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Familial haemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disorder characterised by constitutive defects in cellular cytotoxicity resulting in fever, hepatosplenomegaly and cytopenia, and the outcome is fatal unless treated by chemoimmunotherapy followed by haematopoietic stem-cell transplantation. Since 1999, mutations in the perform gene giving rise to this disease have been identified; however, these account only for 40% of cases. Lack of a genetic marker hampers the diagnosis, suitability for transplantation, selection of familial donors, identification of carriers, genetic counselling and prenatal diagnosis. Mutations in the Munc13-4 gene have recently been described in patients with FHL. We sequenced the Munc13-4 gene in all patients with haemophagocytic lymphohistiocytosis not due to PRF1 mutations. In 15 of the 30 families studied, 12 novel and 4 known Munc13-4 mutations were found, spread throughout the gene. Among novel mutations, 2650C→T introduced a stop codon; 441 del A, 532del C, 3082del C and 3226ins G caused a frameshift, and seven were mis sense mutations. Median age of diagnosis was 4 months, but six patients developed the disease after 5 years of age and one as a young adult of 18 years. Involvement of central nervous system was present in 9 of 15 patients, activity of natural killer cells was markedly reduced or absent in 13 of 1 3 tested patients. Chemo-immunotherapy was effective in all patients. Munc13-4 mutations were found in 15 of 30 patients with FHL without PRF1 mutations. Because these patients may develop the disease during adolescence or even later, haematologists should include FHL2 and FHL3 in the differential diagnosis of young adults with fever, cytopenia, splenomegaly and hypercytokinaemia. [ABSTRACT FROM AUTHOR]

Primary hemophagocytic lymphohistiocytosis (pHLH) is a severe, life-threatening hyperinflammatory syndrome caused by defects in genes of the granule-dependent cytotoxic pathway. Here we investigated the clinical presentation and outcome in a large cohort of 143 patients with pHLH diagnosed in the last 15 years and enrolled in the Italian registry. The median age at diagnosis was 12 months (interquartile range, 2-81), and 92 patients (64%) fulfilled the HLH-2004 criteria. Of 111 patients who received first-line combined therapy (HLH-94, HLH-2004, Euro-HIT protocols), 65 (59%) achieved complete response and 21 (19%) partial response. Thereafter, 33 patients (30%) reactivated, and 92 (64%) received hematopoietic stem cell transplantation, 78 of whom (85%) survived and were alive at a median follow-up from diagnosis of 67 months. Thirty-six patients (25%) died before hematopoietic stem cell transplantation and 14 (10%) after. Overall, 93 patients (65%) were alive after a median follow-up of 30 months. Unadjusted predictors of non-response were age <6 months and high ferritin and bilirubin levels, while predictors of pre-transplant and overall mortality were high ferritin and bilirubin levels. At multivariable analysis, high levels of ferritin predicted non-response, while high levels of bilirubin predicted pre-transplant and overall mortality. Despite recent advances in therapeutic management, pHLH remains a life-threatening condition with significant early mortality. Liver dysfunction is the main predictor of poor prognosis.

Covalent hit identification is a viable approach to identify chemical starting points against difficult-to-drug targets. While most researchers screen libraries of <2k electrophilic fragments, focusing on lead-like compounds can be advantageous in terms of finding hits with improved affinity and with a better chance of identifying cryptic pockets. However, due to the increased molecular complexity, larger numbers of compounds (>10k) are desirable to ensure adequate coverage of chemical space. Herein, the approach taken to build a library of 12k covalent lead-like compounds is reported, utilizing legacy compounds, robust library chemistry, and acquisitions. The lead-like covalent library was screened against the antiapoptotic protein Bfl-1, and six promising hits that displaced the BIM peptide from the PPI interface were identified. Intriguingly, X-ray crystallography of lead-like compound 8 showed that it binds to a previously unobserved conformation of the Bfl-1 protein and is an ideal starting point for the optimization of Bfl-1 inhibitors.

The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 ( 21 ), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.

For oral drugs, medicinal chemists aim to design compounds with high oral bioavailability, of which permeability is a key determinant. Taking advantage of >2000 compounds tested in rat bioavailability studies and >20,000 compounds tested in Caco2 assays at Bayer, we have examined the molecular properties governing bioavailability and permeability. In addition to classical parameters such as logD and molecular weight, we also investigated the relationship between calculated p K a and permeability. We find that neutral compounds retain permeability up to a molecular weight limit of 700, while stronger acids and bases are restricted to weights of 400-500. We also investigate trends for common properties such as hydrogen bond donors and acceptors, polar surface area, aromatic ring count, and rotatable bonds, including compounds which exceed Lipinski's rule of five (Ro5). These property-structure relationships are combined to provide design guidelines for bioavailable drugs in both traditional and "beyond rule of 5" (bRo5) chemical space.

Fragment-based drug discovery is now widely adopted for lead generation in the pharmaceutical industry. However, fragment screening collections are often predominantly populated with flat, 2D molecules. Herein, we report the synthesis of piperidine-based 3D fragment building blocks - 20 regio- and diastereoisomers of methyl substituted pipecolinates using simple and general synthetic methods. cis -Piperidines, accessed through a pyridine hydrogenation were transformed into their trans -diastereoisomers using conformational control and unified reaction conditions. Additionally, diastereoselective lithiation/trapping was utilised to access trans -piperidines. Analysis of a virtual library of fragments derived from the 20 cis - and trans -disubstituted piperidines showed that it consisted of 3D molecules with suitable molecular properties to be used in fragment-based drug discovery programs., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background: Presenting symptoms of childhood cancers might mimic those of rheumatic diseases. However, the evidence available to guide differential diagnosis remains scarce. Preventing wrong or delayed diagnosis is therefore important to avoid incorrect administration of glucocorticoid or immunosuppressive therapy and worsening of prognosis. As such, we aimed to assess the prevalence and characteristics of presenting musculoskeletal manifestations in patients at cancer onset and to identify the factors that differentiate childhood malignancies with arthropathy from juvenile idiopathic arthritis., Methods: We did a multicentre, cross-sectional study at 25 paediatric haemato-oncology centres and 22 paediatric rheumatology centres in Italy. We prospectively recruited patients who were younger than 16 years that were newly diagnosed with cancer or juvenile idiopathic arthritis. We excluded patients with glucocorticoid pre-treatment (>1 mg/kg per day of oral prednisone or equivalent for ≥2 consecutive weeks). We collected data for patients with a new diagnosis of cancer or juvenile idiopathic arthritis using an electronic case report form on a web-based platform powered by the Cineca Interuniversity Consortium. The primary outcome was to describe the frequency and characteristics of musculoskeletal manifestations at cancer onset; and the secondary outcome was to identify factors that could discriminate malignancies presenting with arthropathy, with or without other musculoskeletal symptoms, from juvenile idiopathic arthritis using multivariable logistic regression analysis., Findings: Between May 1, 2015, and May 31, 2018, 1957 patients were eligible, of which 1277 (65%) had cancer and 680 (35%) had juvenile idiopathic arthritis. Musculoskeletal symptoms occurred in 324 (25% [95% CI 23·0-27·8]) of 1277 patients with cancer, of whom 207 had arthropathy. Patients with malignant bone tumours had the highest frequency of musculoskeletal symptoms (53 [80%] of 66), followed by patients with Langerhans histiocytosis (16 [47%] of 34), leukaemia (189 [32%] of 582), soft-tissue sarcomas (16 [24%] of 68), and neuroblastoma (21 [19%] of 109). In the 324 patients with cancer and musculoskeletal symptoms, the most common complaints were joint pain (199 [61%]), followed by limb bone pain (112 [35%]). Joint involvement had a prevalent monoarticular pattern (100 [48%] of 207) and oligoarticular pattern (86 [42%] had 2-4 joints involved and 20 [10%] had >4 joints involved), with the most frequently involved joints being the hip (88 [43%] of 207) and knee (81 [39%]). On multivariable analysis, limb bone pain was the independent variable most strongly associated with cancer (odds ratio [OR] 87·80 [95% CI 18·89-408·12]), followed by weight loss (59·88 [6·34-565·53]), thrombocytopenia (12·67 [2·40-66·92]), monoarticular involvement (11·30 [4·09-31·19]), hip involvement (3·30 [1·13-9·61]), and male sex (2·40 [1·03-5·58]). Factors independently associated with juvenile idiopathic arthritis were morning stiffness (OR 0·04 [95% CI 0·01-0·20]), joint swelling (0·03 [0·01-0·09]), and involvement of the small hand joints (0·02 [0-1·05])., Interpretation: Our study provides detailed information about presenting musculoskeletal manifestations of childhood cancers and highlights the clinical and laboratory features that are most helpful in the differential diagnosis with juvenile idiopathic arthritis., Funding: Associazione Lorenzo Risolo., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

MAT2a is a methionine adenosyltransferase that synthesizes the essential metabolite S -adenosylmethionine (SAM) from methionine and ATP. Tumors bearing the co-deletion of p16 and MTAP genes have been shown to be sensitive to MAT2a inhibition, making it an attractive target for treatment of MTAP-deleted cancers. A fragment-based lead generation campaign identified weak but efficient hits binding in a known allosteric site. By use of structure-guided design and systematic SAR exploration, the hits were elaborated through a merging and growing strategy into an arylquinazolinone series of potent MAT2a inhibitors. The selected in vivo tool compound 28 reduced SAM-dependent methylation events in cells and inhibited proliferation of MTAP-null cells in vitro . In vivo studies showed that 28 was able to induce antitumor response in an MTAP knockout HCT116 xenograft model.

Fragment-based drug discovery is now widely adopted for lead generation in the pharmaceutical industry. However, fragment screening collections are often predominantly populated with flat, 2D molecules. Herein, we describe a workflow for the design and synthesis of 56 3D disubstituted pyrrolidine and piperidine fragments that occupy under-represented areas of fragment space (as demonstrated by a principal moments of inertia (PMI) analysis). A key, and unique, underpinning design feature of this fragment collection is that assessment of fragment shape and conformational diversity (by considering conformations up to 1.5 kcal mol -1 above the energy of the global minimum energy conformer) is carried out prior to synthesis and is also used to select targets for synthesis. The 3D fragments were designed to contain suitable synthetic handles for future fragment elaboration. Finally, by comparing our 3D fragments with six commercial libraries, it is clear that our collection has high three-dimensionality and shape diversity., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

CK2 is a critical cell cycle regulator that also promotes various anti-apoptotic mechanisms. Development of ATP-non-competitive inhibitors of CK2 is a very attractive strategy considering that the ATP binding site is highly conserved among other kinases. We have previously utilised a pocket outside the active site to develop a novel CK2 inhibitor, CAM4066 . Whilst CAM4066 bound to this new pocket it was also interacting with the ATP site: herein, we describe an example of a CK2α inhibitor that binds completely outside the active site. This second generation αD-site binding inhibitor, compound CAM4712 (IC 50 = 7 μM, GI 50 = 10.0 ± 3.6 μM), has numerous advantages over the previously reported CAM4066 , including a reduction in the number of rotatable bonds, the absence of amide groups susceptible to the action of proteases and improved cellular permeability. Unlike with CAM4066 , there was no need to facilitate cellular uptake by making a prodrug. Moreover, CAM4712 displayed no drop off between its ability to inhibit the kinase in vitro (IC 50 ) and the ability to inhibit cell proliferation (GI 50 ).

Osteopontin (OPN) is a multifunctional protein mainly associated with bone metabolism and remodeling. Besides its physiological functions, OPN is implicated in the pathogenesis of a variety of disease states, such as obesity and osteoporosis. Importantly, during the last decades obesity and osteoporosis have become among the main threats to health worldwide. Because OPN is a protein principally expressed in cells with multifaceted effects on bone morphogenesis and remodeling and because it seems to be one of the most overexpressed genes in the adipose tissue of the obese contributing to osteoporosis, this mini review will highlight recent insights about relation between adipose tissue and bone homeostasis., Competing Interests: The authors declare that they have no competing interests.

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 K d 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.

Hypocretin/orexin (ORX) are two hypothalamic neuropeptides discovered in 1998. Since their discovery, they have been one of the most studied neuropeptide systems because of their projecting fields innervating various brain areas. The orexinergic system is tied to sleep-wakefulness cycle, and narcolepsy is a consequence of their system hypofunction. Orexinergic system is also involved in many other autonomic functions such as feeding, thermoregulation, cardiovascular and neuroendocrine regulation. The main aim of this mini review article is to investigate the relationship between ORX and thyroid system regulation. Although knowledge about the ORX system is evolving, its putative effects on hypothalamic-pituitary-thyroid (HPT) axis still appear unclear. We analyzed some studies about ORX control of HPT axis to know better the relationship between them. The studies that were analyzed suggest Hypocretin/ORX to modulate the thyroid regulation, but the nature (excitatory or inhibitory) of this possible interaction remains actually unclear and needs to be confirmed.

Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease affecting mostly children but also adults and characterized by hyperinflammatory features. A subset of patients, referred to as having familial hemophagocytic lymphohistiocytosis (FHL), have various underlying genetic abnormalities, the frequencies of which have not been systematically determined previously., Objective: This work aims to further our understanding of the pathogenic bases of this rare condition based on an analysis of our 25 years of experience., Methods: From our registry, we have analyzed a total of 500 unselected patients with HLH., Results: Biallelic pathogenic mutations defining FHL were found in 171 (34%) patients; the proportion of FHL was much higher (64%) in patients given a diagnosis during the first year of life. Taken together, mutations of the genes PRF1 (FHL2) and UNC13D (FHL3) accounted for 70% of cases of FHL. Overall, a genetic diagnosis was possible in more than 90% of our patients with FHL. Perforin expression and the extent of degranulation have been more useful for diagnosing FHL than hemophagocytosis and the cytotoxicity assay. Of 281 (56%) patients classified as having "sporadic" HLH, 43 had monoallelic mutations in one of the FHL-defining genes. Given this gene dosage effect, FHL is not strictly recessive., Conclusion: We suggest that the clinical syndrome HLH generally results from the combined effects of an exogenous trigger and genetic predisposition. Within this combination, different weights of exogenous and genetic factors account for the wide disease spectrum that ranges from HLH secondary to severe infection to FHL., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The first asymmetric organocatalytic synthesis of helicenes is reported. A novel SPINOL-derived phosphoric acid, bearing extended π-substituents, catalyzes the asymmetric synthesis of helicenes through an enantioselective Fischer indole reaction. A variety of azahelicenes and diazahelicenes could be obtained with good to excellent yields and enantioselectivities., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The first enantioselective α-hydroxylation reaction of α-substituted β-ketoamides has been developed by using the commercially available hydroquinine/TBHP system. The tertiary alcohols are obtained in good to high yield and up to 83% ee, which can be improved by a single crystallization.

A high-yielding and enantioselective access to novel N-Boc terminal aziridines, bearing a quaternary stereogenic center, has been developed via an aza-Michael initiated ring-closure (aza-MIRC) reaction of α-acyl acrylates with an N-tosyloxy tert-butyl carbamate catalyzed by a chiral amino thiourea. The feasibility of the aziridine regioselective ring-opening to valuable α,α-disubstituted α-amino acid esters has been demonstrated.

A dramatic enhancement of the diastereo- and enantioselectivity in the nitro-Michael addition reaction organocatalysed by a commercially available α,α-L-diaryl prolinol was disclosed when performing the reaction in unconventional hexafluorobenzene as a medium. DFT calculations were performed to clarify the origin of stereoselectivity and the role of C(6)F(6)., (This journal is © The Royal Society of Chemistry 2012)

The first diastereospecific and enantioselective epoxidation of trans-2-aroyl-3-arylacrylonitriles by means of the commercially available diaryl L-prolinol/tert-butyl hydroperoxide system has been developed. These diversely functionalized epoxides were obtained in excellent yield (up to 99%), complete diastereoselectivity for the trans-isomer, and good enantioselectivity (up to 84% ee). Highly enantioenriched epoxides can be easily obtained after a single crystallization (ee > 90%).

Mutations of UNC13D have been described in patients affected by familial hemophagocytic lymphohistiocytosis (FHL3). The Munc13-4 protein contributes to the priming of the secretory granules. Mutation in this gene results in defective cellular cytotoxicity and the familial hemophagocytic lymphohistiocytosis clinical picture. Among reported mutations, few are predicted to impair splicing. Yet, functional impact of these mutations has not been addressed. We identified 18 out of 31 familial hemophagocytic lymphohistiocytosis families showing at least one mutation responsible for splicing error. We identified some known and three novel splicing mutations: one falls at the acceptor site of exon 11 and 2 are deep intronic mutations in IVS1 and in IVS30. We demonstrated that these deep intronic mutations affect regulatory sequences causing aberrant splicing. We report that UNC13D mutations leading to splicing errors represent the majority of mutations observed in familial hemophagocytic lymphohistiocytosis. This finding has implications for designing strategies for analysis of the families with suspected familial hemophagocytic lymphohistiocytosis.

Mutations in the perforin gene have been found in patients with hemophagocytic lymphohistiocytosis (HLH), a rare autosomal recessive disease. We describe a patient expressing perforin with amino acid changes A91V and W374X. The ability of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells to lyse target cells is greatly reduced. Here we demonstrate that perforin from this patient is not recognized using an antibody raised against native perforin (deltaG9), but is readily detected using an antibody raised against a peptide epitope (2d4), suggesting that the epitope recognized by deltaG9 is destroyed by the change at A91V. Immunoblotting reveals no protein corresponding to the truncated transcript encoded by W374X, revealing that only perforin with the A91V change is expressed in CTLs from the patient. Patient CTLs show bands corresponding to the immature and intermediate forms of perforin, but the mature active form of perforin is absent or barely detectable. The conformational changes and impaired cleavage of A91V perforin are likely to explain the reduced cytotoxicity in CTLs and NK cells from this patient and are likely to contribute to the pathogenesis of HLH.

The minimum spanning tree (MST) model was applied to identify the history of transmission of hepatitis C virus (HCV) infection in an outbreak involving five children attending a pediatric oncology-hematology outpatient ward between 1992 and 2000. We collected blood samples from all children attending since 1992, all household contacts, and one health care worker positive for antibody to HCV (anti-HCV). HCV RNA detection was performed with these samples and with smears of routinely collected bone marrow samples. For all isolates, we performed sequence analysis and phylogenetic tree analysis of hypervariable region 1 of the E2 gene. The MST model was applied to clinical-epidemiological and molecular data. No additional cases were detected. All children, but not the health care worker, showed genotype 3a. On six occasions, all but one child had shared the medication room with another patient who later seroconverted. HCV RNA detection in bone marrow smears revealed, in some cases, a delay of several months in anti-HCV responses. Sequence analysis and phylogenetic tree analysis revealed a high identity among the isolates. The MST model applied to molecular data, together with the clinical-epidemiological data, allowed us to identify the source of the outbreak and the most probable patient-to-patient chain of transmission. The management of central venous catheters was suspected to be the probable route of transmission. In conclusion, the MST model, supported by an exhaustive clinical-epidemiological investigation, appears to be a useful tool in tracing the history of transmission in outbreaks of HCV infection.

Background and Objectives: Most patients with familial hemophagocytic lymphohistiocytosis (HLH) develop the disease within the first two years of age. In a minority of cases a later occurrence has been reported, with an upper age limit of eight years. A significant concordance of the age at onset within each family has also been observed., Results: We report four cases of families with HLH diagnosed at an unusually late age, comprised between between 9 and 17 years; in each of these families another child developed the disease in infancy. The natural killer activity of the patients was depleted; nevertheless, we had indirect evidence that, in at least two families, mutations of the perforin gene were not causing the disease., Interpretation and Conclusions: Such a late onset is very unusual and suggests that there is a subgroup of families with HLH in which the disease may present early or late in different members. Thus in some families with HLH the siblings might remain at risk of developing the disease for several years. Their actual risk cannot be defined until the genetic mutation is identified in each family and assessed in each member.