Rationalizing Tight Ligand Binding through Cooperative Interaction Networks

Small modifications of the molecular structure of a ligand sometimes cause strong gains in binding affinity to a protein target, rendering a weakly active chemical series suddenly attractive for further optimization. Our goal in this study is to better rationalize and predict the occurrence of such interaction hot-spots in receptor binding sites. To this end, we introduce two new concepts into the computational description of molecular recognition. First, we take a broader view of noncovalent interactions and describe protein–ligand binding with a comprehensive set of favorable and unfavorable contact types, including for example halogen bonding and orthogonal multipolar interactions. Second, we go beyond the commonly used pairwise additive treatment of atomic interactions and use a small world network approach to describe how interactions are modulated by their environment. This approach allows us to capture local cooperativity effects and considerably improves the performance of a newly derived empirical scoring function, ScorpionScore. More importantly, however, we demonstrate how an intuitive visualization of key intermolecular interactions, interaction networks, and binding hot-spots supports the identification and rationalization of tight ligand binding

Torsion Angle Preferences in Druglike Chemical Space: A Comprehensive Guide

Crystal structure databases offer ample opportunities to derive small molecule conformation preferences, but the derived knowledge is not systematically applied in drug discovery research. We address this gap by a comprehensive and extendable expert system enabling quick assessment of the probability of a given conformation to occur. It is based on a hierarchical system of torsion patterns that cover a large part of druglike chemical space. Each torsion pattern has associated frequency histograms generated from CSD and PDB data and, derived from the histograms, traffic-light rules for frequently observed, rare, and highly unlikely torsion ranges. Structures imported into the corresponding software are annotated according to these rules. We present the concept behind the tree of torsion patterns, the design of an intuitive user interface for the management and usage of the torsion library, and we illustrate how the system helps analyze and understand conformation properties of substructures widely used in medicinal chemistry

Torsion Angle Preferences in Druglike Chemical Space: A Comprehensive Guide

Crystal structure databases offer ample opportunities to derive small molecule conformation preferences, but the derived knowledge is not systematically applied in drug discovery research. We address this gap by a comprehensive and extendable expert system enabling quick assessment of the probability of a given conformation to occur. It is based on a hierarchical system of torsion patterns that cover a large part of druglike chemical space. Each torsion pattern has associated frequency histograms generated from CSD and PDB data and, derived from the histograms, traffic-light rules for frequently observed, rare, and highly unlikely torsion ranges. Structures imported into the corresponding software are annotated according to these rules. We present the concept behind the tree of torsion patterns, the design of an intuitive user interface for the management and usage of the torsion library, and we illustrate how the system helps analyze and understand conformation properties of substructures widely used in medicinal chemistry

A Novel Mouse Model of <i>Campylobacter jejuni</i> Gastroenteritis Reveals Key Pro-inflammatory and Tissue Protective Roles for Toll-like Receptor Signaling during Infection

<div><p><i>Campylobacter jejuni</i> is a major source of foodborne illness in the developed world, and a common cause of clinical gastroenteritis. Exactly how <i>C. jejuni</i> colonizes its host's intestines and causes disease is poorly understood. Although it causes severe diarrhea and gastroenteritis in humans, <i>C. jejuni</i> typically dwells as a commensal microbe within the intestines of most animals, including birds, where its colonization is asymptomatic. Pretreatment of C57BL/6 mice with the antibiotic vancomycin facilitated intestinal <i>C. jejuni</i> colonization, albeit with minimal pathology. In contrast, vancomycin pretreatment of mice deficient in SIGIRR (<i>Sigirr^−/−</i>), a negative regulator of MyD88-dependent signaling led to heavy and widespread <i>C. jejuni</i> colonization, accompanied by severe gastroenteritis involving strongly elevated transcription of Th1/Th17 cytokines. <i>C. jejuni</i> heavily colonized the cecal and colonic crypts of <i>Sigirr^−/−</i> mice, adhering to, as well as invading intestinal epithelial cells. This infectivity was dependent on established <i>C. jejuni</i> pathogenicity factors, capsular polysaccharides (<i>kpsM</i>) and motility/flagella (<i>flaA</i>). We also explored the basis for the inflammatory response elicited by <i>C. jejuni</i> in <i>Sigirr^−/−</i> mice, focusing on the roles played by Toll-like receptors (TLR) 2 and 4, as these innate receptors were strongly stimulated by <i>C. jejuni</i>. Despite heavy colonization, <i>Tlr4^−/−/Sigirr^−/−</i> mice were largely unresponsive to infection by <i>C. jejuni</i>, whereas <i>Tlr2^−/−/Sigirr^−/−</i> mice developed exaggerated inflammation and pathology. This indicates that TLR4 signaling underlies the majority of the enteritis seen in this model, whereas TLR2 signaling had a protective role, acting to promote mucosal integrity. Furthermore, we found that loss of the <i>C. jejuni</i> capsule led to increased TLR4 activation and exaggerated inflammation and gastroenteritis. Together, these results validate the use of <i>Sigirr^−/−</i> mice as an exciting and relevant animal model for studying the pathogenesis and innate immune responses to <i>C. jejuni</i>.</p></div

TLR2 and 4 reporter assays.

<p>HEK-Blue hTLR2 (A) and HEK-Blue hTLR4 (B) reporter cell lines were exposed for 4 hrs to either live and heat-killed wildtype <i>C. jejuni</i> 81–176, <i>ΔkpsM</i> or <i>ΔkpsM+kpsM</i>. The wild-type <i>C. jejuni</i> stimulates both TLR2 and TLR4 in a dose-dependent fashion. The <i>ΔkpsM</i> mutant significantly increased the signaling by both TLR2 and TLR4, as indicated by the assay, with the increase in stimulation also being in a dose-dependent manner, except for the TLR4 assay where the readers were near the maximum for both the 20 and 200 MOI readings. The complemented <i>ΔkpsM+kpsM</i> strain completely restored the wild-type phenotype with TLR2 and mostly restored the phenotype with TLR4. Values represent the mean of three independent experiments and statistical significance was determined by a two-way ANOVA with a Bonferroni post-test. (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).</p

Colonization of WT and <i>Sigirr−/−</i> mice by <i>ΔkpsM</i> and <i>ΔflaA</i>.

<p>Colonization of WT and <i>Sigirr^−/−</i> mice by <i>ΔkpsM</i> (A) and <i>ΔflaA</i> (B) and their respective complemented strains (<i>ΔkpsM</i>+<i>kpsM</i> and <i>ΔflaA</i>+<i>flaA</i>), at both 3 and 7 DPI. The <i>ΔkpsM</i> mutant exhibited reduced colonization at 3 DPI only, while the <i>ΔflaA</i> mutant was unable to colonize at either 3 or 7 DPI. The complemented <i>ΔflaA+flaA</i> colonized at high numbers, similar to wild-type. Statistical significance was determined by a Mann-Whitney test, ***p<0.001. n = 7–10 mice for the <i>ΔkpsM</i> mutant and complement, n = 5–7 mice for the <i>ΔflaA</i> mutant and complement. (C) H&E stained histological sections of ceca recovered from WT or <i>Sigirr^−/−</i> mice infected with <i>C. jejuni ΔflaA</i> at ×100 magnification. No noticeable inflammation was evident in either WT or <i>Sigirr^−/−</i> mice infected with <i>C. jejuni ΔflaA</i>. (D) H&E stained histological sections of ceca recovered from WT or <i>Sigirr^−/−</i> mice infected with <i>C. jejuni ΔkpsM</i> 7 DPI. Upper panels are ×100 magnification, while lower panels are ×400 magnification. WT mice did not exhibit signs of inflammation when infected with <i>C. jejuni ΔkpsM</i>, however <i>Sigirr^−/−</i> mice exhibited signs of severe inflammation at 7 DPI.</p

Colonization and pathology of <i>Sigirr^−/−</i> and TLR-deficient mice by <i>C. jejuni ΔkpsM in vivo</i>.

<p>(A) H&E stained histological sections of ceca recovered from <i>Sigirr^−/−</i>, <i>Tlr2^−/−/Sigirr^−/−</i> and <i>Tlr4^−/−/Sigirr^−/−</i> mice, colonized with <i>C. jejuni ΔkpsM</i> 7 DPI, at 100× magnification. Very severe inflammation is evident in the <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice, however once again, no significant pathology was evident in the <i>Tlr4^−/−/Sigirr^−/−</i> mice. (B) Immunofluorescence of <i>Sigirr^−/−</i> mice infected by <i>C. jejuni ΔkpsM</i>, 7 DPI. Sections are stained for DAPI (blue), β-actin (green), and <i>C. jejuni</i> (red). <i>Sigirr^−/−</i> mice exhibit significant neutrophil infiltration, hyperplasia, and <i>C. jejuni ΔkpsM</i> is clearly visible in large masses within the cecal crypts. (C) Pathological scoring was done by two blinded observers, using H&E stained, formalin-fixed cecal tissue sections. Each condition represents a minimum of three separate experimental replicates, with 2–3 mice per experiment. Control mice were used as a reference and consisted of 3 uninfected mice, pre-treated with a single dose of 5 mg/100 µl vancomycin, and euthanized 3 days post-treatment. Only <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice at 7 DPI showed a significant increase in pathology (****p<0.0001), relative to the uninfected <i>Sigirr^−/−</i> control. The <i>Tlr2^−/−/Sigirr^−/−</i> mice also exhibited statistically significantly higher inflammation at 7 DPI relative to <i>Sigirr^−/−</i> mice also at 7 DPI (**p<0.001). In contrast, none of the mouse strains at 3 DPI showed any statistically significant increase in pathology, relative to control mice. Statistical significance was determined using a two-way ANOVA and a Bonferroni post-test.</p

Immunofluorescent staining of intracellular <i>C. jejuni in vivo</i>.

<p>(A) Intracellular <i>C. jejuni</i> are visible in the cecal epithelium. <i>C. jejuni</i> (red), are visible against the β-actin (green) and the nuclei (DAPI, blue) of the cecal epithelium of a <i>Sigirr^−/−</i> mouse, ×1000 magnification. (B) Confocal image of <i>C. jejuni</i> (red) present within epithelial cells of the colon of a <i>Sigirr^−/−</i> mouse, highlighted against the Cytokeratin 19 of the cytoskeleton (green) and the nuclei (blue), with the z-stack cross-section indicating the <i>C. jejuni</i> within the cell. (C) Cross-section of a Z-stack, of a colonic epithelial cell of a <i>Sigirr^−/−</i> mouse. The internalized <i>C. jejuni</i> (red) are clearly visible within the cytoplasm of the cell, as outlined by the β-actin (green) along the edge of the cell. (D) Internalized <i>C. jejuni</i> (red) co-localize with LAMP-1 positive (green) vesicles present within epithelial cells of a <i>Sigirr^−/−</i> mouse colon.</p

Cytokine production in infected mice.

<p>(A–H) RT-qPCR conducted on RNA extracted from the ceca of uninfected control or infected mice. Controls are the pooled results of 9, vancomycin pre-treated, but uninfected mice, euthanized 3 days post-treatment. All infected mice represent the average results of 3 independent experiments, each of which include the pooled RNA of 2–3 mice, for 6–9 mice total for each mouse strain, euthanized either 3 or 7 DPI. Statistical significance was determined using a One way ANOVA with a Bonferroni post-test. * p<0.05 relative to WT (B6) or <i>Sigirr^−/−</i> uninfected control mice. ** p<0.05 relative to the infected WT (B6) mice euthanized on the same DPI in addition to the uninfected control mice.</p

Colonization of WT and <i>Sigirr−/−</i> mice by <i>C. jejuni</i> 81–176, 3 and 7 DPI.

<p>(A) High numbers (∼10⁹ CFUs/g) of <i>C. jejuni</i> were recovered at both 3 and 7 DPI from the ceca of infected mice that were pre-treated with 5 mg of vancomycin. No statistically significant differences in numbers were found between WT and <i>Sigirr^−/−</i> mice as indicated by a t-test, p>0.05. n = 10 or 11 WT mice, and 12 or 13 <i>Sigirr^−/−</i> mice for 3 and 7 DPI respectively. (B) H&E stained, formalin-fixed histological sections of ceca recovered from WT or <i>Sigirr^−/−</i> mice 3 and 7 DPI. Upper panels are ×100 magnification, while lower panels are ×400 magnification. (C) Pathological scoring was done by two blinded observers, using H&E stained, formalin-fixed cecal tissue sections. Each condition represents a minimum of three separate experimental replicates, with 2–3 mice per experiment for a total of 6–9 mice per group. Control mice were used as a reference and consisted of 3 uninfected mice, pre-treated with a single dose of 5 mg/100 µl vancomycin, and euthanized 3 days post-treatment. WT (B6) mice did not exhibit any significant signs of inflammation, while <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice showed a significant increase relative to the uninfected <i>Sigirr^−/−</i> control, both 3 and 7 DPI. <i>Tlr4^−/−/Sigirr^−/−</i> mice showed a statistically significant increase, relative to control mice at 3 DPI only, but even at 3 DPI, were significantly less than either <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice. Statistical significance was determined using a two-way ANOVA and a Bonferroni post-test (NS p>0.05, *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).</p