Hit-to-lead evaluation of a novel class of sphingosine 1-phosphate lyase inhibitors.

Inhibition of sphingosine-1-phosphate lyase has recently been proposed as a potential treatment option for inflammatory disorders such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. In this report we describe our hit-to-lead evaluation of the isoxazolecarboxamide 6, a high-throughput screening hit (in vitro IC50=1.0 µM, cell IC50=1.8 µM), as a novel S1P lyase inhibitor. We were able to establish basic structure-activity relationships around 6 and succeeded in obtaining X-ray structural information which enabled structure-based design. With the discovery of 28, enzyme activity was quickly improved to IC50=120 nM and cell potency to IC50=230 nM. The main liability in the established isoxazolecarboxamide hit series was determined to be metabolic stability. In particular we identified that future lead-optimization efforts to overcome this problem should focus on blocking the N-dealkylation on the secondary amine.

Importance of PLD2 in an IL-23 driven psoriasiform dermatitis model and potential link to human psoriasis.

Phospholipase D2 (PLD2), a major isoform of the PLD family, has been reported to regulate inflammatory responses. Thus far, the relevance of PLD2 in psoriasis, an inflammatory skin disease, has not been explored. In the current study, we examined PLD2 expression in the skin of psoriasis patients and the role of PLD2 in an interleukin (IL)-23-induced mouse model of psoriasiform dermatitis. Both in situ hybridization and bulk RNA sequencing showed PLD2 gene expression is significantly higher in lesional relative to non-lesional skin of psoriasis patients or the skin of healthy subjects. PLD2 expression is also enriched in residual lesions from patients on biologic therapies. Murine in vivo studies showed that PLD2 deficiency significantly reduced psoriasiform inflammation in IL-23-injected ears, as reflected by decreases in ear thickness, expression of defensin beta 4A and the S100 calcium binding protein A7A, macrophage infiltrate, and expression of CXCL10 and IL-6. However, the expression of type 17 cytokines, IL-17A and IL-17F, were not reduced. Dual knockout of PLD1 and PLD2 offered little additional protection compared to PLD2 knockout alone in the IL-23 model. In addition, pharmacological inhibition with a pan-PLD1/PLD2 inhibitor also suppressed IL-23-induced psoriasiform dermatitis. Bone-marrow-derived macrophages from wild type (WT) and PLD2 knockout (KO) mice exhibited little difference in viability and sensitivity to lipopolysaccharide and/or interferon gamma, or resiquimod (R848). PLD2 deficiency did not alter the differentiation and function of Th17 cells in an ex vivo study with splenocytes isolated from WT and PLD2 KO mice. Overall, these data suggest that PLD2 may play a role in the pathophysiology of psoriasis. Reducing macrophage infiltrate and cytokine/chemokine production might contribute to an anti-inflammatory effect observed in PLD2 knockout mice. Further studies are required to better understand the mechanisms by which PLD2 contributes to skin lesions in psoriasis patients and psoriasiform dermatitis models.

ACT1 Is Required for Murine IL-23-Induced Psoriasiform Inflammation Potentially Independent of E3 Ligase Activity.

Psoriasis is a debilitating skin disease characterized by epidermal thickening, abnormal keratinocyte differentiation, and proinflammatory immune cell infiltrate into the affected skin. IL-17A plays a critical role in the etiology of psoriasis. ACT1, an intracellular adaptor protein and a putative ubiquitin E3 ligase, is essential for signal transduction downstream of the IL-17A receptor. Thus, IL-17A signaling in general, and ACT1 specifically, represent attractive targets for the treatment of psoriasis. We generated Act1 knockout and Act1 L286G knockin (ligase domain) mice to investigate the potential therapeutic effects of targeting ACT1 and its U-box domain, respectively. Act1 knockout, but not Act1 L286G knockin, mice were resistant to increases in CXCL1 plasma levels induced by subcutaneous injection of recombinant IL-17A. Moreover, in a mouse model of psoriasiform dermatitis induced by intradermal IL-23 injection, Act1 knockout, but not Act1 L286G knockin, was protective against increases in ear thickness, keratinocyte hyperproliferation, expression of genes for antimicrobial peptides and chemokines, and infiltration of monocytes and macrophages. Our studies highlight the critical contribution of ACT1 to proinflammatory skin changes mediated by the IL-23/IL-17 signaling axis and illustrate the need for further insight into ACT1 E3 ligase activity.

A non-clinical comparative study of IL-23 antibodies in psoriasis.

Four antibodies that inhibit interleukin (IL)-23 are approved for the treatment of moderate-to-severe plaque psoriasis. Here, we present non-clinical data comparing ustekinumab, guselkumab, tildrakizumab and risankizumab with regard to thermostability, IL-23 binding affinity, inhibitory-binding mode, in vitro potency and in vivo efficacy. Risankizumab and guselkumab exhibited 5-fold higher affinity for IL-23 and showed more potent inhibition of IL-23 signaling than ustekinumab and tildrakizumab. Risankizumab and guselkumab completely blocked the binding of IL-23 to IL-23Rα as expected, whereas tildrakizumab did not. In vitro, risankizumab and guselkumab blocked the terminal differentiation of TH17 cells in a similar manner, while tildrakizumab had minimal impact on TH17 differentiation. In a human IL-23-induced ear-swelling mouse model, risankizumab and guselkumab were more effective than ustekinumab and tildrakizumab at reducing IL-17, IL-22, and keratinocyte gene expression. Our results indicate that the four clinically approved antibodies targeting IL-23 differ in affinity and binding epitope. These attributes contribute to differences in in vitro potency, receptor interaction inhibition mode and in vivo efficacy in preclinical studies as described in this report, and similarly may affect the clinical performance of these drugs.

2,4-Diaminopyrimidine MK2 inhibitors. Part II: Structure-based inhibitor optimization.

We describe structure-based optimization of a series of novel 2,4-diaminopyrimidine MK2 inhibitors. Co-crystal structures (see accompanying Letter) demonstrated a unique inhibitor binding mode. Resulting inhibitors had IC(50) values as low as 19nM and moderate selectivity against a kinase panel. Compounds 15, 31a, and 31b inhibit TNFalpha production in peripheral human monocytes.

CD40/anti-CD40 antibody complexes which illustrate agonist and antagonist structural switches.

BACKGROUND: CD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases. CD40 antagonists are currently being explored for the treatment of autoimmune diseases and several anti-CD40 agonist mAbs have entered clinical development for oncological indications. RESULTS: To better understand the mode of action of anti-CD40 mAbs, we have determined the x-ray crystal structures of the ABBV-323 (anti-CD40 antagonist, ravagalimab) Fab alone, ABBV-323 Fab complexed to human CD40 and FAB516 (anti-CD40 agonist) complexed to human CD40. These three crystals structures 1) identify the conformational CD40 epitope for ABBV-323 recognition 2) illustrate conformational changes which occur in the CDRs of ABBV-323 Fab upon CD40 binding and 3) develop a structural hypothesis for an agonist/antagonist switch in the LCDR1 of this proprietary class of CD40 antibodies. CONCLUSIONS: The structure of ABBV-323 Fab demonstrates a unique method for antagonism by stabilizing the proposed functional antiparallel dimer for CD40 receptor via novel contacts to LCDR1, namely residue position R32 which is further supported by a closely related agonist antibody FAB516 which shows only monomeric recognition and no contacts with LCDR1 due to a mutation to L32 on LCDR1. These data provide a structural basis for the full antagonist activity of ABBV-323.

Upright static pelvic posture as rotations and translations in 3-dimensional from three 2-dimensional digital images: validation of a computerized analysis.

PURPOSE: The aim of this study was to determine the accuracy in measuring the pelvic orientations of a phantom model using the PosturePrint method. METHODS: In the Université du Québec à Trois-Rivières biomechanics laboratory, Trois-Rivières, Quebec, Canada, a mannequin was fixed on a rotating platform. For a set of 3 photographs (left lateral, anterior to posterior, right lateral) of each position, the mannequin pelvis was placed in 68 different postures on a stand, 61 cm from a wall, in front of a digital camera. The camera was at 83.8 cm in height and at 3.35 m from a calibrated wall grid. Mannequin postures were in 5 degrees of freedom: lateral translation (Tx), lateral flexion (Rz), axial rotation (Ry), flexion-extension (Rx), and anterior-posterior translation (Tz). Average errors were the differences of the positioned postures to the PosturePrint computed values. RESULTS: Mean and SD of computational errors for rotation displacements were Rx = 0.5 degrees +/- 0.8 degrees , Ry = 1.3 degrees +/- 0.8 degrees , and Rz = 0.5 degrees +/- 0.3 degrees , and for translation, Tz = 1.2 +/- 0.6 mm and Tx = 0.9 +/- 0.5 mm. CONCLUSIONS: The PosturePrint system allowed for accurate postural measurement of rotations and translations of a mannequin pelvis. The next step in evaluation of this product would be a reliability study on human subjects.

Validity of a computer postural analysis to estimate 3-dimensional rotations and translations of the head from three 2-dimensional digital images.

OBJECTIVE: The purpose of this study is to describe and evaluate the validity/accuracy of the computerized system PosturePrint for measuring head posture. METHODS: Computer analysis was compared with 125 measured positions of a mannequin head in 5 degrees of freedom. For each mannequin position, 3 digital photographs were obtained (left lateral, anteroposterior, and right lateral) and were processed through the PosturePrint computer system. For the head analysis, a headgear with 3 reflective markers was placed on a subject; and there were additional click-on markers at the ear tragus, upper lip, acromioclavicular joints, and episternal notch. Head postures were calculated as lateral translation (T(x)), lateral flexion (R(z)), axial rotation (R(y)), flexion-extension (R(x)), and anterior-posterior translation (T(z)). For an error analysis, PosturePrint algorithm calculations were compared with the true mannequin head positions. Furthermore, average head posture was determined in student volunteers (n = 40). RESULTS: Mean computational errors were R(x) = 1.3 degrees (SD 0.6 degrees) and T(z) = 1.1 mm (SD 0.5 mm) for sagittal displacements and R(y) = 1.1 degrees (SD 0.7 degrees), R(z) = 0.6 degrees (SD 0.4 degrees), and T(x) = 1.1 mm (SD 0.5 mm) for frontal view displacements. For the normal group, mean head displacements were 1.1 degrees or less for all rotations and 1 mm or less for lateral translations (T(x)); and forward head posture (T(z)) averaged 3 cm. CONCLUSION: From the mannequin positions, small mean errors indicate that the PosturePrint system is accurate. In the future, statistical research determining the correlation between head displacements, neck pain, function, and health status should be performed.

Three dimensional evaluation of posture in standing with the PosturePrint: an intra- and inter-examiner reliability study.

BACKGROUND: Few digitizers can measure the complexity of upright human postural displacements in six degrees of freedom of the head, rib cage, and pelvis. METHODS: In a University laboratory, three examiners performed delayed repeated postural measurements on forty subjects over two days. Three digital photographs (left lateral, AP, right lateral) of each of 40 volunteer participants were obtained, twice, by three examiners. Examiners placed 13 markers on the subjects before photography and chose 16 points on the photographic images. Using the PosturePrint internet computer system, head, rib cage, and pelvic postures were calculated as rotations (Rx, Ry, Rz) in degrees and translations (Tx, Tz) in millimeters. For reliability, two different types (liberal = ICC(3,1) & conservative = ICC(2,1)) of inter- and intra-examiner correlation coefficients (ICC) were calculated. Standard error of measurements (SEM) and mean absolute differences within and between observers' measurements were also determined. RESULTS: All of the "liberal" ICCs were in the excellent range (> 0.84). For the more "conservative" type ICCs, four Inter-examiner ICCs were in the interval (0.5-0.6), 10 ICCs were in the interval (0.61-0.74), and the remainder were greater than 0.75. SEMs were 2.7 degrees or less for all rotations and 5.9 mm or less for all translations. Mean absolute differences within examiners and between examiners were 3.5 degrees or less for all rotations and 8.4 mm or less for all translations. CONCLUSION: For the PosturePrint system, the combined inter-examiner and intra-examiner correlation coefficients were in the good (14/44) and excellent (30/44) ranges. SEMs and mean absolute differences within and between examiners' measurements were small. Thus, this posture digitizer is reliable for clinical use.

Validation of a computer analysis to determine 3-D rotations and translations of the rib cage in upright posture from three 2-D digital images.

Since thoracic cage posture affects lumbar spine coupling and loads on the spinal tissues and extremities, a scientific analysis of upright posture is needed. Common posture analyzers measure human posture as displacements from a plumb line, while the PosturePrint claims to measure head, rib cage, and pelvic postures as rotations and translations. In this study, it was decided to evaluate the validity of the PosturePrint Internet computer system's analysis of thoracic cage postures. In a university biomechanics laboratory, photographs of a mannequin thoracic cage were obtained in different postures on a stand in front of a digital camera. For each mannequin posture, three photographs were obtained (left lateral, right lateral, and AP). The mannequin thoracic cage was placed in 68 different single and combined postures (requiring 204 photographs) in five degrees of freedom: lateral translation (Tx), lateral flexion (Rz), axial rotation (Ry), flexion-extension (Rx), and anterior-posterior translation (Tz). The PosturePrint system requires 13 reflective markers to be placed on the subject (mannequin) during photography and 16 additional "click-on" markers via computer mouse before a set of three photographs is analyzed by the PosturePrint computer system over the Internet. Errors were the differences between the positioned mannequin and the calculated positions from the computer system. Average absolute value errors were obtained by comparing the exact inputted posture to the PosturePrint computed values. Mean and standard deviation of computational errors for sagittal displacements of the thoracic cage were Rx=0.3+/-0.1 degrees , Tz=1.6+/-0.7 mm, and for frontal view displacements were Ry=1.2+/-1.0 degrees , Rz=0.6+/-0.4 degrees , and Tx=1.5+/-0.6 mm. The PosturePrint system is sufficiently accurate in measuring thoracic cage postures in five degrees of freedom on a mannequin indicating the need for a further study on human subjects.