Steven Sheriff

The collectins in innate immunity

The collectins are proteins with collagen tails and globular lectin domains that appear to play an important role in mammalian first line host defense. Recent insights have clarified the structural basis of ligand recognition, the interactions of collectins with complement cascades, and the association with disease susceptibility.

Redefining the minimal antigen-binding fragment.

The X-ray structures of a lysozyme-complexed camel VH domain and an antigen-free llama VH domain reveal novel features that open up new possibilities for generating and engineering minimal antigen recognition units.

Structure and Specificity of the Anti-Digoxin Antibody 40-50

We determined the sequence, specificity for structurally related cardenolides, and three-dimensional structure of the anti-digoxin antibody 40-50 Fab in complex with ouabain. The 40-50 antibody does not share close sequence homology with other high-affinity anti-digoxin antibodies. Measurement of the binding constants of structurally distinct digoxin analogs indicated a well-defined specificity pattern also distinct from other anti-digoxin antibodies. The 40-50-ouabain Fab complex crystallizes in space group C2 with cell dimensions of a = 93.7 A, b = 84.8 A, c = 70.1 A, beta = 128.0 degrees. The structure of the complex was determined by X-ray crystallography and refined at a resolution of 2.7 A. The hapten is bound in a pocket extending as a groove from the center of the combining site across the light chain variable domain, with five of the six complementarity-determining regions involved in interactions with the hapten. Approximately three-quarters of the hapten surface area is buried in the complex; two hydrogen bonds are formed between the antibody and hapten. The surface area of the antibody combining site buried by ouabain is contributed equally by the light and heavy chain variable domains. Over half of the surface area buried on the Fab consists of the aromatic side-chains. The surface complementarity between hapten and antibody is sufficient to make the complex specific for only one lactone ring conformation in the hapten. The crystal structure of the 40-50-ouabain complex allows qualitative explanation of the observed fine specificities of 40-50, including that for the binding of haptens substituted at the 16 and 12 positions. Comparison of the crystal structures of 40-50 complexed with ouabain and the previously determined 26-10 anti-digoxin Fab complexed with digoxin, demonstrates that the antibodies bind these structurally related haptens in different orientations, consistent with their different fine specificities. These results demonstrate that the immune system can generate antibodies that provide diverse structural solutions to the binding of even small molecules.

Structures of adnectin/protein complexes reveal an expanded binding footprint.

Adnectins are targeted biologics derived from the tenth type III domain of human fibronectin (¹⁰Fn3), a member of the immunoglobulin superfamily. Target-specific binders are selected from libraries generated by diversifying the three ¹⁰Fn3 loops that are analogous to the complementarity determining regions of antibodies. The crystal structures of two Adnectins were determined, each in complex with its therapeutic target, EGFR or IL-23. Both Adnectins bind different epitopes than those bound by known monoclonal antibodies. Molecular modeling suggests that some of these epitopes might not be accessible to antibodies because of the size and concave shape of the antibody combining site. In addition to interactions from the Adnectin diversified loops, residues from the N terminus and/or the β strands interact with the target proteins in both complexes. Alanine-scanning mutagenesis confirmed the calculated binding energies of these β strand interactions, indicating that these nonloop residues can expand the available binding footprint.

Discovery and Preclinical Characterization of the Cyclopropylindolobenzazepine BMS-791325, A Potent Allosteric Inhibitor of the Hepatitis C Virus NS5B Polymerase.

Described herein are structure-activity relationship studies that resulted in the optimization of the activity of members of a class of cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors. Subsequent iterations of analogue design and syntheses successfully addressed off-target activities, most notably human pregnane X receptor (hPXR) transactivation, and led to significant improvements in the physicochemical properties of lead compounds. Those analogues exhibiting improved solubility and membrane permeability were shown to have notably enhanced pharmacokinetic profiles. Additionally, a series of alkyl bridged piperazine carboxamides was identified as being of particular interest, and from which the compound BMS-791325 (2) was found to have distinguishing antiviral, safety, and pharmacokinetic properties that resulted in its selection for clinical evaluation.

Refined Structure of the Monoclonal Antibody HyHEL-5 with its Antigen Hen Egg-White Lysozyme

The crystal structure of the complex of the antibody Fab, HyHEL-5, with its antigen, hen egg-white lysozyme, has been refined at 2.65 A resolution to an R value of 0.196. The resulting model has significantly better stereochemistry than the previously reported model of the complex, PDB reference 2HFL, and sufficiently improved phases, permitting the reliable location of a number of water molecules. No major conformational differences are observed between this structure and that previously reported, although small differences occur throughout the complex. 82 water molecules have been assigned, of which three are in the antibody-antigen interface involved in a hydrogen-bonding network. Three other waters are trapped within the interface between V(H) and V(L) and a fourth water molecule is observed near the interface but buried below the lysozyme surface as observed in crystal structures of lysozyme alone.

Implementation of a six-dimensional search using the AMoRe translation function for difficult molecular-replacement problems

A six-dimensional molecular-replacement procedure has been implemented using Perl scripts and the CCP4 version of the translation function of the molecular-replacement program AMoRe. These tools have allowed the structure determination of CTLA-4 monomer from NMR-derived coordinates, and of a potential complex of MurD with a substrate. In both cases other molecular-replacement programs, X-PLOR and AMoRe, when used in a conventional manner, either failed or did not yield an obvious solution.

Small molecule antagonist of leukocyte function associated antigen-1 (LFA-1): structure-activity relationships leading to the identification of 6-((5S,9R)-9-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]nonan-7-yl)nicotinic acid (BMS-688521).

Leukocyte function-associated antigen-1 (LFA-1), also known as CD11a/CD18 or alpha(L)beta(2), belongs to the beta(2) integrin subfamily and is constitutively expressed on all leukocytes. The major ligands of LFA-1 include three intercellular adhesion molecules 1, 2, and 3 (ICAM 1, 2, and 3). The interactions between LFA-1 and the ICAMs are critical for cell adhesion, and preclinical animal studies and clinical data from the humanized anti-LFA-1 antibody efalizumab have provided proof-of-concept for LFA-1 as an immunological target. This article will detail the structure-activity relationships (SAR) leading to a novel second generation series of highly potent spirocyclic hydantoin antagonists of LFA-1. With significantly enhanced in vitro and ex vivo potency relative to our first clinical compound (1), as well as demonstrated in vivo activity and an acceptable pharmacokinetic and safety profile, 6-((5S,9R)-9-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro-[4.4]nonan-7-yl)nicotinic acid (2e) was selected to advance into clinical trials.

Refined structures of bobwhite quail lysozyme uncomplexed and complexed with the HyHEL-5 Fab fragment.

The HyHEL‐5 antibody has more than a thousandfold lower affinity for bobwhite quail lysozyme (BWQL) than for hen egg‐white lysozyme (HEL). Four sequence differences exist between BWQL and HEL, of which only one is involved in the interface with the Fab. The structure of bobwhite quail lysozyme has been determined in the uncomplexed state in two different crystal forms and in the complexed state with HyHEL‐5, an anti‐hen egg‐white lysozyme Fab. Similar backbone conformations are observed in the three molecules of the two crystal forms of uncomplexed BWQL, although they show considerable variability in side‐chain conformation. A relatively mobile segment in uncomplexed BWQL is observed to be part of the HyHEL‐5 epitope. No major backbone conformational differences are observed in the lysozyme upon complex formation, but side‐chain conformational differences are seen in surface residues that are involved in the interface with the antibody. The hydrogen bonding in the interface between BWQL and HyHEL‐5 is similar to that in previously determined lysozyme‐HyHEL‐5 complexes.

Syntheses and initial evaluation of a series of indolo-fused heterocyclic inhibitors of the polymerase enzyme (NS5B) of the hepatitis C virus.

Herein, we present initial SAR studies on a series of bridged 2-arylindole-based NS5B inhibitors. The introduction of bridging elements between the indole N1 and the ortho-position of the 2-aryl moiety resulted in conformationally constrained heterocycles that possess multiple additional vectors for further exploration. The binding mode and pharmacokinetic (PK) properties of select examples, including: 13-cyclohexyl-6-oxo-6,7-dihydro-5H-indolo[2,1-d][1,4]benzodiazepine-10-carboxylic acid (7) (IC(50)=0.07 μM, %F=18), are reported.