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Abstract Background Haemophilus ducreyi, the causative agent of the sexually transmitted disease chancroid, contains a flp (fimbria like protein) operon that encodes proteins predicted to contribute to adherence and pathogenesis. H. ducreyi mutants that lack expression of Flp1 and Flp2 or TadA, which has homology to NTPases of type IV secretion systems, have decreased abilities to attach to and form microcolonies on human foreskin fibroblasts (HFF). A tadA mutant is attenuated in its ability to cause disease in human volunteers and in the temperature dependent rabbit model, but a flp1flp2 mutant is virulent in rabbits. Whether a flp deletion mutant would cause disease in humans is not clear. Results We constructed 35000HPΔflp1-3, a deletion mutant that lacks expression of all three Flp proteins but has an intact tad secretion system. 35000HPΔflp1-3 was impaired in its ability to form microcolonies and to attach to HFF in vitro when compared to its parent (35000HP). Complementation of the mutant with flp1-3 in trans restored the parental phenotype. To test whether expression of Flp1-3 was necessary for virulence in humans, ten healthy adult volunteers were experimentally infected with a fixed dose of 35000HP (ranging from 54 to 67 CFU) on one arm and three doses of 35000HPΔflp1-3 (ranging from 63 to 961 CFU) on the other arm. The overall papule formation rate for the parent was 80% (95% confidence interval, CI, 55.2%-99.9%) and for the mutant was 70.0% (95% CI, 50.5%-89.5%) (P = 0.52). Mutant papules were significantly smaller (mean, 11.2 mm2) than were parent papules (21.8 mm2) 24 h after inoculation (P = 0.018). The overall pustule formation rates were 46.7% (95% CI 23.7-69.7%) at 30 parent sites and 6.7% (95% CI, 0.1-19.1%) at 30 mutant sites (P = 0.001). Conclusion These data suggest that production and secretion of the Flp proteins contributes to microcolony formation and attachment to HFF cells in vitro. Expression of flp1-3 is also necessary for H. ducreyi to initiate disease and progress to pustule formation in humans. Future studies will focus on how Flp proteins contribute to microcolony formation and attachment in vivo.

Background: Efforts to identify risk and resilience factors for anxiety severity and course during the COVID-19 pandemic have focused primarily on demographic rather than psychological variables. Intolerance of uncertainty (IU), a transdiagnostic risk factor for anxiety, may be a particularly relevant vulnerability factor., Method: N = 641 adults with pre-pandemic anxiety data reported their anxiety, IU, and other pandemic and mental health-related variables at least once and up to four times during the COVID-19 pandemic, with assessments beginning in May 2020 through March 2021., Results: In preregistered analyses using latent growth models, higher IU at the first pandemic timepoint predicted more severe anxiety, but also a sharper decline in anxiety, across timepoints. This finding was robust to the addition of pre-pandemic anxiety and demographic predictors as covariates (in the full sample) as well as pre-pandemic depression severity (in participants for whom pre-pandemic depression data were available). Younger age, lower self/parent education, and self-reported history of COVID-19 illness at the first pandemic timepoint predicted more severe anxiety across timepoints with strong model fit, but did not predict anxiety trajectory., Conclusions: IU prospectively predicted more severe anxiety but a sharper decrease in anxiety over time during the pandemic, including after adjustment for covariates. IU therefore appears to have unique and specific predictive utility with respect to anxiety in the context of the COVID-19 pandemic., Competing Interests: Declaration of Competing Interest Lauren S. Hallion and Gordon J. G. Asmundson are Associate Editor and Editor-in-Chief at Journal of Anxiety Disorders, respectively, and receive financial support through payments for their editorial work on the journal. Neither author was involved in the review of the manuscript or the decision regarding its acceptance. We have no additional conflicts of interest to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Sexual-minority adolescents frequently endure peer rejection, yet scant research has investigated sexual-orientation differences in behavioral and neural reactions to peer rejection and acceptance. In a community sample of adolescents approximately 15 years old (47.2% female; same-sex attracted: n = 36, exclusively other-sex attracted: n = 310), we examined associations among sexual orientation and behavioral and neural reactivity to peer feedback and the moderating role of family support. Participants completed a social-interaction task while electroencephalogram data were recorded in which they voted to accept/reject peers and, in turn, received peer acceptance/rejection feedback. Compared with heterosexual adolescents, sexual-minority adolescents engaged in more behavioral efforts to ingratiate after peer rejection and demonstrated more blunted neural reactivity to peer acceptance at low, but not medium or high, levels of family support. By using a simulated real-world social-interaction task, these results demonstrate that sexual-minority adolescents display distinct behavioral and neural reactions to peer acceptance and rejection., Competing Interests: Declaration of Conflicting Interests The author(s) declared that there were no conflicts of interest with respect to the authorship or the publication of this article.

Background: Offspring of depressed mothers have elevated risk of developing depression because they are exposed to greater stress. While generally assumed that youth's increased exposure to stress is due to the environmental effects of living with a depressed parent, youth's genes may influence stress exposure through gene-environment correlations (rGEs). To understand the relationship between risk for depression and stress, we examined the effects of polygenic risk for depression on youth stress exposure., Methods: We examined the relations of a polygenic risk score (PRS) for depression (DEP-PRS), as well as PRSs for 5 other disorders, with youth stress exposure. Data were from a longitudinal study of a community sample of youth and their parents (n = 377) focusing on data collected at youth's aged 12 and 15 assessments., Results: Elevated youth DEP-PRS was robustly associated with increased dependent stress, particularly interpersonal events. Exploratory analyses indicated that findings were driven by major stress and were not moderated by maternal nor paternal history of depression, and of the 5 additional PRSs tested, only elevated genetic liability for bipolar I was associated with increased dependent stress-particularly non-interpersonal events., Limitations: Like other PRS studies, we focused on those of European ancestry thus, generalizability of findings is limited., Conclusion: Polygenic risk contributes to youth experiencing stressful life events which are dependent on their behavior. This rGE appears to be specific to genetic risk for mood disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

The COVID-19 pandemic has had medical, economic and behavioral implications on a global scale, with research emerging to indicate that it negatively impacted the population's mental health as well. The current study utilizes longitudinal data to assess whether the pandemic led to an increase in depression and anxiety across participants or whether a diathesis-stress model would be more appropriate. An international group of 218 participants completed measures of depression, anxiety, rumination and distress intolerance at two baselines six months apart as well as during the onset of the COVID-19 pandemic exactly 12 months later. Contrary to expectations, depression, rumination, and distress intolerance were at equivalent levels during the pandemic as they were at baseline. Anxiety was reduced by a trivial degree (d = .10). Furthermore, a comparison of quantitative explanatory models indicated that symptom severity and pandemic-related environmental stressors predicted pandemic-related distress. Pandemic-related distress did not predict symptom severity. These findings underscore the necessity of longitudinal designs and diathesis-stress models in the study of mental health during the COVID-19 pandemic. They also emphasize that individuals with higher rates of baseline psychopathology are as particularly at risk for higher levels of distress in response to disaster-related stressors., Competing Interests: The authors have declared that no competing interests exist.

The current studies systematically examined a new version of the Questionnaire-Based Implicit Association Test (qIAT), which minimizes the differences between direct and indirect modes of assessment. Studies 1a ( N  = 276) and 1 b ( N  = 238) tested a method that enables an indirect assessment of questionnaires that include only non-reversed items. Studies 2a ( N  = 255) and 2 b ( N  = 284) tested a task that substitutes the problematic construct-related category labels with generic, universal categories. These studies, which focused on extraversion, supported the feasibility, reliability and validity of this procedure. Studies 3a-3c ( N  = 159, 154 and 151, respectively) supported the internal consistency, test-retest reliability and convergent validity of these methods, assessing three well-researched, semantically complex personality scales: Aggressive Humor Style, Need for Closure and Anxiety Sensitivity. Studies 4a ( N  = 195) and 4 b ( N  = 283) supported the implicitness of the qIAT, as most respondents were not aware of this task's purpose. In Study 4c ( N  = 598), participants who reported using antidepressants had lower self-esteem qIAT scores compared to a control group, thus supporting the criterion validity of this task. Taken together, findings suggest that the new qIAT substantially increases the scope of indirect personality assessment.

The current article presents a model wherein reinforcement sensitivity predicts depression and anxiety via trait preferences for concomitant emotion regulation strategies. In Study 1 (N = 593), BAS sensitivity positively predicted reappraisal and BIS sensitivity negatively predicted it. Reappraisal then negatively predicted depression. BIS sensitivity also predicted rumination, which predicted both depression and anxiety. Study 2a confirmed the model developed in Study 1 with an independent sample (N = 513) and examined the relationships longitudinally. While the cross-sectional relationships were generally maintained, reinforcement sensitivity did not predict reappraisal. In Study 2b, participants (N = 218) were assessed a third time one year later, at the onset of the COVID-19 pandemic. During this stressful time, BAS sensitivity did longitudinally predict reappraisal. These studies highlight the role of emotion regulation in mediating the relationship between reinforcement sensitivity and affective pathology, particularly during times of high stress., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The self-esteem Questionnaire-based Implicit Association Test (SE-qIAT) provides an indirect assessment of general self-worth that is based on the items of the well-validated Rosenberg Self-Esteem Scale (RSES), and the structure of this variant of the IAT enables a clearer interpretation, compared with the conventional self-esteem IAT. Study 1 (N = 224) provided support for the internal consistency, test-retest reliability, and implicit-explicit convergent validity of the SE-qIAT. In Study 2 (N = 305), the correlation of the SE-qIAT with the explicit RSES was replicated, and it was larger than the correlations of the SE-qIAT with other self-reports. As to criterion validity, the SE-qIAT moderated the effect of a mild social threat (being excluded in the Cyberball game) on participants' performance in a subsequent anagram task, and this effect was incremental to the explicit self-esteem assessment. In Study 3 (N = 334), the SE-qIAT correlated positively with the self-esteem IAT and negatively with a measure of depression. The two implicit tasks correlated uniquely with each other, above and beyond the variance they each shared with the explicit RSES. Taken together, these findings provide initial support for the reliability and validity of the SE-qIAT., (© 2021 British Psychological Society.)

This study examines relationships among different aspects of therapeutic alliance with treatment outcome, adherence and attrition in internet delivered cognitive behavioral therapy (ICBT) for panic disorder., We examined alliance-outcome relationships in ICBT ( N  = 74) using a newly developed self-report alliance measure that disentangles alliance with program content (Internet Patient's Experience of Attunement and Responsiveness with the program; I-PEARp) and with the therapist (I-PEARt). We compared ICBT outcomes of patient rated and therapist-rated alliance with conventional alliance scales (WAI-6 and WAI-T)., Consistent with our hypothesis, I-PEARp and I-PEARt distinguished between different aspects of the alliance and predicted outcomes better than standard alliance scales. Furthermore, higher ratings of I-PEARp were associated with subsequent lower symptoms and lower symptoms were associated with higher subsequent alliance. In contrast, I-PEARt predicted adherence, but not symptoms. Although therapists' ratings of alliance (thI-PEAR) improved significantly during treatment, they did not predict subsequent symptoms, adherence, or dropout., Results indicate that the patient experience of the alliance in ICBT includes two aspects, each of which uniquely contributes to outcomes; patient connection to the program is related to symptom outcomes whereas the dyadic relationship with the therapist serves as the glue to allow the treatment to hold.

Self-report questionnaires can only yield information that people are able and willing to report, but implicit assessment methods are not commonly used in mainstream personality research. The Questionnaire-based Implicit Association Test (qIAT) was designed to address the limitations associated with the conventional self-concept IAT, and it enables an indirect assessment that is based on the items of standard self-reports. The present studies examined the psychometric properties of the qIAT across two personality constructs. Study 1 (N = 528) provided support for the internal consistency, test-retest reliability and convergent and discriminant validity of the qIAT that measured extraversion. Study 2 (N = 164) supported the reliability and validity of the qIAT assessment of conscientiousness, which predicted who returned to complete the second session of the study two weeks later, for which participants were paid in advance. This same prediction effect was marginally significant in Study 3 (N = 200), and across both Studies 2 and 3 the qIAT prediction of the criterion behavior was incremental to the parallel self-report questionnaire. Taken together, findings support the reliability and validity of the qIAT, which enables the indirect measurement of a wide variety of distinct personality constructs, currently measured only by self-report scales.

Bipolar spectrum disorders are characterized by alternating intervals of extreme positive and negative affect. We performed a meta-analysis to test the hypothesis that such disorders would be related to dysregulated reinforcement sensitivity. First, we reviewed 23 studies that reported the correlation between self-report measures of (hypo)manic personality and measures of reinforcement sensitivity. A large relationship was found between (hypo)manic personality and BAS sensitivity (g = .74), but not with BIS sensitivity (g = -.08). This stands in contrast to self-reported depression which has a small, negative relationship with BAS sensitivity and a large positive one with BIS sensitivity (Katz et al., 2020). Next, we reviewed 33 studies that compared reinforcement sensitivity between euthymic, bipolar participants and healthy controls. There, bipolar disorder had a small, positive relationship with BAS sensitivity (g = .20) and a medium, positive relationship with BIS sensitivity (g = .64). These findings support a dualsystem theory of bipolar disorders, wherein BAS sensitivity is more closely related to mania and BIS sensitivity more closely to bipolar depression. Bipolar disorders show diatheses for both states with euthymic participants being BAS- and BIS- hypersensitive. Implications for further theory and research practice are expounded upon in the discussion., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Reinforcement Sensitivity Theory (RST) posits that individual differences in reward and punishment processing predict differences in cognition, behavior, and psychopathology. We performed a quantitative review of the relationships between reinforcement sensitivity, depression and anxiety, in two separate sets of analyses. First, we reviewed 204 studies that reported either correlations between reinforcement sensitivity and self-reported symptom severity or differences in reinforcement sensitivity between diagnosed and healthy participants, yielding 483 effect sizes. Both depression (Hedges' g = .99) and anxiety (g = 1.21) were found to be high on punishment sensitivity. Reward sensitivity negatively predicted only depressive disorders (g = -.21). More severe clinical states (e.g., acute vs remission) predicted larger effect sizes for depression but not anxiety. Next, we reviewed an additional 39 studies that reported correlations between reinforcement sensitivity and both depression and anxiety, yielding 156 effect sizes. We then performed meta-analytic structural equation modeling to simultaneously estimate all covariances and control for comorbidity. Again we found punishment sensitivity to predict depression (β = .37) and anxiety (β = .35), with reward sensitivity only predicting depression (β = -.07). The transdiagnostic role of punishment sensitivity and the discriminatory role of reward sensitivity support a hierarchical approach to RST and psychopathology., (Copyright © 2020. Published by Elsevier Ltd.)

Recent variants of exposure therapy ask clients to directly engage with the distress associated with avoided experiences in order to become more resilient to future anxiety-provoking situations. In this study, we consider how this engagement impacts behavioral willingness. Forty-eight participants with high fear of cockroaches completed in vivo exposures while either mindfully attending externally to the feared object (Ext), or to both the object and their internal distress (Int/Ext). While both groups showed improvement, behavioral, subjective and physiological measures revealed different patterns of change. Immediate testing showed that participants in the Ext condition improved more in subjective distress, with no other differences between groups. A second testing a week later in an ecologically valid environment showed that participants the Int/Ext intervention continued to improve behaviorally, regardless of their reported subjective discomfort. These findings highlight the importance of explicit engagement with distress during exposures, that forego immediate subjective relief for long-term behavioral improvement., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The relationship between context and emotion regulation is currently at the center of a burgeoning area of research. Commonly used emotion regulation questionnaires, however, are predominantly trait-based, and insensitive to situational choice of regulatory strategy. The current work describes the development and validation of the State Emotion Regulation Inventory (SERI), a brief measure of situational use of distraction, reappraisal, brooding and acceptance. In Study 1, an initial item pool was constructed, based on commonly used trait-based emotion regulation surveys. Then, the psychometric properties of the items were examined with a group of 181 participants who recalled a saddening autobiographical event, identified a distressing thought it triggered, and then waited for 3 minutes without instruction, as an opportunity to allow for spontaneous emotion regulation. Participants then completed the initial item pool, and other relevant trait-based scales. Exploratory factor analysis suggested a 4-factor solution, corresponding to the 4 regulatory strategies measured in the SERI. The 4 items to exclusively load highest on each factor were selected for the final measure. Assembled subscales correlated with relevant trait-based subscales in the expected directions. In Study 2, another sample of 155 participants completed the same procedure and the new SERI, and confirmatory factor analysis supported the 4-factor structure of this instrument. As a brief, validated instrument, the SERI may be a useful measure for studies of state emotion regulation, in protocols that use repeated measures in a single session, over the course of multiple sessions, or via ecological momentary assessments. (PsycINFO Database Record, ((c) 2017 APA, all rights reserved).)

While many therapies focus on the reduction of disturbing symptoms, others pursue behavior consistent with personally held values. Based on regulatory focus theory (Higgins, 1997), reducing symptoms is a type of prevention goal while pursuing values is a promotion goal. In the current study, 123 undergraduate students elicited a negative, self-focused emotion-laden cognition. They were then randomly assigned to construe their negative thought as either (a) an impediment to valued behaviors, (b) a cause of unpleasant symptoms, or to one of two control conditions: (c) distraction or (d) no intervention. Then, participants in all groups completed a series of repetitive therapeutic tasks that targeted their elicited negative cognitions. Results showed that participants who construed treatment in terms of valued behavior promotion spent more time on a therapeutic task than all other groups. The group in the unpleasant symptom promotion condition did not differ from either control group. The motivational advantage of value promotion was not accounted for by differences in mood. The present findings suggest that clients may be better motivated through value promotion goals, as opposed to symptom prevention goals., (Copyright © 2015. Published by Elsevier Ltd.)

The discovery and development of 5-azaindole factor VIIa inhibitors will be described.

Improved peptide-based inhibitors of human beta tryptase were discovered using information gleaned from tripeptide library screening and structure-guided design methods, including fragment screening. Our efforts sought to improve this class of inhibitors by replacing the traditional Lys or Arg P1 element. The optimized compounds display low nanomolar potency against the mast cell target and several hundred-fold selectivity with respect to serine protease off targets. Thus, replacement of Lys/Arg at P1 in a peptide-like scaffold does not need to be accompanied by a loss in target affinity.

Synthesis and biological data of a novel selective and efficacious factor IXa inhibitor are described along with its crystal structure in factor VIIa.

Structure-activity relationships and binding mode of novel heterocyclic factor VIIa inhibitors will be described. In these inhibitors, a highly basic 5-amidinoindole moiety has been successfully replaced with a less basic 5-aminopyrrolo[3,2-b]pyridine scaffold.

Efforts to improve the potency and pharmacokinetic properties of small molecule factor VIIa inhibitors are described. Small structural modifications to existing leads allow the modulation of half-life and clearance, potentially making these compounds suitable candidates for drug development.

Highly selective and potent factor VIIa-tissue factor (fVIIa.TF) complex inhibitors were generated through structure-based design. The pharmacokinetic properties of an optimized analog (9) were characterized in several preclinical species, demonstrating pharmacokinetic characteristics suitable for once-a-day dosing in humans. Analog 9 inhibited platelet and fibrin deposition in a dose-dependent manner after intravenous administration in a baboon thrombosis model, and a pharmacodynamic concentration-response model was developed to describe the platelet deposition data. Results for heparin and enoxaparin (Lovenox) in the baboon model are also presented.

Plasma kallikrein is a serine protease that is involved in pathways of inflammation, complement fixation, coagulation, and fibrinolysis. Herein, we describe the SAR and structural binding modes of a series of inhibitors of plasma kallikrein as well as the pharmacokinetics of a lead analog 11 in rat.

Within the trypsin family of coagulation proteases, obtaining highly selective inhibitors of factor VIIa has been challenging. We report a series of factor VIIa (fVIIa) inhibitors based on the 5-amidino-2-(2-hydroxy-biphenyl-3-yl)-benzimidazole (1) scaffold with potency for fVIIa and high selectivity against factors IIa, Xa, and trypsin. With this scaffold class, we propose that a unique hydrogen bond interaction between a hydroxyl on the distal ring of the biaryl system and the backbone carbonyl of fVIIa lysine-192 provides a basis for enhanced selectivity and potency for fVIIa.

Plasma kallikrein is a serine protease that has many important functions, including modulation of blood pressure, complement activation, and mediation and maintenance of inflammatory responses. Although plasma kallikrein has been purified for 40 years, its structure has not been elucidated. In this report, we described two systems (Pichia pastoris and baculovirus/Sf9 cells) for expression of the protease domain of plasma kallikrein, along with the purification and high resolution crystal structures of the two recombinant forms. In the Pichia pastoris system, the protease domain was expressed as a heterogeneously glycosylated zymogen that was activated by limited trypsin digestion and treated with endoglycosidase H deglycosidase to reduce heterogeneity from the glycosylation. The resulting protein was chromatographically resolved into four components, one of which was crystallized. In the baculovirus/Sf9 system, homogeneous, crystallizable, and nonglycosylated protein was expressed after mutagenizing three asparagines (the glycosylation sites) to glutamates. When assayed against the peptide substrates, pefachrome-PK and oxidized insulin B chain, both forms of the protease domain were found to have catalytic activity similar to that of the full-length protein. Crystallization and x-ray crystal structure determination of both forms have yielded the first three-dimensional views of the catalytic domain of plasma kallikrein. The structures, determined at 1.85 A for the endoglycosidase H-deglycosylated protease domain produced from P. pastoris and at 1.40 A for the mutagenically deglycosylated form produced from Sf9 cells, show that the protease domain adopts a typical chymotrypsin-like serine protease conformation. The structural information provides insights into the biochemical and enzymatic properties of plasma kallikrein and paves the way for structure-based design of protease inhibitors that are selective either for or against plasma kallikrein.

A site-directed mutant of the serine protease urokinase-type plasminogen activator (uPA), was produced to assess the contribution of the Ser190 side-chain to the affinity and selectivity of lead uPA inhibitors in the absence of other differences present in comparisons of natural proteases. Crystallography and enzymology involving WT and Ala190 uPA were used to calculate free energy binding contributions of hydrogen bonds involving the Ser190 hydroxyl group (O(gamma)(Ser190)) responsible for the remarkable selectivity of 6-halo-5-amidinoindole and 6-halo-5-amidinobenzimidazole inhibitors toward uPA and against natural Ala190 protease anti-targets. Crystal structures of uPA complexes of novel, active site-directed arylguanidine and 2-aminobenzimidazole inhibitors of WT uPA, together with associated K(i) values for WT and Ala190 uPA, also indicate a significant role of Ser190 in the binding of these classes of uPA inhibitors. Structures and associated K(i) values for a lead inhibitor (CA-11) bound to uPA and to five other proteases, as well as for other leads bound to multiple proteases, help reveal the features responsible for the potency (K(i)=11nM) and selectivity of the remarkably small inhibitor, CA-11. The 6-fluoro-5-amidinobenzimidzole, CA-11, is more than 1000-fold selective against natural Ala190 protease anti-targets, and more than 100-fold selective against other Ser190 anti-targets.

Modulation of the acetylation state of histones plays a pivotal role in the regulation of gene expression. Histone deacetylases (HDACs) catalyze the removal of acetyl groups from lysines near the N termini of histones. This reaction promotes the condensation of chromatin, leading to repression of transcription. HDAC deregulation has been linked to several types of cancer, suggesting a potential use for HDAC inhibitors in oncology. Here we describe the first crystal structures of a human HDAC: the structures of human HDAC8 complexed with four structurally diverse hydroxamate inhibitors. This work sheds light on the catalytic mechanism of the HDACs, and on differences in substrate specificity across the HDAC family. The structure also suggests how phosphorylation of Ser39 affects HDAC8 activity.

Hepsin is an integral membrane protein that may participate in cell growth and in maintaining proper cell morphology and is overexpressed in a number of primary tumors. We have determined the 1.75 A resolution structure of the extracellular component of human hepsin. This structure includes a 255-residue trypsin-like serine protease domain and a 109-residue region that forms a novel, poorly conserved, scavenger receptor cysteine-rich (SRCR) domain. The two domains are associated with each other through a single disulfide bond and an extensive network of noncovalent interactions. The structure suggests how the extracellular region of hepsin may be positioned with respect to the plasma membrane.

An extensive structural manifold of short hydrogen bond-mediated, active site-directed, serine protease inhibition motifs is revealed in a set of over 300 crystal structures involving a large suite of small molecule inhibitors (2-(2-phenol)-indoles and 2-(2-phenol)-benzimidazoles) determined over a wide range of pH (3.5-11.4). The active site hydrogen-bonding mode was found to vary markedly with pH, with the steric and electronic properties of the inhibitor, and with the type of protease (trypsin, thrombin or urokinase type plasminogen activator (uPA)). The pH dependence of the active site hydrogen-bonding motif is often intricate, constituting a distinct fingerprint of each complex. Isosteric replacements or minor substitutions within the inhibitor that modulate the pK(a) of the phenol hydroxyl involved in short hydrogen bonding, or that affect steric interactions distal to the active site, can significantly shift the pH-dependent structural profile characteristic of the parent scaffold, or produce active site-binding motifs unique to the bound analog. Ionization equilibria at the active site associated with inhibitor binding are probed in a series of the protease-inhibitor complexes through analysis of the pH dependence of the structure and environment of the active site-binding groups involved in short hydrogen bond arrays. Structures determined at high pH (>11), suggest that the pK(a) of His57 is dramatically elevated, to a value as high as approximately 11 in certain complexes. K(i) values involving uPA and trypsin determined as a function of pH for a set of inhibitors show pronounced parabolic pH dependence, the pH for optimal inhibition governed by the pK(a) of the inhibitor phenol involved in short hydrogen bonds. Comparison of structures of trypsin, thrombin and uPA, each bound by the same inhibitor, highlights important structural variations in the S1 and active sites accessible for engineering notable selectivity into remarkably small molecules with low nanomolar K(i) values.

We describe and compare the pH dependencies of the potencies and of the bound structures of two inhibitor isosteres that form multicentered short hydrogen bond arrays at the active sites of trypsin, thrombin, and urokinase type plasminogen activator (urokinase or uPA) over certain ranges of pH. Depending on the pH, short hydrogen bond arrays at the active site are mediated by two waters, one in the oxyanion hole (H(2)O(oxy)) and one on the other (S2) side of the inhibitor (H(2)O(S2)), by one water (H(2)O(oxy)), or by no water. The dramatic variation in the length of the active site hydrogen bonds as a function of pH, of inhibitor, and of enzyme, along with the involvement or absence of ordered water, produces a large structural manifold of active site hydrogen bond motifs. Diverse examples of multicentered and two-centered short hydrogen bond arrays, both at and away from the active site, recently discovered in several protein crystal systems, suggest that short hydrogen bonds in proteins may be more common than has been recognized. The short hydrogen bond arrays resemble one another with respect to ionic nature, highly polar environment, multitude of associated ordinary hydrogen bonds, and disparate pK(a) values of participating groups. Comparison of structures and K(i) values of trypsin complexes at pH values where the multicentered short hydrogen bond arrays mediating inhibitor binding are present or absent indicate that these arrays have a minor effect on inhibitor potency. These features suggest little covalent nature within the short hydrogen bonds, despite their extraordinary shortness (as short as 2.0 A).

Stromal-derived factor (SDF)-1 and its G protein-coupled receptor, CXCR4, regulate stem/progenitor cell migration and retention in the marrow and are required for hematopoiesis. We show here an interaction between CXCR4 and the Src-related kinase, Lyn, in normal progenitors. We demonstrate that CXCR4-dependent stimulation of Lyn is associated with the activation of phosphatidylinositol 3-kinase (PI3-kinase). This chemokine signaling, which involves a Src-related kinase and PI3-kinase, appears to be a target for BCR/ABL, a fusion oncoprotein expressed only in leukemia cells. We show that the binding of phosphorylated BCR/ABL to Lyn results in the constitutive activation of Lyn and PI3-kinase, along with a total loss of responsiveness of these kinases to SDF-1 stimulation. Inhibition of BCR/ABL tyrosine kinase with STI571 restores Lyn responsiveness to SDF-1 signaling. Thus, BCR/ABL perturbs Lyn function through a tyrosine kinase-dependent mechanism. Accordingly, the blockade of Lyn tyrosine kinase inhibits both BCR/ABL-dependent and CXCR4-dependent cell movements. Our results demonstrate, for the first time, that Lyn-mediated pathological crosstalk exists between BCR/ABL and the CXCR4 pathway in leukemia cells, which disrupts chemokine signaling and chemotaxis, and increases the ability of immature cells to escape from the marrow. These results define a Src tyrosine kinases-dependent mechanism whereby BCR/ABL (and potentially other oncoproteins) dysregulates G protein-coupled receptor signaling and function of mammalian precursors.

The development of potent and selective urokinase-type plasminogen activator (uPA) inhibitors based on the lead molecule 2-(2-hydroxy-3-ethoxyphenyl)-1H-benzimidazole-5-carboxamidine (3a) is described.

The structure-based design of potent and selective urokinase-type plasminogen activator (uPA) inhibitors with 4-aminoarylamidine or 4-aminoarylguanidine S1 binding groups, is described.

A nonselective inhibitor of trypsin-like serine proteases, 2-(2-hydroxybiphenyl-3-yl)-1H-indole-5-carboxamidine (1) (Verner, E.; Katz, B. A.; Spencer, J.; Allen, D.; Hataye, J.; Hruzewicz, W.; Hui, H. C.; Kolesnikov, A.; Li, Y.; Luong, C.; Martelli, A.; Radika. K.; Rai, R.; She, M.; Shrader, W.; Sprengeler, P. A.; Trapp, S.; Wang, J.; Young, W. B.; Mackman, R. L. J. Med. Chem. 2001, 44, 2753-2771) has been optimized through minor structural changes on the S1 binding group to afford remarkably selective and potent inhibitors of urokinase-type plasminogen activator (uPA). The trypsin-like serine proteases(1) that comprise drug targets can be broadly categorized into two subfamilies, those with Ser190 and those with Ala190. A single-atom modification, for example, replacement of hydrogen for chlorine at the 6-position of the 5-amidinoindole P1 group on 1, generated up to 6700-fold selectivity toward the Ser190 enzymes and against the Ala190 enzymes. The larger chlorine atom displaces a water molecule (H(2)O1(S1)) that binds near residue 190 in all the complexes of 1, and related inhibitors, in uPA, thrombin, and trypsin. The water molecule, H(2)O1(S1), in both the Ser190 or Ala190 enzymes, hydrogen bonds with the amidine N1 nitrogen of the inhibitor. When it is displaced, a reduction in affinity toward the Ala190 enzymes is observed due to the amidine N1 nitrogen of the bound inhibitor being deprived of a key hydrogen-bonding partner. In the Ser190 enzymes the affinity is maintained since the serine hydroxyl oxygen O gamma(Ser190) compensates for the displaced water molecule. High-resolution crystallography provided evidence for the displacement of the water molecule and validated the design rationale. In summation, a novel and powerful method for engineering selectivity toward Ser190 proteases and against Ala190 proteases without substantially increasing molecular weight is described.

Background: Involved or implicated in a wide spectrum of diseases, trypsin-like serine proteases comprise well studied drug targets and anti-targets that can be subdivided into two major classes. In one class there is a serine at position 190 at the S1 site, as in urokinase type plasminogen activator (urokinase or uPA) and factor VIIa, and in the other there is an alanine at 190, as in tissue type plasminogen activator (tPA) and factor Xa. A hydrogen bond unique to Ser190 protease-arylamidine complexes between O gamma(Ser190) and the inhibitor amidine confers an intrinsic preference for such inhibitors toward Ser190 proteases over Ala190 counterparts., Results: Based on the structural differences between the S1 sites of Ser190 and Ala190 protease-arylamidine complexes, we amplified the selectivity of amidine inhibitors toward uPA and against tPA, by factors as high as 220-fold, by incorporating a halo group ortho to the amidine of a lead inhibitor scaffold. Comparison of K(i) values of such halo-substituted and parent inhibitors toward a panel of Ser190 and Ala190 proteases demonstrates pronounced selectivity of the halo analogs for Ser190 proteases over Ala190 counterparts. Crystal structures of Ser190 proteases, uPA and trypsin, and of an Ala190 counterpart, thrombin, bound by a set of ortho (halo, amidino) aryl inhibitors and of non-halo parents reveal the structural basis of the exquisite selectivity and validate the design principle., Conclusions: Remarkable selectivity enhancements of exceptionally small inhibitors are achieved toward the uPA target over the highly similar tPA anti-target through a single atom substitution on an otherwise relatively non-selective scaffold. Overall selectivities for uPA over tPA as high as 980-fold at physiological pH were realized. The increase in selectivity results from the displacement of a single bound water molecule common to the S1 site of both the uPA target and the tPA anti-target because of the ensuing deficit in hydrogen bonding of the arylamidine inhibitor when bound in the Ala190 protease anti-target.

Novel scaffolds that bind to serine proteases through a unique network of short hydrogen bonds to the catalytic Ser195 have been developed. The resulting potent serine protease inhibitors were designed from lead molecule 2-(2-hydroxyphenyl)1H-benzoimidazole-5-carboxamidine, 6b, which is known to display several modes of binding. For instance, 6b can recruit zinc and bind in a manner similar to that reported by bis(5-amidino-2-benzimidazolyl)methane (BABIM) (Nature 1998, 391, 608-612).(1) Alternatively, 6b can bind in the absence of zinc through a multicentered network of short (<2.3 A) hydrogen bonds. The lead structure was optimized in the zinc-independent binding mode toward a panel of six human serine proteases to yield optimized inhibitors such as 2-(3-bromo-2-hydroxy-5-methylphenyl)-1H-indole-5-carboxamidine, 22a, and 2-(2-hydroxybiphenyl-3-yl)-1H-indole-5-carboxamidine, 22f. Structure-activity relationships determined that, apart from the amidine function, an indole or benzimidazole and an ortho substituted phenol group were also essential components for optimal potency. The affinities (K(i)) of 22a and 22f, for example, bearing these groups ranged from 8 to 600 nM toward a panel of six human serine proteases. High-resolution crystal structures revealed that the binding mode of these molecules in several of the enzymes was identical to that of 6b and involved short (<2.3 A) hydrogen bonds among the inhibitor hydroxyl oxygen, Ser195, and a water molecule trapped in the oxyanion hole. In summation, novel and potent trypsin-like serine protease inhibitors possessing a unique mode of binding have been discovered.

The development of potent and selective small molecule inhibitors of factor Xa is described.

We describe a new serine protease inhibition motif in which binding is mediated by a cluster of very short hydrogen bonds (<2.3 A) at the active site. This protease-inhibitor binding paradigm is observed at high resolution in a large set of crystal structures of trypsin, thrombin, and urokinase-type plasminogen activator (uPA) bound with a series of small molecule inhibitors (2-(2-phenol)indoles and 2-(2-phenol)benzimidazoles). In each complex there are eight enzyme-inhibitor or enzyme-water-inhibitor hydrogen bonds at the active site, three of which are very short. These short hydrogen bonds connect a triangle of oxygen atoms comprising O(gamma)(Ser195), a water molecule co-bound in the oxyanion hole (H(2)O(oxy)), and the phenolate oxygen atom of the inhibitor (O6'). Two of the other hydrogen bonds between the inhibitor and active site of the trypsin and uPA complexes become short in the thrombin counterparts, extending the three-centered short hydrogen-bonding array into a tetrahedral array of atoms (three oxygen and one nitrogen) involved in short hydrogen bonds. In the uPA complexes, the extensive hydrogen-bonding interactions at the active site prevent the inhibitor S1 amidine from forming direct hydrogen bonds with Asp189 because the S1 site is deeper in uPA than in trypsin or thrombin. Ionization equilibria at the active site associated with inhibitor binding are probed through determination and comparison of structures over a wide range of pH (3.5 to 11.4) of thrombin complexes and of trypsin complexes in three different crystal forms. The high-pH trypsin-inhibitor structures suggest that His57 is protonated at pH values as high as 9.5. The pH-dependent inhibition of trypsin, thrombin, uPA and factor Xa by 2-(2-phenol)benzimidazole analogs in which the pK(a) of the phenol group is modulated is shown to be consistent with a binding process involving ionization of both the inhibitor and the enzyme. These data further suggest that the pK(a) of His57 of each protease in the unbound state in solution is about the same, approximately 6.8. By comparing inhibition constants (K(i) values), inhibitor solubilities, inhibitor conformational energies and corresponding structures of short and normal hydrogen bond-mediated complexes, we have estimated the contribution of the short hydrogen bond networks to inhibitor affinity ( approximately 1.7 kcal/mol). The structures and K(i) values associated with the short hydrogen-bonding motif are compared with those corresponding to an alternate, Zn(2+)-mediated inhibition motif at the active site. Structural differences among apo-enzymes, enzyme-inhibitor and enzyme-inhibitor-Zn(2+) complexes are discussed in the context of affinity determinants, selectivity development, and structure-based inhibitor design., (Copyright 2001 Academic Press.)

Serine proteases play a role in a variety of disease states and thus are attractive targets for therapeutic intervention. We report the kinetic characterization of a class of serine protease inhibitors whose potencies and selectivities are dramatically enhanced in the presence of Zn(II). The structural basis for Zn(II)-mediated inhibition of trypsin-like proteases has recently been reported [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612]. A case study of the kinetic behavior of human tryptase inhibitors is provided to illustrate the general phenomenon of Zn(II)-mediated inhibition. Tryptase, Zn(II), and the inhibitor form a ternary complex which exhibits classic tight-binding inhibition. The half-life for release of inhibitor from the tryptase-Zn(II)-inhibitor complex has been measured for a number of inhibitors. Consistent with tight-binding behavior, potent tryptase inhibitors are characterized by extremely slow rates of dissociation from the ternary complex with half-lives on the order of hours. A model of human serum, designed to reproduce physiological levels of Zn(II), has been employed to evaluate the performance of Zn(II)-potentiated tryptase inhibitors under physiological conditions. We demonstrate that Zn(II)-mediated inhibition can be achieved at physiological Zn(II) levels.

Background: Urokinase-type plasminogen activator (uPA) is a protease associated with tumor metastasis and invasion. Inhibitors of uPA may have potential as drugs for prostate, breast and other cancers. Therapeutically useful inhibitors must be selective for uPA and not appreciably inhibit the related, and structurally and functionally similar enzyme, tissue-type plasminogen activator (tPA), involved in the vital blood-clotting cascade., Results: We produced mutagenically deglycosylated low molecular weight uPA and determined the crystal structure of its complex with 4-iodobenzo[b]thiophene 2-carboxamidine (K(i) = 0.21 +/- 0.02 microM). To probe the structural determinants of the affinity and selectivity of this inhibitor for uPA we also determined the structures of its trypsin and thrombin complexes, of apo-trypsin, apo-thrombin and apo-factor Xa, and of uPA, trypsin and thrombin bound by compounds that are less effective uPA inhibitors, benzo[b]thiophene-2-carboxamidine, thieno[2,3-b]-pyridine-2-carboxamidine and benzamidine. The K(i) values of each inhibitor toward uPA, tPA, trypsin, tryptase, thrombin and factor Xa were determined and compared. One selectivity determinant of the benzo[b]thiophene-2-carboxamidines for uPA involves a hydrogen bond at the S1 site to Ogamma(Ser190) that is absent in the Ala190 proteases, tPA, thrombin and factor Xa. Other subtle differences in the architecture of the S1 site also influence inhibitor affinity and enzyme-bound structure., Conclusions: Subtle structural differences in the S1 site of uPA compared with that of related proteases, which result in part from the presence of a serine residue at position 190, account for the selectivity of small thiophene-2-carboxamidines for uPA, and afford a framework for structure-based design of small, potent, selective uPA inhibitors.

Structural and mechanistic determinants of affinity of streptavidin-binding peptide ligands discovered by phage display are reviewed along with the use of streptavidin as a paradigm for structure-based design. A novel way of producing protein-dimerizing ligands in the streptavidin model system is discussed, in which crystal packing topochemically mediates or even catalyzes dimerization of adjacent bound ligands whose reactive ligating groups are presented toward one another in productive orientations in the crystal lattice. Finally, through crystallography on a set of streptavidin complexes with small molecule and peptide ligands at multiple pHs in two space groups, the mechanism by which ligands enhance intersubunit stabilization of the streptavidin tetramer is probed.

As regulators of ubiquitous biological processes, serine proteases can cause disease states when inappropriately expressed or regulated, and are thus rational targets for inhibition by drugs. Recently we described a new inhibition mechanism applicable for the development of potent, selective small molecule serine protease inhibitors that recruit physiological Zn2+ to mediate high affinity (sub-nanomolar) binding. To demonstrate some of the structural principles by which the selectivity of Zn2+-mediated serine protease inhibitors can be developed toward or against a particular target, here we determine and describe the structures of thrombin-BABIM-Zn2+, -keto-BABIM-Zn2+, and -hemi-BABIM-Zn2+ (where BABIM is bis(5-amidino-2-benzimidazolyl)methane, keto-BABIM is bis(5-amidino-2-benzimidazolyl)methane ketone, and hemi-BABIM is (5-amidino-2-benzimidazolyl)(2-benzimidazolyl)methane), and compare them with the corresponding trypsin-inhibitor-Zn2+ complexes. Inhibitor binding is mediated by a Zn ion tetrahedrally coordinated by two benzimidazole nitrogen atoms of the inhibitor, by N(epsilon2)His57, and by O(gamma)Ser195. The structures of Zn2+-free trypsin-BABIM and -hemi-BABIM were also determined at selected pH values for comparison with the corresponding Zn2+-mediated complexes. To assess some of the physiological parameters important for harnessing Zn2+ as a co-inhibitor, crystal structures at multiple pH and [Zn2+] values were determined for trypsin-keto-BABIM. The Kdvalue of Zn2+ for the binary trypsin-keto-BABIM complex was estimated to be <12 nM at pH 7.06 by crystallographic determination of the occupancy of bound Zn2+ in trypsin-keto-BABIM crystals soaked at this pH in synthetic mother liquor containing inhibitor and 100 nM Zn2+. In synthetic mother liquor saturated in Zn2+, trypsin-bound keto-BABIM is unhydrated at pH 9.00 and 9.93, and has an sp2 hybridized ketone carbon bridging the 5-amidinobenzimidazoles, whereas at pH 7.00 and 8.00 it undergoes hydration and a change in geometry upon addition of water to the bridging carbonyl group. To show how Zn2+ could be recruited as a co-inhibitor of other enzymes, a method was developed for locating in protein crystals Zn2+ binding sites where design of Zn2+-mediated ligands can be attempted. Thus, by soaking trypsin crystals in high concentrations of Zn2+ in the absence of a molecular inhibitor, the site where Zn2+ mediates binding of BABIM and analogs was identified, as well as another Zn2+ binding site., (Copyright 1999 Academic Press.)

The extracellular ligand-binding domain (EPObp) of the human EPO receptor (EPOR) was expressed both in CHO (Chinese Hamster Ovary) cells and in Pichia pastoris. The CHO and yeast expressed receptors showed identical affinity for EPO binding. Expression levels in P. pastoris were significantly higher, favoring its use as an expression and scale-up production system. Incubation of EPO with a fourfold molar excess of receptor at high protein concentrations yielded stable EPO-EPObp complexes. Quantification of EPO and EPObp in the complex yielded a molar ratio of one EPO molecule to two receptor molecules. Residues that are responsible for EPOR glycosylation and isomerization in Pichia were identified and eliminated by site-specific mutagenesis. A thiol modification was identified and a method was developed to remove the modified species from EPObp. EPObp was complexed with erythropoietin (EPO) and purified. The complex crystallized in two crystal forms that diffracted to 2.8 and 1.9 A respectively. (Form 1 and form 2 crystals were independently obtained at AxyS Pharmaceuticals, Inc. and Amgen, Inc. respectively.) Both contained one complex per asymmetric unit with a stoichiometry of two EPObps to one EPO.

Human erythropoietin is a haematopoietic cytokine required for the differentiation and proliferation of precursor cells into red blood cells. It activates cells by binding and orientating two cell-surface erythropoietin receptors (EPORs) which trigger an intracellular phosphorylation cascade. The half-maximal response in a cellular proliferation assay is evoked at an erythropoietin concentration of 10 pM, 10(-2) of its Kd value for erythropoietin-EPOR binding site 1 (Kd approximately equal to nM), and 10(-5) of the Kd for erythropoietin-EPOR binding site 2 (Kd approximately equal to 1 microM). Overall half-maximal binding (IC50) of cell-surface receptors is produced with approximately 0.18 nM erythropoietin, indicating that only approximately 6% of the receptors would be bound in the presence of 10 pM erythropoietin. Other effective erythropoietin-mimetic ligands that dimerize receptors can evoke the same cellular responses but much less efficiently, requiring concentrations close to their Kd values (approximately 0.1 microM). The crystal structure of erythropoietin complexed to the extracellular ligand-binding domains of the erythropoietin receptor, determined at 1.9 A from two crystal forms, shows that erythropoietin imposes a unique 120 degrees angular relationship and orientation that is responsible for optimal signalling through intracellular kinase pathways.