the presence of the other to effect protein protection. Molecular Dynamics (MD) simulations of homology-modeled monomers and heterooligomers of these sHsps identify a possible mechanism for cooperation between IbpA and IbpB.
Cite: Eamonn F. Healy, "A model for heterooligomer formation in the heat shock response of E. coli.", Biochem. Biophys. Res. Commun.,2012, 420, 639-643.
Polyglutamine (polyQ) repeat expansions that lead to the formation of amyloid aggregates are linked to several devastating neurodegenerative disorders. While molecular chaperones, including the small heat shock proteins (sHsp), play an important role in protecting against protein misfolding, the aberrant protein folding that accompanies these polyQ diseases overwhelms the chaperone network. By generating a model structure to explain the observed suppression of spinocerebellar ataxia 3 (SCA3) by the sHsp alphaB-crystallin we have identified key vulnerabilities that provide a possible mechanism to explain this heat shock response. A docking study involving a small bioactive peptide should also aid in the development of new drug targets for the prevention of polyQ-based aggregation.
Cite: Eamonn F. Healy , Carley Little† ,and Peter King “A model for small heat shock protein inhibition of polyglutamine aggregation.” Cell Biochem. Biophys. 2014, 69, 275-28.
† student author
Amyotrophic Lateral Sclerosis I
Amyotrophic Lateral Sclerosis (ALS) is linked to the misfolding and aggregation of superoxide dismutase (SOD1), with over 90% of cases sporadic and the remaining familial cases associated with a wide array of inherited mutations. While it is known that oligomeric assemblies of both wild type (wt) and mutant SOD1 are precursors to the larger and detergent-insoluble aggregates, the structural events that trigger oligomerization remain elusive. Furthermore studies have found that mutant, misfolded SOD1 can convert wtSOD1 in a prion-like fashion, and that misfolded wtSOD1 can be propagated by release and uptake of protein aggregates. Using solvent-exposed intramolecular backbone hydrogen bonds as physico-chemical descriptors for protein packing, a role for transient, non-obligate oligomers in the formation of aberrant protein aggregates is presented. Oligomeric models of the both wild type (wt) and select mutant variants of superoxide dismutase (SOD1) are proposed to provide a structural basis for investigating the etiology of ALS.
Cite: Eamonn F. Healy “A role for non-obligate oligomer formation in protein aggregation”, Biochem. Biophys. Res. Commun. 2015, 465 , 523-527.
Amyotrophic Lateral Sclerosis II
Superoxide dismutase [Cu-Zn], or SOD1, is a homo-dimeric protein that functions as an antioxidant by scavenging for superoxide. A wide range of SOD1 variants are linked to inherited, or familial, amyotrophic lateral sclerosis (FALS), a progressive and fatal neurodegenerative disease. Aberrant SOD1 oligomerization has been strongly implicated in disease causation, even for sporadic ALS , or SALS, which accounts for ~90% of ALS cases. Small heat shock proteins (sHSP) have been shown to protect against amyloid fibril formation in vitro, and the sHSP aB-crystallin suppresses in vitro aggregation of SOD1. We are seeking to elucidate the structural features of both SOD1 amyloid formation and alphaB-crystallin amyloid suppression. Specifically we have used a flexible docking protocol to refine our model of a SOD1 non-obligate tetramer, postulated to function as a transient desolvating complex. Homology modeling and molecular dynamics (MD) are used to supply the missing structural elements of a previously characterized SOD1 amyloid filament, thereby providing a structural analysis for the observed gain of interaction (GOI). This completed filament is then further modified using MD to provide a structural model for protfibril capping of SOD1 filaments by aB-crystallin.
Cite: Eamonn F. Healy and Luis Cervantes† “An in silico study of the effect of SOD1 electrostatic loop dynamics on amyloid-like filament formation ”, Euro. Biophys. J. 2016, 45, 853-859.
† student author
Amyloid-like filament formation
Application of landscape theory and the dehydron hypothesis to a crystal structure of a G85R mutant superoxide dismutase (SOD1) tetrameric complex allows for the description of a prion-like hypothesis that serves to explain propagated SOD1 misfolding. We have developed two conformational-change scenarios, one local to the ESL at the complex interface, and a second displacement at the ESL of the another dimeric subunit. When taken together these provide for a prion-like mechanism that can serve to explain the observed conversion of wtSOD1 to a misfolded form by the G85R mutant.
Cite: Eamonn F. Healy “A mechanism for propagated SOD1 misfolding from frustration analysis of a G85R mutant protein assembly”, Biochem. Biophys. Res. Commun. 2016, 478, 1634-1639.
A prion-like mechanism in ALS
A prion-like mechanism has been developed to explain the observed promotion of amyloid aggregation caused by conversion of structurally intact SOD1 to a misfolded form. Superoxide dismutase [Cu-Zn], or SOD1, is a homo-dimeric protein that functions as an antioxidant by scavenging for superoxide. The misfolding and aggregation of SOD1 is linked to inherited, or familial, amyotrophic lateral sclerosis (FALS), a progressive and fatal neurodegenerative disease. Aberrant SOD1 folding has also been strongly implicated in disease causation for sporadic ALS, or SALS, which accounts for ~90% of ALS cases. Studies have found that mutant, misfolded SOD1 can convert wtSOD1 in a prion-like fashion, and that misfolded wtSOD1 can be propagated by release and uptake of protein aggregates. Here it is demonstrated that enervating the SOD1 electrostatic loop can lead to an experimentally observed gain of interaction (GOI) responsible for the formation of SOD1 amyloid-like filaments. This enervation is caused in turn by the formation of transient, non-obligate oligomers between pathogenic SOD1 mutants and wt SOD1.
Cite: Eamonn F. Healy “A prion-like mechanism for the propagated misfolding of SOD1 ”, PLoS ONE .2017, , 12 (5), e0177284.
Biosynthesis of Resveratrol
The polyketide synthases found in a variety of plants and fungi provide a varied source of biologically active compounds of pharmacological and medicinal interest. Stilbene synthase and chalcone synthase catalyze the formation of a common tetraketide intermediate, but use different cyclization mechanisms to produce distinct and separate natural products. While key structural differences have been identified to explain this functional diversity, a fuller explication of the factors responsible for this mechanistic disparity is required. Based on the energetics of our models of the bound tetraketides, and our structural analysis of the active sites we propose that a key tautomeric conversion provides a mechanistic framework common to both cyclizations. A previously unidentified active water molecule facilitates cyclization in chalcone synthase through a Claisen mechanism. Such a “Claisen switch” is comparable to the previously characterized “aldol switch” mechanism proposed for the biosynthesis of resveratrol in stilbene synthase.
Cite: Eamonn F Healy, Luis Cervantes†, Barret Nabona†, Jacob Williams† , Biochemical and Biophysical Research Communications, 2018, 497, 1123-1128
† student author