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The N-Terminal Peptide Of The Syntaxin Tlg2p Modulates Binding Of Its Closed Conformation To Vps45p, Melonnie Furgason, Chris Macdonald, Scott Shanks, Sean Ryder, Nia Bryant, Mary Munson
The N-Terminal Peptide Of The Syntaxin Tlg2p Modulates Binding Of Its Closed Conformation To Vps45p, Melonnie Furgason, Chris Macdonald, Scott Shanks, Sean Ryder, Nia Bryant, Mary Munson
Sean P. Ryder
The Sec1/Munc18 (SM) protein family regulates intracellular trafficking through interactions with individual SNARE proteins and assembled SNARE complexes. Revealing a common mechanism of this regulation has been challenging, largely because of the multiple modes of interaction observed between SM proteins and their cognate syntaxin-type SNAREs. These modes include binding of the SM to a closed conformation of syntaxin, binding to the N-terminal peptide of syntaxin, binding to assembled SNARE complexes, and/or binding to nonsyntaxin SNAREs. The SM protein Vps45p, which regulates endosomal trafficking in yeast, binds the conserved N-terminal peptide of the syntaxin Tlg2p. We used size exclusion chromatography and …
Crystal Structures Of Human Ctbp In Complex With Substrate Mtob Reveal Active Site Features Useful For Inhibitor Design, Brendan Hilbert, Steven Grossman, Celia Schiffer, William Royer
Crystal Structures Of Human Ctbp In Complex With Substrate Mtob Reveal Active Site Features Useful For Inhibitor Design, Brendan Hilbert, Steven Grossman, Celia Schiffer, William Royer
Celia A. Schiffer
The oncogenic corepressors C-terminal Binding Protein (CtBP) 1 and 2 harbor regulatory d-isomer specific 2-hydroxyacid dehydrogenase (d2-HDH) domains. 4-Methylthio 2-oxobutyric acid (MTOB) exhibits substrate inhibition and can interfere with CtBP oncogenic activity in cell culture and mice. Crystal structures of human CtBP1 and CtBP2 in complex with MTOB and NAD(+) revealed two key features: a conserved tryptophan that likely contributes to substrate specificity and a hydrophilic cavity that links MTOB with an NAD(+) phosphate. Neither feature is present in other d2-HDH enzymes. These structures thus offer key opportunities for the development of highly selective anti-neoplastic CtBP inhibitors. Elsevier B.V. All …
Structural And Thermodynamic Basis Of Amprenavir/Darunavir And Atazanavir Resistance In Hiv-1 Protease With Mutations At Residue 50, Seema Mittal, Rajintha Bandaranayake, Nancy King, Moses Prabu-Jeyabalan, Madhavi Nalam, Ellen Nalivaika, Nese Yilmaz, Celia Schiffer
Structural And Thermodynamic Basis Of Amprenavir/Darunavir And Atazanavir Resistance In Hiv-1 Protease With Mutations At Residue 50, Seema Mittal, Rajintha Bandaranayake, Nancy King, Moses Prabu-Jeyabalan, Madhavi Nalam, Ellen Nalivaika, Nese Yilmaz, Celia Schiffer
Celia A. Schiffer
Drug resistance occurs through a series of subtle changes that maintain substrate recognition but no longer permit inhibitor binding. In HIV-1 protease, mutations at I50 are associated with such subtle changes that confer differential resistance to specific inhibitors. Residue I50 is located at the protease flap tips, closing the active site upon ligand binding. Under selective drug pressure, I50V/L substitutions emerge in patients, compromising drug susceptibility and leading to treatment failure. The I50V substitution is often associated with amprenavir (APV) and darunavir (DRV) resistance, while the I50L substitution is observed in patients failing atazanavir (ATV) therapy. To explain how APV, …
Hydrophobic Core Flexibility Modulates Enzyme Activity In Hiv-1 Protease, Seema Mittal, Yufeng Cai, Madhavi Nalam, Daniel Bolon, Celia Schiffer
Hydrophobic Core Flexibility Modulates Enzyme Activity In Hiv-1 Protease, Seema Mittal, Yufeng Cai, Madhavi Nalam, Daniel Bolon, Celia Schiffer
Celia A. Schiffer
Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the …
Crystal Structure Of Human Thymidylate Synthase: A Structural Mechanism For Guiding Substrates Into The Active Site, Celia Schiffer, Ian Clifton, V. Jo Davisson, Daniel Santi, Robert Stroud
Crystal Structure Of Human Thymidylate Synthase: A Structural Mechanism For Guiding Substrates Into The Active Site, Celia Schiffer, Ian Clifton, V. Jo Davisson, Daniel Santi, Robert Stroud
Celia A. Schiffer
The crystal structure of human thymidylate synthase, a target for anti-cancer drugs, is determined to 3.0 A resolution and refined to a crystallographic residual of 17.8%. The structure implicates the enzyme in a mechanism for facilitating the docking of substrates into the active site. This mechanism involves a twist of approximately 180 degrees of the active site loop, pivoted around the neighboring residues 184 and 204, and implicates ordering of external, eukaryote specific loops along with the well-characterized closure of the active site upon substrate binding. The highly conserved, but eukaryote-specific insertion of twelve residues 90-101 (h117-128), and of eight …
Evaluation Of The Substrate Envelope Hypothesis For Inhibitors Of Hiv-1 Protease, Sripriya Chellappan, Visvaldas Kairys, Miguel Fernandes, Celia Schiffer, Michael Gilson
Evaluation Of The Substrate Envelope Hypothesis For Inhibitors Of Hiv-1 Protease, Sripriya Chellappan, Visvaldas Kairys, Miguel Fernandes, Celia Schiffer, Michael Gilson
Celia A. Schiffer
Crystallographic data show that various substrates of HIV protease occupy a remarkably uniform region within the binding site; this region has been termed the substrate envelope. It has been suggested that an inhibitor that fits within the substrate envelope should tend to evade viral resistance because a protease mutation that reduces the affinity of the inhibitor will also tend to reduce the affinity of substrate, and will hence decrease the activity of the enzyme. Accordingly, inhibitors that fit the substrate envelope better should be less susceptible to clinically observed resistant mutations, since these must also allow substrates to bind. The …
Expression, Purification, And Characterization Of Thymidylate Synthase From Lactococcus Lactis, Patricia Greene, Pak-Lam Yu, Jia Zhao, Celia Schiffer, Daniel Santi
Expression, Purification, And Characterization Of Thymidylate Synthase From Lactococcus Lactis, Patricia Greene, Pak-Lam Yu, Jia Zhao, Celia Schiffer, Daniel Santi
Celia A. Schiffer
The thymidylate synthase (TS) gene from Lactococcus lactis has been highly expressed in Escherichia coli. The TS protein was purified by sequential chromatography on Q-Sepharose and phenyl-Sepharose. Six grams of cell pellet yielded 140 mg of homogeneous TS. TS is a highly conserved enzyme, and several of the conserved amino acid residues that have been implicated in catalytic function are altered in L. lactis TS. By use of a 3-dimensional homology model, we have predicted covariant changes that might compensate for these differences. With the large amounts of L. lactis TS now available, studies can be pursued to understand the …
The Role Of Protein-Solvent Interactions In Protein Unfolding, Celia Schiffer, Volker Dötsch
The Role Of Protein-Solvent Interactions In Protein Unfolding, Celia Schiffer, Volker Dötsch
Celia A. Schiffer
Protein unfolding occurs when the balance of forces between the protein's interaction with itself and the protein's interaction with its environment is disrupted. The disruption of this balance of forces may be as simple as a perturbance of the normal water structure around the protein. A decrease in the normal water-water interaction will result in an increase in the relative interaction of water with the protein. An increase in the number of interactions between water and the protein may initiate a protein's unfolding. This model for protein unfolding is supported by a range of recent experimental and computational data.
Association Of A Novel Human Immunodeficiency Virus Type 1 Protease Substrate Cleft Mutation, L23i, With Protease Inhibitor Therapy And In Vitro Drug Resistance, Elizabeth Johnston, Mark Winters, Soo-Yon Rhee, Thomas Merigan, Celia Schiffer, Robert Shafer
Association Of A Novel Human Immunodeficiency Virus Type 1 Protease Substrate Cleft Mutation, L23i, With Protease Inhibitor Therapy And In Vitro Drug Resistance, Elizabeth Johnston, Mark Winters, Soo-Yon Rhee, Thomas Merigan, Celia Schiffer, Robert Shafer
Celia A. Schiffer
We observed a previously uncharacterized mutation in the protease substrate cleft, L23I, in 31 of 4,303 persons undergoing human immunodeficiency virus type 1 genotypic resistance testing. In combination with V82I, L23I was associated with a sevenfold reduction in nelfinavir susceptibility and a decrease in replication capacity. In combination with other drug resistance mutations, L23I was associated with multidrug resistance and a compensatory increase in replication capacity.
Substrate Shape Determines Specificity Of Recognition For Hiv-1 Protease: Analysis Of Crystal Structures Of Six Substrate Complexes, Moses Prabu-Jeyabalan, Ellen Nalivaika, Celia Schiffer
Substrate Shape Determines Specificity Of Recognition For Hiv-1 Protease: Analysis Of Crystal Structures Of Six Substrate Complexes, Moses Prabu-Jeyabalan, Ellen Nalivaika, Celia Schiffer
Celia A. Schiffer
The homodimeric HIV-1 protease is the target of some of the most effective antiviral AIDS therapy, as it facilitates viral maturation by cleaving ten asymmetric and nonhomologous sequences in the Gag and Pol polyproteins. Since the specificity of this enzyme is not easily determined from the sequences of these cleavage sites alone, we solved the crystal structures of complexes of an inactive variant (D25N) of HIV-1 protease with six peptides that correspond to the natural substrate cleavage sites. When the protease binds to its substrate and buries nearly 1000 A2 of surface area, the symmetry of the protease is broken, …
Rationale For More Diverse Inhibitors In Competition With Substrates In Hiv-1 Protease, Nevra Ozer, Celia Schiffer, Turkan Haliloglu
Rationale For More Diverse Inhibitors In Competition With Substrates In Hiv-1 Protease, Nevra Ozer, Celia Schiffer, Turkan Haliloglu
Celia A. Schiffer
The structural fluctuations of HIV-1 protease in interaction with its substrates versus inhibitors were analyzed using the anisotropic network model. The directions of fluctuations in the most cooperative functional modes differ mainly around the dynamically key regions, i.e., the hinge axes, which appear to be more flexible in substrate complexes. The flexibility of HIV-1 protease is likely optimized for the substrates' turnover, resulting in substrate complexes being dynamic. In contrast, in an inhibitor complex, the inhibitor should bind and lock down to inactivate the active site. Protease and ligands are not independent. Substrates are also more flexible than inhibitors and …
Molecular Mechanisms Of Viral And Host Cell Substrate Recognition By Hepatitis C Virus Ns3/4a Protease, Keith Romano, Jennifer Laine, Laura Deveau, Hong Cao, Francesca Massi, Celia Schiffer
Molecular Mechanisms Of Viral And Host Cell Substrate Recognition By Hepatitis C Virus Ns3/4a Protease, Keith Romano, Jennifer Laine, Laura Deveau, Hong Cao, Francesca Massi, Celia Schiffer
Celia A. Schiffer
Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and …
Crystal Structure Of Lysine Sulfonamide Inhibitor Reveals The Displacement Of The Conserved Flap Water Molecule In Human Immunodeficiency Virus Type 1 Protease, Madhavi Nalam, Anik Peeters, Tim Jonckers, Inge Dierynck, Celia Schiffer
Crystal Structure Of Lysine Sulfonamide Inhibitor Reveals The Displacement Of The Conserved Flap Water Molecule In Human Immunodeficiency Virus Type 1 Protease, Madhavi Nalam, Anik Peeters, Tim Jonckers, Inge Dierynck, Celia Schiffer
Celia A. Schiffer
Human immunodeficiency virus type 1 (HIV-1) protease has been continuously evolving and developing resistance to all of the protease inhibitors. This requires the development of new inhibitors that bind to the protease in a novel fashion. Most of the inhibitors that are on the market are peptidomimetics, where a conserved water molecule mediates hydrogen bonding interactions between the inhibitors and the flaps of the protease. Recently a new class of inhibitors, lysine sulfonamides, was developed to combat the resistant variants of HIV protease. Here we report the crystal structure of a lysine sulfonamide. This inhibitor binds to the active site …
Additivity In The Analysis And Design Of Hiv Protease Inhibitors, Robert Jorissen, G. S. Kiran Kumar Reddy, Akbar Ali, Michael Altman, Sripriya Chellappan, Saima Anjum, Bruce Tidor, Celia Schiffer, Tariq Rana, Michael Gilson
Additivity In The Analysis And Design Of Hiv Protease Inhibitors, Robert Jorissen, G. S. Kiran Kumar Reddy, Akbar Ali, Michael Altman, Sripriya Chellappan, Saima Anjum, Bruce Tidor, Celia Schiffer, Tariq Rana, Michael Gilson
Celia A. Schiffer
We explore the applicability of an additive treatment of substituent effects to the analysis and design of HIV protease inhibitors. Affinity data for a set of inhibitors with a common chemical framework were analyzed to provide estimates of the free energy contribution of each chemical substituent. These estimates were then used to design new inhibitors whose high affinities were confirmed by synthesis and experimental testing. Derivations of additive models by least-squares and ridge-regression methods were found to yield statistically similar results. The additivity approach was also compared with standard molecular descriptor-based QSAR; the latter was not found to provide superior …
Insights Into Interferon Regulatory Factor Activation From The Crystal Structure Of Dimeric Irf5, Weijun Chen, Suvana Lam, Hema Srinath, Zhaozhao Jiang, John Correia, Celia Schiffer, Katherine Fitzgerald, Kai Lin, William Royer
Insights Into Interferon Regulatory Factor Activation From The Crystal Structure Of Dimeric Irf5, Weijun Chen, Suvana Lam, Hema Srinath, Zhaozhao Jiang, John Correia, Celia Schiffer, Katherine Fitzgerald, Kai Lin, William Royer
Celia A. Schiffer
Interferon regulatory factors (IRFs) are essential in the innate immune response and other physiological processes. Activation of these proteins in the cytoplasm is triggered by phosphorylation of serine and threonine residues in a C-terminal autoinhibitory region, which stimulates dimerization, transport into the nucleus, assembly with the coactivator CBP/p300 and initiation of transcription. The crystal structure of the transactivation domain of pseudophosphorylated human IRF5 strikingly reveals a dimer in which the bulk of intersubunit interactions involve a highly extended C-terminal region. The corresponding region has previously been shown to block CBP/p300 binding to unphosphorylated IRF3. Mutation of key interface residues supports …
How Does A Symmetric Dimer Recognize An Asymmetric Substrate? A Substrate Complex Of Hiv-1 Protease, Moses Prabu-Jeyabalan, Ellen Nalivaika, Celia Schiffer
How Does A Symmetric Dimer Recognize An Asymmetric Substrate? A Substrate Complex Of Hiv-1 Protease, Moses Prabu-Jeyabalan, Ellen Nalivaika, Celia Schiffer
Celia A. Schiffer
The crystal structure of an actual HIV-1 protease-substrate complex is presented at 2.0 A resolution (R-value of 19.7 % (R(free) 23.3 %)) between an inactive variant (D25N) of HIV-1 protease and a long substrate peptide, Lys-Ala-Arg-Val-Leu-Ala-Glu-Ala-Met-Ser, which covers a full binding epitope of capsid(CA)-p2, cleavage site. The substrate peptide is asymmetric in both size and charge distribution. To accommodate this asymmetry the two protease monomers adopt different conformations burying a total of 1038 A(2) of surface area at the protease-substrate interface. The specificity for the CA-p2 substrate peptide is mainly hydrophobic, as most of the hydrogen bonds are made with …
Design Of Hiv-1 Protease Inhibitors Active On Multidrug-Resistant Virus, Dominique Surleraux, Herman De Kock, Wim Verschueren, Geert Pille, Louis Maes, Anik Peeters, Sandrine Vendeville, Sandra De Meyer, Hilde Azijn, Rudi Pauwels, Marie-Pierre De Bethune, Nancy King, Moses Prabu-Jeyabalan, Celia Schiffer, Piet Wigerinck
Design Of Hiv-1 Protease Inhibitors Active On Multidrug-Resistant Virus, Dominique Surleraux, Herman De Kock, Wim Verschueren, Geert Pille, Louis Maes, Anik Peeters, Sandrine Vendeville, Sandra De Meyer, Hilde Azijn, Rudi Pauwels, Marie-Pierre De Bethune, Nancy King, Moses Prabu-Jeyabalan, Celia Schiffer, Piet Wigerinck
Celia A. Schiffer
On the basis of structural data gathered during our ongoing HIV-1 protease inhibitors program, from which our clinical candidate TMC114 9 was selected, we have discovered new series of fused heteroaromatic sulfonamides. The further extension into the P2' region was aimed at identifying new classes of compounds with an improved broad spectrum activity and acceptable pharmacokinetic properties. Several of these compounds display an exceptional broad spectrum activity against a panel of highly cross-resistant mutants. Certain members of these series exhibit favorable pharmacokinetic profiles in rat and dog. Crystal structures and molecular modeling were used to rationalize the broad spectrum profile …
Design And Synthesis Of Hiv-1 Protease Inhibitors Incorporating Oxazolidinones As P2/P2' Ligands In Pseudosymmetric Dipeptide Isosteres, G. S. Kiran Kumar Reddy, Akbar Ali, Madhavi Nalam, Saima Anjum, Hong Cao, Robin Nathans, Celia Schiffer, Tariq Rana
Design And Synthesis Of Hiv-1 Protease Inhibitors Incorporating Oxazolidinones As P2/P2' Ligands In Pseudosymmetric Dipeptide Isosteres, G. S. Kiran Kumar Reddy, Akbar Ali, Madhavi Nalam, Saima Anjum, Hong Cao, Robin Nathans, Celia Schiffer, Tariq Rana
Celia A. Schiffer
A series of novel HIV-1 protease inhibitors based on two pseudosymmetric dipeptide isosteres have been synthesized and evaluated. The inhibitors were designed by incorporating N-phenyloxazolidinone-5-carboxamides into the hydroxyethylene and (hydroxyethyl)hydrazine dipeptide isosteres as P2 and P2' ligands. Compounds with (S)-phenyloxazolidinones attached at a position proximal to the central hydroxyl group showed low nM inhibitory activities against wild-type HIV-1 protease. Selected compounds were further evaluated for their inhibitory activities against a panel of multidrug-resistant protease variants and for their antiviral potencies in MT-4 cells. The crystal structures of lopinavir (LPV) and two new inhibitors containing phenyloxazolidinone-based ligands in complex with wild-type …
Structural Stability Of Disulfide Mutants Of Basic Pancreatic Trypsin Inhibitor: A Molecular Dynamics Study, Celia Schiffer, Wilfred Van Gunsteren
Structural Stability Of Disulfide Mutants Of Basic Pancreatic Trypsin Inhibitor: A Molecular Dynamics Study, Celia Schiffer, Wilfred Van Gunsteren
Celia A. Schiffer
The structure and folding of basic pancreatic trypsin inhibitor (BPTI) has been studied extensively by experimental means. We report a computer simulation study of the structural stability of various disulfide mutants of BPTI, involving eight 250-psec molecular dynamics simulations of the proteins in water, with and without a phosphate counterion. The presence of the latter alters the relative stability of the single disulfide species [5-55] and [30-51]. This conclusion can explain results of mutational studies and the conservation of residues in homologues of BPTI, and suggests a possible role of ions in stabilizing one intermediate over another in unfolding or …
Evaluating The Substrate-Envelope Hypothesis: Structural Analysis Of Novel Hiv-1 Protease Inhibitors Designed To Be Robust Against Drug Resistance, Madhavi Nalam, Akbar Ali, Michael Altman, G. S. Kiran Kumar Reddy, Sripriya Chellappan, Visvaldas Kairys, Aysegul Ozen, Hong Cao, Michael Gilson, Bruce Tidor, Tariq Rana, Celia Schiffer
Evaluating The Substrate-Envelope Hypothesis: Structural Analysis Of Novel Hiv-1 Protease Inhibitors Designed To Be Robust Against Drug Resistance, Madhavi Nalam, Akbar Ali, Michael Altman, G. S. Kiran Kumar Reddy, Sripriya Chellappan, Visvaldas Kairys, Aysegul Ozen, Hong Cao, Michael Gilson, Bruce Tidor, Tariq Rana, Celia Schiffer
Celia A. Schiffer
Drug resistance mutations in HIV-1 protease selectively alter inhibitor binding without significantly affecting substrate recognition and cleavage. This alteration in molecular recognition led us to develop the substrate-envelope hypothesis which predicts that HIV-1 protease inhibitors that fit within the overlapping consensus volume of the substrates are less likely to be susceptible to drug-resistant mutations, as a mutation impacting such inhibitors would simultaneously impact the processing of substrates. To evaluate this hypothesis, over 130 HIV-1 protease inhibitors were designed and synthesized using three different approaches with and without substrate-envelope constraints. A subset of 16 representative inhibitors with binding affinities to wild-type …
Structure Of A Phage Display-Derived Variant Of Human Growth Hormone Complexed To Two Copies Of The Extracellular Domain Of Its Receptor: Evidence For Strong Structural Coupling Between Receptor Binding Sites, Celia Schiffer, Mark Ultsch, Scott Walsh, William Somers, Abraham De Vos, Anthony Kossiakoff
Structure Of A Phage Display-Derived Variant Of Human Growth Hormone Complexed To Two Copies Of The Extracellular Domain Of Its Receptor: Evidence For Strong Structural Coupling Between Receptor Binding Sites, Celia Schiffer, Mark Ultsch, Scott Walsh, William Somers, Abraham De Vos, Anthony Kossiakoff
Celia A. Schiffer
The structure of the ternary complex between the phage display- optimized, high-affinity Site 1 variant of human growth hormone (hGH) and two copies of the extracellular domain (ECD) of the hGH receptor (hGHR) has been determined at 2.6 A resolution. There are widespread and significant structural differences compared to the wild-type ternary hGH hGHR complex. The hGH variant (hGH(v)) contains 15 Site 1 mutations and binds>10(2) tighter to the hGHR ECD (hGH(R1)) at Site 1. It is biologically active and specific to hGHR. The hGH(v) Site 1 interface is somewhat smaller and 20% more hydrophobic compared to the wild-type …
Hiv-1 Protease Inhibitors From Inverse Design In The Substrate Envelope Exhibit Subnanomolar Binding To Drug-Resistant Variants, Michael Altman, Akbar Ali, G. S. Kiran Kumar Reddy, Madhavi Nalam, Saima Anjum, Hong Cao, Sripriya Chellappan, Visvaldas Kairys, Miguel Fernandes, Michael Gilson, Celia Schiffer, Tariq Rana, Bruce Tidor
Hiv-1 Protease Inhibitors From Inverse Design In The Substrate Envelope Exhibit Subnanomolar Binding To Drug-Resistant Variants, Michael Altman, Akbar Ali, G. S. Kiran Kumar Reddy, Madhavi Nalam, Saima Anjum, Hong Cao, Sripriya Chellappan, Visvaldas Kairys, Miguel Fernandes, Michael Gilson, Celia Schiffer, Tariq Rana, Bruce Tidor
Celia A. Schiffer
The acquisition of drug-resistant mutations by infectious pathogens remains a pressing health concern, and the development of strategies to combat this threat is a priority. Here we have applied a general strategy, inverse design using the substrate envelope, to develop inhibitors of HIV-1 protease. Structure-based computation was used to design inhibitors predicted to stay within a consensus substrate volume in the binding site. Two rounds of design, synthesis, experimental testing, and structural analysis were carried out, resulting in a total of 51 compounds. Improvements in design methodology led to a roughly 1000-fold affinity enhancement to a wild-type protease for the …
Point Mutants Of Ehec Intimin That Diminish Tir Recognition And Actin Pedestal Formation Highlight A Putative Tir Binding Pocket, Hui Liu, Padhma Radhakrishnan, Loranne Magoun, Moses Prabu-Jeyabalan, Kenneth Campellone, Pamela Savage, Feng He, Celia Schiffer, John Leong
Point Mutants Of Ehec Intimin That Diminish Tir Recognition And Actin Pedestal Formation Highlight A Putative Tir Binding Pocket, Hui Liu, Padhma Radhakrishnan, Loranne Magoun, Moses Prabu-Jeyabalan, Kenneth Campellone, Pamela Savage, Feng He, Celia Schiffer, John Leong
Celia A. Schiffer
Attachment to host cells by enterohaemorrhagic Escherichia coli (EHEC) is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed an attaching and effacing (AE) lesion. Intimin, an outer membrane protein of EHEC, is required for the formation of AE lesions, as is Tir, a bacterial protein that is translocated into the host cell to function as a receptor for intimin. We established a yeast two-hybrid assay for intimin-Tir interaction and, after random mutagenesis, isolated 24 point mutants in intimin, which disrupted Tir recognition in this system. Analysis of 11 point mutants revealed a correlation between …
The Effect Of Clade-Specific Sequence Polymorphisms On Hiv-1 Protease Activity And Inhibitor Resistance Pathways, Rajintha Bandaranayake, Madhavi Kolli, Nancy King, Ellen Nalivaika, Annie Heroux, Junko Kakizawa, Wataru Sugiura, Celia Schiffer
The Effect Of Clade-Specific Sequence Polymorphisms On Hiv-1 Protease Activity And Inhibitor Resistance Pathways, Rajintha Bandaranayake, Madhavi Kolli, Nancy King, Ellen Nalivaika, Annie Heroux, Junko Kakizawa, Wataru Sugiura, Celia Schiffer
Celia A. Schiffer
The majority of HIV-1 infections around the world result from non-B clade HIV-1 strains. The CRF01_AE (AE) strain is seen principally in Southeast Asia. AE protease differs by approximately 10% in amino acid sequence from clade B protease and carries several naturally occurring polymorphisms that are associated with drug resistance in clade B. AE protease has been observed to develop resistance through a nonactive-site N88S mutation in response to nelfinavir (NFV) therapy, whereas clade B protease develops both the active-site mutation D30N and the nonactive-site mutation N88D. Structural and biochemical studies were carried out with wild-type and NFV-resistant clade B …
Human Immunodeficiency Virus Type 1 Protease-Correlated Cleavage Site Mutations Enhance Inhibitor Resistance, Madhavi Kolli, Eric Stawiski, Colombe Chappey, Celia Schiffer
Human Immunodeficiency Virus Type 1 Protease-Correlated Cleavage Site Mutations Enhance Inhibitor Resistance, Madhavi Kolli, Eric Stawiski, Colombe Chappey, Celia Schiffer
Celia A. Schiffer
Drug resistance is an important cause of antiretroviral therapy failure in human immunodeficiency virus (HIV)-infected patients. Mutations in the protease render the virus resistant to protease inhibitors (PIs). Gag cleavage sites also mutate, sometimes correlating with resistance mutations in the protease, but their contribution to resistance has not been systematically analyzed. The present study examines mutations in Gag cleavage sites that associate with protease mutations and the impact of these associations on drug susceptibilities. Significant associations were observed between mutations in the nucleocapsid-p1 (NC-p1) and p1-p6 cleavage sites and various PI resistance-associated mutations in the protease. Several patterns were frequently …
Discovery And Selection Of Tmc114, A Next Generation Hiv-1 Protease Inhibitor, Dominique Surleraux, Abdellah Tahri, Wim Verschueren, Geert Pille, Herman De Kock, Tim Jonckers, Anik Peeters, Sandra De Meyer, Hilde Azijn, Rudi Pauwels, Marie-Pierre De Bethune, Nancy King, Moses Prabu-Jeyabalan, Celia Schiffer, Piet Wigerinck
Discovery And Selection Of Tmc114, A Next Generation Hiv-1 Protease Inhibitor, Dominique Surleraux, Abdellah Tahri, Wim Verschueren, Geert Pille, Herman De Kock, Tim Jonckers, Anik Peeters, Sandra De Meyer, Hilde Azijn, Rudi Pauwels, Marie-Pierre De Bethune, Nancy King, Moses Prabu-Jeyabalan, Celia Schiffer, Piet Wigerinck
Celia A. Schiffer
The screening of known HIV-1 protease inhibitors against a panel of multi-drug-resistant viruses revealed the potent activity of TMC126 on drug-resistant mutants. In comparison to amprenavir, the improved affinity of TMC126 is largely the result of one extra hydrogen bond to the backbone of the protein in the P2 pocket. Modification of the substitution pattern on the phenylsulfonamide P2' substituent of TMC126 created an interesting SAR, with the close analogue TMC114 being found to have a similar antiviral activity against the mutant and the wild-type viruses. X-ray and thermodynamic studies on both wild-type and mutant enzymes showed an extremely high …
Accessibility And Order Of Water Sites In And Around Proteins: A Crystallographic Time-Averaging Study, Celia Schiffer, Wilfred Van Gunsteren
Accessibility And Order Of Water Sites In And Around Proteins: A Crystallographic Time-Averaging Study, Celia Schiffer, Wilfred Van Gunsteren
Celia A. Schiffer
Water plays an essential role in most biological processes. Water molecules solvating biomolecules are generally in fast exchange with the environment. Nevertheless, well-defined electron density is seen for water associated with proteins whose crystal structure is determined to high resolution. The relative accessibility of these water sites is likely to be relevant to their biological role but is difficult to assess. A time-averaging crystallographic refinement simulation on basic pancreatic trypsin inhibitor successfully characterizes the relative accessibility of the crystallographic water sites. In such a refinement simulation water diffuses through the crystal lattice in a manner that is consistent with the …
Contribution Of Ser386 And Ser396 To Activation Of Interferon Regulatory Factor 3, Weijun Chen, Hema Srinath, Suvana Lam, Celia Schiffer, William Royer, Kai Lin
Contribution Of Ser386 And Ser396 To Activation Of Interferon Regulatory Factor 3, Weijun Chen, Hema Srinath, Suvana Lam, Celia Schiffer, William Royer, Kai Lin
Celia A. Schiffer
IRF-3, a member of the interferon regulatory factor (IRF) family of transcription factors, functions in innate immune defense against viral infection. Upon infection, host cell IRF-3 is activated by phosphorylation at its seven C-terminal Ser/Thr residues: (385)SSLENTVDLHISNSHPLSLTS(405). This phosphoactivation triggers IRF-3 to react with the coactivators, CREB-binding protein (CBP)/p300, to form a complex that activates target genes in the nucleus. However, the role of each phosphorylation site for IRF-3 phosphoactivation remains unresolved. To address this issue, all seven Ser/Thr potential phosphorylation sites were screened by mutational studies, size-exclusion chromatography, and isothermal titration calorimetry. Using purified proteins, we show that CBP …