Barry Finzel - Academia.edu (original) (raw)
Papers by Barry Finzel
Acta Crystallographica Section A Foundations and Advances
The exact cause of Alzheimer's disease (AD) has yet to be completely described despite the diseas... more The exact cause of Alzheimer's disease (AD) has yet to be completely described despite the disease being defined over 100 years ago. A potential approach to better understand the pathogenesis of AD could be the development of selective caspase-2 (Casp2) probes, as we have shown that a Casp2-mediated cleavage product of tau (Δtau314) reversibly impairs cognitive and synaptic function in animal models of tauopathies. We have taken a multi-pronged approach to studying this target and are currently developing peptide inhibitors as well as characterizing both electrophilic and non-covalent fragments. Due to limitations with Casp2 protein production, we have expressed and characterized a recently published circularly permuted Casp2 (cpCasp2) to use as a surrogate for the wild type protein. cpCasp2 is both enzymatically and structurally similar to Casp2, but cpCasp2 does not appear to be conducive to the crystallographic studies needed to support our medicinal chemistry endeavors. The design of cpCasp2 involves linking loop 2 (L2) and L2', the N-and C-terminus, of Casp2 with a GS moiety, creating L2 of cpCasp2. This loop is not well ordered and appears to create challenges for crystal growth, stability, and data resolution. We have therefore designed six L2 mutants of cpCasp2 with the goal of eliminating structural clashes we have observed in its crystal packing. We expect that these mutations will stabilize crystal growth while preserving the enzymatic activity profile. Work from our recent publications and a closer look at the structural biology data generated to date on this project will be presented.
Journal of Medicinal Chemistry, 1996
From a broad screening program, the 4-hydroxycoumarin phenprocoumon (I) was previously identified... more From a broad screening program, the 4-hydroxycoumarin phenprocoumon (I) was previously identified as a lead template with HIV protease inhibitory activity. The crystal structure of phenprocoumon/HIV protease complex initiated a structure-based design effort that initially identified the 4-hydroxy-2-pyrone U-96988 (II) as a first-generation clinical candidate for the potential treatment of HIV infection. Based upon the crystal structure of the 4-hydroxy-2pyrone III/HIV protease complex, a series of analogues incorporating a 5,6-dihydro-4-hydroxy-2-pyrone template were studied. It was recognized that in addition to having the required pharmacophore (the 4-hydroxy group with hydrogen-bonding interaction with the two catalytic aspartic acid residues and the lactone moiety replacing the ubiquitous water molecule in the active site), these 5,6-dihydro-4-hydroxy-2-pyrones incorporated side chains at the C-6 position that appropriately extended into the S 1 ′ and S 2 ′ subsites of the enzyme active site. The crystal structures of a number of representative 5,6-dihydro-4-hydroxy-2-pyrones complexed with the HIV protease were also determined to provide better understanding of the interaction between the enzyme and these inhibitors to aid the structure-based drug design effort. The crystal structures of the ligands in the enzyme active site did not always agree with the conformations expected from experience with previous pyrone inhibitors. This is likely due to the increased flexibility of the dihydropyrone ring. From this study, compound XIX exhibited reasonably high enzyme inhibitory activity (K i) 15 nM) and showed antiviral activity (IC 50) 5 µM) in the cell-culture assay. This result provided a research direction which led to the discovery of active 5,6-dihydro-4-hydroxy-2-pyrones as potential agents for the treatment of HIV infection.
ACS Chemical Neuroscience, 2019
Introduction With the rapid increase in the rate at which protein structures are solved in recent... more Introduction With the rapid increase in the rate at which protein structures are solved in recent times, we are challenged as crystallographers to try and make as much use of previous structural data as possible in the completion of our own work. To do so may be advantageous for several reasons. The tasks of density interpretation, refinement, and structure analysis could all be simplified with easy access to prior work. Making use of earlier data in a more analytical way should also help to provide more uniformity in structure determinations from laboratory to laboratory. Furthermore, if we have convenient tools for identifying and retrieving protein structural data, there is no telling what generalizations about structure may be revealed. But as crystallographers, we have a number of unique needs that must be addressed in the design of software to help us utilize structural information derived by others. Simply knowing that one structure is related to another
bioRxiv, 2020
Antimicrobial agents that target a specific pathogen of interest is the gold standard in drug des... more Antimicrobial agents that target a specific pathogen of interest is the gold standard in drug design. para-Aminosalicylic acid (PAS), remains a cornerstone therapy, in the treatment against Mycobacterium tuberculosis, owing to its high level of selectivity. Despite its high level of selectivity, PAS has been reassigned to treat drug-resistant strains of M. tuberculosis because it causes severe gastrointestinal (GI) distress that results in poor patient compliance. We have previously shown PAS inhibits the folate biosynthetic pathway specifically inhibiting dihydrofolate reductase1,2. In this study, we sought to determine the mechanistic basis of PAS selectivity and determined that PAS can be utilized in folate biosynthesis by other bacterial pathogens. The utilization of PAS ultimately led to the antagonism of key antibiotics, specifically the sulfonamides, used to prophylactically treat individuals with HIV-AIDS3. In addition, we found many bacteria in the GI tract could also utili...
Acta Crystallographica Section A Foundations and Advances
Biochemical and Biophysical Research Communications
Human histidine triad nucleotide binding protein 1 (hHint1) is a purine nucleoside phosphoramidas... more Human histidine triad nucleotide binding protein 1 (hHint1) is a purine nucleoside phosphoramidase and adenylate hydrolase that has emerged as a potential therapeutic target for the management of pain. However, the molecular mechanism of Hint1 in the signaling pathway has remained less clear. The role of metal ions in regulating postsynaptic transmission is well known, and the active site of hHint1 contains multiple histidines. Here we have investigated the effect of divalent metal ions (Cd(2+), Cu(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+)) on the structural integrity and catalytic activity of hHint1. With the exception of Mg(2+), all the divalent ions inhibited hHint1, the rank of order was found to be Cu(2+) >Zn(2+) >Cd(2+) ≥Ni(2+) >Mn(2+) based on their IC50 and kin/KI values. A crystal structure of hHint1 with bound Cu(2+) is described to explain the competitive reversible inactivation of hHint1 by divalent cations. All the metal ions exhibited time- and concentration- dependent inhibition, with the rate of inactivation highly dependent on alterations of the C-terminus. With the exception of Cu(2+); restoration of inhibition was observed for all the metal ions after treatment with EDTA. Our studies reveal a loss in secondary structure and aggregation of hHint1 upon incubation with 10-fold excess of copper. Thus, hHint1 appears to be structurally sensitive to irreversible inactivation by copper, which may be of neurotoxicological and pharmacological significance.
Biochemistry, Jul 18, 2017
Human histidine triad nucleotide binding protein 1 (hHint1) is classified as an efficient nucleos... more Human histidine triad nucleotide binding protein 1 (hHint1) is classified as an efficient nucleoside phosphoramidase and acyl-adenosine monophosphate hydrolase. Human Hint1 has been shown to be essential for the metabolic activation of nucleotide antiviral pronucleotides (i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir. The active site of hHint1 comprises an ensemble of strictly conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHint1 catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nucleotidylated-His112 intermediate (E*) using time-dependent crystallography. Utilizing a catalytically inactive hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme-substrate (ES(1)) and product (EP(2)) complexes were also cocrystallized, respectively, thus providing a structural map of the reaction trajectory. On the basis of these observa...
Journal of medicinal chemistry, Jan 22, 2017
The pyridoxal 5'-phosphate (PLP)-dependent transaminase BioA catalyzes the second step in the... more The pyridoxal 5'-phosphate (PLP)-dependent transaminase BioA catalyzes the second step in the biosynthesis of biotin in Mycobacterium tuberculosis (Mtb) and is an essential enzyme for bacterial survival and persistence in vivo. A promising BioA inhibitor 6 containing an N-aryl, N'-benzoylpiperazine scaffold was previously identified by target-based whole-cell screening. Here, we explore the structure-activity relationships (SAR) through the design, synthesis, and biological evaluation of a systematic series of analogues of the original hit using a structure-based drug design strategy, which was enabled by cocrystallization of several analogues with BioA. To confirm target engagement and discern analogues with off-target activity, each compound was evaluated against wild-type (WT) Mtb in biotin-free and -containing medium as well as BioA under- and overexpressing Mtb strains. Conformationally constrained derivative 36 emerged as the most potent analogue with a KD of 76 nM aga...
Nature, 1985
Atomic temperature factors (B-values) obtained from X-ray refinement experiments provide empirica... more Atomic temperature factors (B-values) obtained from X-ray refinement experiments provide empirical estimates of protein mobility that have been correlated with both theoretical simulations of protein dynamics and experimental studies of antibody reactivity. The comparison of B-values with protein solution properties requires adjustment of the apparent atomic mobilities to compensate for the effects of the crystal environment. Here we compare crystallographically independent subunits of the dimeric cytochrome c' from the bacterium Rhodospirillum molischianum to examine how lattice effects influence refined B-values. In addition to local effects on protein mobility at crystal contacts, we show that B-value differences up to 12 A between subunits result from lattice disordering effects that approximate to concerted rotations of the molecules about a crystal symmetry axis.
ACS Medicinal Chemistry Letters, 2016
General Methods and Materials. Guanosine was purchased from Acros Organics. Chloroacetaldehyde so... more General Methods and Materials. Guanosine was purchased from Acros Organics. Chloroacetaldehyde solution (50% wt in water), Triphenylphosphine (cat no: T84409-1004), Methyl triphenoxy phosphonium iodide (MTPI, cat no: 226432-10), Chlorosulfonyl Isocynate (cat no: 142662-254), Sulfamide (cat no: 277310) was purchased from Sigma-Aldrich. All solvents were purchased from Fischer Scientific and used as received unless otherwise noted. Anhydrous solvents such as DMF, Acetonitrile were used directly from solvent dispensing system (J. C. Meyer) packed with two columns of neutral alumina and dispensed under argon. DMA and Pyridine was purchased in a sure seal bottle from Sigma-Aldrich. Thin-layer chromatography was performed using EMD pre-coated silica gel 60 F-254 plates. All preparative separations were performed using Teledyne Isco combiflash system and using RediSepRf high performance gold silica pre-packed columns. Analytical HPLC for the stability studies were performed on Agilent C18 zorbax SB-Aq column (3.5 µm, 4.6 x 150 mm) using water (solvent A) and acetonitrile (solvent B) with 0.1% TEA as additive. High-resolution mass spectrometry was performed LTQ Orbitrap Velos (Thermo ScientificTM). Samples and compounds during synthesis were freeze-dried with a lyophilizer available from Labonaco. All 1 Hand 13 C-NMR spectra were collected in d 6-DMSO (Cambridge Isotope Laboratories, Cambridge, MA) at 25 °C using AscendTM Bruker spectrometer 500 MHz at the Department of Medicinal Chemistry CCRB NMR facility at the University of Minnesota unless otherwise stated. All NMR chemical shifts were recorded in δ parts per million using d 6-DMSO as internal reference. Thermodynamic measurements for protein-ligand association were performed in 96-well plates (Nunc 260251 U96 DeepWell 96-Well x 1.3 ml from Thermo Scientific) using MicroCal Auto-ITC200 system (GE Healthcare life sciences). Nickel nitrilotriacetic acid (Ni-NTA) was purchased from Qiagen and cobalt column agarose from Thermofishcer Scientific. Biological buffers were purchased from Sigma-Aldrich. Protease inhibitor tablets were obtained from Roche. Protein Expression and Purification The full-length sequence of hHint1 was expressed from the pMCSG7 vector (N-terminal, tobacco etch virus (TEV) protease cleavable His6 tag) in Rosetta2 pLysS cells. The cells were grown in 2 x 1L LB (Fisher Scientific) media with ampicillin (100 mg/L, Sigma-Aldrich), chloramphenicol (34 mg/L, Sigma-Aldrich), and glucose (0.1% w/v, BD Difco) at 37 °C with shaking at 250 rpm. At OD 600 = 0.7, cultures were induced to a final concentration of 1 mM IPTG (Denville Scientific Inc) and incubated at 25°C overnight. The cultures were harvested by centrifugation at 7,500 g at 4 °C for 10 min and the pellets were collected, then resuspended in buffer A (50 mM HEPES pH 7.0, 300 mM NaCl, 10% glycerol, 10 mM imidazole), which was then adjusted to 1 mg•mL-1 lysozyme and Benzonase nuclease (20 µl). The resuspended cells were lysed by sonication (eight cycles of 30 s on, 30 s off) at 4 °C. The cell debris was removed from the lysate by centrifugation at 16,000 g at 4 °C for 45 min. The supernatant was loaded onto a nickel affinity column, washed with buffer A, and then eluted with an imidazole gradient using buffer B (50 mM HEPES pH 7.0, 300 mM NaCl, 10% glycerol, 500 mm imidazole). Fractions containing desired protein was combined and to it was added N-terminally His-tagged TEV protease 2% (w/w). The resulting solutions was transferred to a dialysis tubing (molecular weight cutoff of 6000-7000 Da) and dialyzed against 2 L of TEV cleavage buffer (50 mM HEPES pH 7.0, 300 mM NaCl, 10% glycerol, 0.5 mM EDTA and 1 mM DTT) overnight at 4 °C. The dialyzed protein was then buffer exchanged into buffer A and passed through cobalt affinity chromatography to remove TEV protease. The flow through obtained was concentrated to 5 mL and further purified using size exclusion chromatography (SEC buffer, 20 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol). Pure fractions were collected and concentrated. The protein concentration was then determined using A 280 absorbance in nanodrop using calculated extinction coefficient of 8480 M-1 cm-1 and molecular weight of 14000 Da. The final protein was stored at −80 °C until in use. Protein Crystallography Crystals were grown via hanging drop vapor diffusion, with drops comprised of 2 µL of protein (A280 = 6-10, in 50mM HEPES, 250 mM NaCl, 10% glycerol v/v, pH 7.5 buffer) and 2 µL of well solution. Well solutions contained 25-35% PEG 8K, and 100 mM MES at pH 6.1-6.5. Crystals formed after 3 days of incubation at 20 °C. Co-crystals with inhibitors were prepared by soaking preformed crystals in mother liquor containing 2.5 mM 7 or 5 mM BioAMS for 15-60 minutes. After soaking, crystals were cryoprotected using 20% PEG 400 and flash vitrified with liquid nitrogen. Diffraction data were collected at 100K at beamline 17-ID (IMCA-CAT) using a Dectris Pilatus 6M Pixel Array Dectector at the Advanced Photon Source of Argonne National Laboratories in Argonne, IL. Molecular replacement was conducted with hHint1 coordinates (PDB ID 3TW2) using Phaser1 within PHENIX.2 Modeling and molecular visualization were performed in Coot.3 Ligand restraints were calculated using JLigand,4 and refinement was performed using PHENIX. Data processing and refinement statistics are presented in Table 2.
Methods in Enzymology, Feb 1, 1997
The collection of known protein structures contains a wealth of information, not only about small... more The collection of known protein structures contains a wealth of information, not only about small building blocks of proteins, but also about the way these elements assemble in real structures. Both types of information can be valuable to the modeler or crystallographer. With the software known as LORE, we provide substructure search and coordinate manipulation tools that place a wide variety of different types of structural information at the user's fingertips. Substructures, varying in complexity from small segments to complicated elements of a protein fold, can be identified and used in a wide range of applications, from density interpretation to protein engineering. To our knowledge, no other database modeling software provides such generalized coordinate manipulation capabilities. We have found the software to be a valuable addition to our molecular modeling tools.
FEBS Letters, 2015
The Bacillus anthracis lethal factor (LF) is one component of a tripartite exotoxin partly respon... more The Bacillus anthracis lethal factor (LF) is one component of a tripartite exotoxin partly responsible for persistent anthrax cytotoxicity after initial bacterial infection. Inhibitors of the zinc metalloproteinase have been investigated as potential therapeutic agents, but LF is a challenging target because inhibitors lack sufficient selectivity or possess poor pharmaceutical properties. These structural studies reveal an alternate conformation of the enzyme, induced upon binding of specific inhibitors, that opens a previously unobserved deep pocket termed S1'(∗) which might afford new opportunities to design selective inhibitors that target this subsite.
Acta Crystallographica Section A Foundations and Advances
The exact cause of Alzheimer's disease (AD) has yet to be completely described despite the diseas... more The exact cause of Alzheimer's disease (AD) has yet to be completely described despite the disease being defined over 100 years ago. A potential approach to better understand the pathogenesis of AD could be the development of selective caspase-2 (Casp2) probes, as we have shown that a Casp2-mediated cleavage product of tau (Δtau314) reversibly impairs cognitive and synaptic function in animal models of tauopathies. We have taken a multi-pronged approach to studying this target and are currently developing peptide inhibitors as well as characterizing both electrophilic and non-covalent fragments. Due to limitations with Casp2 protein production, we have expressed and characterized a recently published circularly permuted Casp2 (cpCasp2) to use as a surrogate for the wild type protein. cpCasp2 is both enzymatically and structurally similar to Casp2, but cpCasp2 does not appear to be conducive to the crystallographic studies needed to support our medicinal chemistry endeavors. The design of cpCasp2 involves linking loop 2 (L2) and L2', the N-and C-terminus, of Casp2 with a GS moiety, creating L2 of cpCasp2. This loop is not well ordered and appears to create challenges for crystal growth, stability, and data resolution. We have therefore designed six L2 mutants of cpCasp2 with the goal of eliminating structural clashes we have observed in its crystal packing. We expect that these mutations will stabilize crystal growth while preserving the enzymatic activity profile. Work from our recent publications and a closer look at the structural biology data generated to date on this project will be presented.
Journal of Medicinal Chemistry, 1996
From a broad screening program, the 4-hydroxycoumarin phenprocoumon (I) was previously identified... more From a broad screening program, the 4-hydroxycoumarin phenprocoumon (I) was previously identified as a lead template with HIV protease inhibitory activity. The crystal structure of phenprocoumon/HIV protease complex initiated a structure-based design effort that initially identified the 4-hydroxy-2-pyrone U-96988 (II) as a first-generation clinical candidate for the potential treatment of HIV infection. Based upon the crystal structure of the 4-hydroxy-2pyrone III/HIV protease complex, a series of analogues incorporating a 5,6-dihydro-4-hydroxy-2-pyrone template were studied. It was recognized that in addition to having the required pharmacophore (the 4-hydroxy group with hydrogen-bonding interaction with the two catalytic aspartic acid residues and the lactone moiety replacing the ubiquitous water molecule in the active site), these 5,6-dihydro-4-hydroxy-2-pyrones incorporated side chains at the C-6 position that appropriately extended into the S 1 ′ and S 2 ′ subsites of the enzyme active site. The crystal structures of a number of representative 5,6-dihydro-4-hydroxy-2-pyrones complexed with the HIV protease were also determined to provide better understanding of the interaction between the enzyme and these inhibitors to aid the structure-based drug design effort. The crystal structures of the ligands in the enzyme active site did not always agree with the conformations expected from experience with previous pyrone inhibitors. This is likely due to the increased flexibility of the dihydropyrone ring. From this study, compound XIX exhibited reasonably high enzyme inhibitory activity (K i) 15 nM) and showed antiviral activity (IC 50) 5 µM) in the cell-culture assay. This result provided a research direction which led to the discovery of active 5,6-dihydro-4-hydroxy-2-pyrones as potential agents for the treatment of HIV infection.
ACS Chemical Neuroscience, 2019
Introduction With the rapid increase in the rate at which protein structures are solved in recent... more Introduction With the rapid increase in the rate at which protein structures are solved in recent times, we are challenged as crystallographers to try and make as much use of previous structural data as possible in the completion of our own work. To do so may be advantageous for several reasons. The tasks of density interpretation, refinement, and structure analysis could all be simplified with easy access to prior work. Making use of earlier data in a more analytical way should also help to provide more uniformity in structure determinations from laboratory to laboratory. Furthermore, if we have convenient tools for identifying and retrieving protein structural data, there is no telling what generalizations about structure may be revealed. But as crystallographers, we have a number of unique needs that must be addressed in the design of software to help us utilize structural information derived by others. Simply knowing that one structure is related to another
bioRxiv, 2020
Antimicrobial agents that target a specific pathogen of interest is the gold standard in drug des... more Antimicrobial agents that target a specific pathogen of interest is the gold standard in drug design. para-Aminosalicylic acid (PAS), remains a cornerstone therapy, in the treatment against Mycobacterium tuberculosis, owing to its high level of selectivity. Despite its high level of selectivity, PAS has been reassigned to treat drug-resistant strains of M. tuberculosis because it causes severe gastrointestinal (GI) distress that results in poor patient compliance. We have previously shown PAS inhibits the folate biosynthetic pathway specifically inhibiting dihydrofolate reductase1,2. In this study, we sought to determine the mechanistic basis of PAS selectivity and determined that PAS can be utilized in folate biosynthesis by other bacterial pathogens. The utilization of PAS ultimately led to the antagonism of key antibiotics, specifically the sulfonamides, used to prophylactically treat individuals with HIV-AIDS3. In addition, we found many bacteria in the GI tract could also utili...
Acta Crystallographica Section A Foundations and Advances
Biochemical and Biophysical Research Communications
Human histidine triad nucleotide binding protein 1 (hHint1) is a purine nucleoside phosphoramidas... more Human histidine triad nucleotide binding protein 1 (hHint1) is a purine nucleoside phosphoramidase and adenylate hydrolase that has emerged as a potential therapeutic target for the management of pain. However, the molecular mechanism of Hint1 in the signaling pathway has remained less clear. The role of metal ions in regulating postsynaptic transmission is well known, and the active site of hHint1 contains multiple histidines. Here we have investigated the effect of divalent metal ions (Cd(2+), Cu(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+)) on the structural integrity and catalytic activity of hHint1. With the exception of Mg(2+), all the divalent ions inhibited hHint1, the rank of order was found to be Cu(2+) >Zn(2+) >Cd(2+) ≥Ni(2+) >Mn(2+) based on their IC50 and kin/KI values. A crystal structure of hHint1 with bound Cu(2+) is described to explain the competitive reversible inactivation of hHint1 by divalent cations. All the metal ions exhibited time- and concentration- dependent inhibition, with the rate of inactivation highly dependent on alterations of the C-terminus. With the exception of Cu(2+); restoration of inhibition was observed for all the metal ions after treatment with EDTA. Our studies reveal a loss in secondary structure and aggregation of hHint1 upon incubation with 10-fold excess of copper. Thus, hHint1 appears to be structurally sensitive to irreversible inactivation by copper, which may be of neurotoxicological and pharmacological significance.
Biochemistry, Jul 18, 2017
Human histidine triad nucleotide binding protein 1 (hHint1) is classified as an efficient nucleos... more Human histidine triad nucleotide binding protein 1 (hHint1) is classified as an efficient nucleoside phosphoramidase and acyl-adenosine monophosphate hydrolase. Human Hint1 has been shown to be essential for the metabolic activation of nucleotide antiviral pronucleotides (i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir. The active site of hHint1 comprises an ensemble of strictly conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHint1 catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nucleotidylated-His112 intermediate (E*) using time-dependent crystallography. Utilizing a catalytically inactive hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme-substrate (ES(1)) and product (EP(2)) complexes were also cocrystallized, respectively, thus providing a structural map of the reaction trajectory. On the basis of these observa...
Journal of medicinal chemistry, Jan 22, 2017
The pyridoxal 5'-phosphate (PLP)-dependent transaminase BioA catalyzes the second step in the... more The pyridoxal 5'-phosphate (PLP)-dependent transaminase BioA catalyzes the second step in the biosynthesis of biotin in Mycobacterium tuberculosis (Mtb) and is an essential enzyme for bacterial survival and persistence in vivo. A promising BioA inhibitor 6 containing an N-aryl, N'-benzoylpiperazine scaffold was previously identified by target-based whole-cell screening. Here, we explore the structure-activity relationships (SAR) through the design, synthesis, and biological evaluation of a systematic series of analogues of the original hit using a structure-based drug design strategy, which was enabled by cocrystallization of several analogues with BioA. To confirm target engagement and discern analogues with off-target activity, each compound was evaluated against wild-type (WT) Mtb in biotin-free and -containing medium as well as BioA under- and overexpressing Mtb strains. Conformationally constrained derivative 36 emerged as the most potent analogue with a KD of 76 nM aga...
Nature, 1985
Atomic temperature factors (B-values) obtained from X-ray refinement experiments provide empirica... more Atomic temperature factors (B-values) obtained from X-ray refinement experiments provide empirical estimates of protein mobility that have been correlated with both theoretical simulations of protein dynamics and experimental studies of antibody reactivity. The comparison of B-values with protein solution properties requires adjustment of the apparent atomic mobilities to compensate for the effects of the crystal environment. Here we compare crystallographically independent subunits of the dimeric cytochrome c' from the bacterium Rhodospirillum molischianum to examine how lattice effects influence refined B-values. In addition to local effects on protein mobility at crystal contacts, we show that B-value differences up to 12 A between subunits result from lattice disordering effects that approximate to concerted rotations of the molecules about a crystal symmetry axis.
ACS Medicinal Chemistry Letters, 2016
General Methods and Materials. Guanosine was purchased from Acros Organics. Chloroacetaldehyde so... more General Methods and Materials. Guanosine was purchased from Acros Organics. Chloroacetaldehyde solution (50% wt in water), Triphenylphosphine (cat no: T84409-1004), Methyl triphenoxy phosphonium iodide (MTPI, cat no: 226432-10), Chlorosulfonyl Isocynate (cat no: 142662-254), Sulfamide (cat no: 277310) was purchased from Sigma-Aldrich. All solvents were purchased from Fischer Scientific and used as received unless otherwise noted. Anhydrous solvents such as DMF, Acetonitrile were used directly from solvent dispensing system (J. C. Meyer) packed with two columns of neutral alumina and dispensed under argon. DMA and Pyridine was purchased in a sure seal bottle from Sigma-Aldrich. Thin-layer chromatography was performed using EMD pre-coated silica gel 60 F-254 plates. All preparative separations were performed using Teledyne Isco combiflash system and using RediSepRf high performance gold silica pre-packed columns. Analytical HPLC for the stability studies were performed on Agilent C18 zorbax SB-Aq column (3.5 µm, 4.6 x 150 mm) using water (solvent A) and acetonitrile (solvent B) with 0.1% TEA as additive. High-resolution mass spectrometry was performed LTQ Orbitrap Velos (Thermo ScientificTM). Samples and compounds during synthesis were freeze-dried with a lyophilizer available from Labonaco. All 1 Hand 13 C-NMR spectra were collected in d 6-DMSO (Cambridge Isotope Laboratories, Cambridge, MA) at 25 °C using AscendTM Bruker spectrometer 500 MHz at the Department of Medicinal Chemistry CCRB NMR facility at the University of Minnesota unless otherwise stated. All NMR chemical shifts were recorded in δ parts per million using d 6-DMSO as internal reference. Thermodynamic measurements for protein-ligand association were performed in 96-well plates (Nunc 260251 U96 DeepWell 96-Well x 1.3 ml from Thermo Scientific) using MicroCal Auto-ITC200 system (GE Healthcare life sciences). Nickel nitrilotriacetic acid (Ni-NTA) was purchased from Qiagen and cobalt column agarose from Thermofishcer Scientific. Biological buffers were purchased from Sigma-Aldrich. Protease inhibitor tablets were obtained from Roche. Protein Expression and Purification The full-length sequence of hHint1 was expressed from the pMCSG7 vector (N-terminal, tobacco etch virus (TEV) protease cleavable His6 tag) in Rosetta2 pLysS cells. The cells were grown in 2 x 1L LB (Fisher Scientific) media with ampicillin (100 mg/L, Sigma-Aldrich), chloramphenicol (34 mg/L, Sigma-Aldrich), and glucose (0.1% w/v, BD Difco) at 37 °C with shaking at 250 rpm. At OD 600 = 0.7, cultures were induced to a final concentration of 1 mM IPTG (Denville Scientific Inc) and incubated at 25°C overnight. The cultures were harvested by centrifugation at 7,500 g at 4 °C for 10 min and the pellets were collected, then resuspended in buffer A (50 mM HEPES pH 7.0, 300 mM NaCl, 10% glycerol, 10 mM imidazole), which was then adjusted to 1 mg•mL-1 lysozyme and Benzonase nuclease (20 µl). The resuspended cells were lysed by sonication (eight cycles of 30 s on, 30 s off) at 4 °C. The cell debris was removed from the lysate by centrifugation at 16,000 g at 4 °C for 45 min. The supernatant was loaded onto a nickel affinity column, washed with buffer A, and then eluted with an imidazole gradient using buffer B (50 mM HEPES pH 7.0, 300 mM NaCl, 10% glycerol, 500 mm imidazole). Fractions containing desired protein was combined and to it was added N-terminally His-tagged TEV protease 2% (w/w). The resulting solutions was transferred to a dialysis tubing (molecular weight cutoff of 6000-7000 Da) and dialyzed against 2 L of TEV cleavage buffer (50 mM HEPES pH 7.0, 300 mM NaCl, 10% glycerol, 0.5 mM EDTA and 1 mM DTT) overnight at 4 °C. The dialyzed protein was then buffer exchanged into buffer A and passed through cobalt affinity chromatography to remove TEV protease. The flow through obtained was concentrated to 5 mL and further purified using size exclusion chromatography (SEC buffer, 20 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol). Pure fractions were collected and concentrated. The protein concentration was then determined using A 280 absorbance in nanodrop using calculated extinction coefficient of 8480 M-1 cm-1 and molecular weight of 14000 Da. The final protein was stored at −80 °C until in use. Protein Crystallography Crystals were grown via hanging drop vapor diffusion, with drops comprised of 2 µL of protein (A280 = 6-10, in 50mM HEPES, 250 mM NaCl, 10% glycerol v/v, pH 7.5 buffer) and 2 µL of well solution. Well solutions contained 25-35% PEG 8K, and 100 mM MES at pH 6.1-6.5. Crystals formed after 3 days of incubation at 20 °C. Co-crystals with inhibitors were prepared by soaking preformed crystals in mother liquor containing 2.5 mM 7 or 5 mM BioAMS for 15-60 minutes. After soaking, crystals were cryoprotected using 20% PEG 400 and flash vitrified with liquid nitrogen. Diffraction data were collected at 100K at beamline 17-ID (IMCA-CAT) using a Dectris Pilatus 6M Pixel Array Dectector at the Advanced Photon Source of Argonne National Laboratories in Argonne, IL. Molecular replacement was conducted with hHint1 coordinates (PDB ID 3TW2) using Phaser1 within PHENIX.2 Modeling and molecular visualization were performed in Coot.3 Ligand restraints were calculated using JLigand,4 and refinement was performed using PHENIX. Data processing and refinement statistics are presented in Table 2.
Methods in Enzymology, Feb 1, 1997
The collection of known protein structures contains a wealth of information, not only about small... more The collection of known protein structures contains a wealth of information, not only about small building blocks of proteins, but also about the way these elements assemble in real structures. Both types of information can be valuable to the modeler or crystallographer. With the software known as LORE, we provide substructure search and coordinate manipulation tools that place a wide variety of different types of structural information at the user's fingertips. Substructures, varying in complexity from small segments to complicated elements of a protein fold, can be identified and used in a wide range of applications, from density interpretation to protein engineering. To our knowledge, no other database modeling software provides such generalized coordinate manipulation capabilities. We have found the software to be a valuable addition to our molecular modeling tools.
FEBS Letters, 2015
The Bacillus anthracis lethal factor (LF) is one component of a tripartite exotoxin partly respon... more The Bacillus anthracis lethal factor (LF) is one component of a tripartite exotoxin partly responsible for persistent anthrax cytotoxicity after initial bacterial infection. Inhibitors of the zinc metalloproteinase have been investigated as potential therapeutic agents, but LF is a challenging target because inhibitors lack sufficient selectivity or possess poor pharmaceutical properties. These structural studies reveal an alternate conformation of the enzyme, induced upon binding of specific inhibitors, that opens a previously unobserved deep pocket termed S1'(∗) which might afford new opportunities to design selective inhibitors that target this subsite.