A fragment-based approach leading to the discovery of inhibitors of CK2α with a novel mechanism of action.

CK2 is a ubiquitous protein kinase with an anti-apoptotic role and is found to be overexpressed in multiple cancer types. To this end, the inhibition of CK2 is of great interest with regard to the development of novel anti-cancer therapeutics. ATP-site inhibition of CK2 is possible; however, this typically results in poor selectivity due to the highly conserved nature of the catalytic site amongst kinases. An alternative methodology for the modulation of CK2 activity is through allosteric inhibition. The recently identified αD site represents a promising binding site for allosteric inhibition of CK2α. The work presented herein describes the development of a series of CK2α allosteric inhibitors through iterative cycles of X-ray crystallography and enzymatic assays, in addition to both fragment growing and fragment merging design strategies. The lead fragment developed, fragment 8, exhibits a high ligand efficiency, displays no drop off in activity between enzymatic and cellular assays, and successfully engages CK2α in cells. Furthermore, X-ray crystallographic analysis provided indications towards a novel mechanism of allosteric inhibition through αD site binding. Fragments described in this paper therefore represent promising starting points for the development of highly selective allosteric CK2 inhibitors.

A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death.

BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.

Study of Tourniquet Use in Simulated First Aid: User Judgment.

BACKGROUND: The purpose of this study was to survey the judgments of tourniquet users in simulation to discern opportunities for further study. METHODS: The study design constituted two parts: questions posed to four tourniquet users and then their tourniquet use was surveyed in simulated first aid, where the users had to decide how to perform among five different cases. The questions addressed judged confidence, blood volumes, a reason bleeding resumes, regret of preventable death, hemorrhage assessment, need for side-by-side use of tourniquets, shock severity, predicting reliability, and difference in blood losses. The mechanical performance was tested on a manikin. Case 1 had no bleeding. Case 2 had limb-wound bleeding that indicated tourniquet use in first aid. Case 3 was like case 2, except the patient was a child. Case 4 was like case 2, except caregiving was under gunfire. Case 5 was like case 4, but two tourniquets were to be used side by side. Each user made tests of the five cases to constitute a block. Each user had three blocks. Case order was randomized within blocks. The study had 60 tests. RESULTS: In answering questions relevant to first-aid use of limb tourniquets, judgments were in line with previous studies of judgment science, and thus were plausibly applicable. Mechanical performance results on the manikin were as follows: 38 satisfactory, 10 unsatisfactory (a loose tourniquet and nine incorrect tourniquet placements), and 12 not applicable (case 1 needed no mechanical intervention). For cases 1 to 5, satisfactory results were: 100%, 83%, 100%, 75%, and 58%, respectively. For blocks 1 to 3, satisfactory results were 50%, 83%, and 83%, respectively. CONCLUSION: For tourniquet use in simulated first aid, the results are plausibly applicable because user judgments were coherent with those in previous studies of judgment science. However, the opportunities for further studies were noted.

New and Established Models of Limb Tourniquet Compared in Simulated First Aid.

BACKGROUND: The performance of a new tourniquet model was compared with that of an established model in simulated first aid. METHODS: Four users applied the Combat Application Tourniquet (C-A-T), an established model that served as the control tourniquet, and the new SAM Extremity Tourniquet (SXT) model, which was the study tourniquet. RESULTS: The performance of the C-A-T was better than that of the SXT for seven measured parameters versus two, respectively; metrics were statistically tied 12 times. The degree of difference, when present, was often small. For pretime, a period of uncontrolled bleeding from the start to a time point when the tourniquet first contacts the manikin, the bleeding rate was uncontrolled at approximately 10.4mL/s, and for an overall average of 39 seconds of pretime, 406mL of blood loss was calculated. The mean time to determination of bleeding control (± standard deviation [SD]) was 66 seconds (SXT, 70 ± 30 seconds; C-A-T, 62 ± 18 seconds; p = .0075). The mean ease-of-use score was 4 (indicating easy) on a scale of 1 to 5, with 5 indicating very easy (mean ± SD: SXT, 4 ± 1; C-A-T, 5 ± 0; p < .0001). C-A-T also performed better for total trial time, manikin damage, blood loss rate, pressure, and composite score. SXT was better for pretime and unwrap time. All users intuitively self-selected the speed at which they applied the tourniquets and that speed was similar in all of the required steps. However, by time segments, one user went slowest in each segment while the other three generally went faster. CONCLUSIONS: In simulated first aid with tourniquets, better results generally were seen with the C-A-T than with the SXT in terms of performance metrics. However, the degree of difference, when present, was often small.

Specific inhibition of CK2α from an anchor outside the active site.

The development of selective inhibitors of protein kinases is challenging because of the significant conservation of the ATP binding site. Here, we describe a new mechanism by which the protein kinase CK2α can be selectively inhibited using features outside the ATP site. We have identified a new binding site for small molecules on CK2α adjacent to the ATP site and behind the αD loop, termed the αD pocket. An elaborated fragment anchored in this site has been linked with a low affinity fragment binding in the ATP site, creating a novel and selective inhibitor (CAM4066) that binds CK2α with a Kd of 320 nM and shows significantly improved selectivity compared to other CK2α inhibitors. CAM4066 shows target engagement in several cell lines and similar potency to clinical trial candidate CX4945. Our data demonstrate that targeting a poorly conserved, cryptic pocket allows inhibition of CK2α via a novel mechanism, enabling the development of a new generation of selective CK2α inhibitors.

The discovery of potent, selective, and reversible inhibitors of the house dust mite peptidase allergen Der p 1: an innovative approach to the treatment of allergic asthma.

Blocking the bioactivity of allergens is conceptually attractive as a small-molecule therapy for allergic diseases but has not been attempted previously. Group 1 allergens of house dust mites (HDM) are meaningful targets in this quest because they are globally prevalent and clinically important triggers of allergic asthma. Group 1 HDM allergens are cysteine peptidases whose proteolytic activity triggers essential steps in the allergy cascade. Using the HDM allergen Der p 1 as an archetype for structure-based drug discovery, we have identified a series of novel, reversible inhibitors. Potency and selectivity were manipulated by optimizing drug interactions with enzyme binding pockets, while variation of terminal groups conferred the physicochemical and pharmacokinetic attributes required for inhaled delivery. Studies in animals challenged with the gamut of HDM allergens showed an attenuation of allergic responses by targeting just a single component, namely, Der p 1. Our findings suggest that these inhibitors may be used as novel therapies for allergic asthma.

Narcissism in midlife: longitudinal changes in and correlates of women's narcissistic personality traits.

We examined changes in and correlates of 3 kinds of narcissism--hypersensitivity, willfulness, and autonomy--during middle adulthood. Few studies have examined narcissistic personality traits beyond young adulthood, and none has assessed longitudinal changes in narcissism during midlife. In a sample of 70 college-educated women, we found that observer ratings of hypersensitive narcissism were associated with more negative outcomes at ages 43 and 53 (i.e., more depressive symptoms and physical health problems, lower life satisfaction and well-being). Ratings of willfulness and autonomy predicted more positive outcomes. All 3 kinds of narcissism showed considerable rank-order stability over 10 years, but there were also mean-level changes: Hypersensitivity and autonomy decreased, whereas willfulness increased. More positive outcomes were associated with decreases in hypersensitivity and increases in willfulness and autonomy. However, in multivariate analyses, autonomy did not show any significant associations with women's health and well-being outcomes, suggesting that it may have less predictive utility compared to hypersensitivity and willfulness. Our findings highlight developmental changes in and correlates of women's narcissistic personality traits and the importance of assessing different aspects of narcissism in midlife.