Identification of Potential Binding Sites of Sialic Acids on the RBD Domain of SARS-CoV-2 Spike Protein.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still an emergent pandemic for humans. The virus infection is achieved by penetrating its spike protein to host cells via binding with ACE2. Moreover, recent studies show that SARS-CoV-2 may have multiple receptors that need to be further revealed. SARS-CoV-2 shares similar sequences of the spike protein with the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which can invade host cells by binding to either DPP4 or sialic acids. Sialic acids can be linked to the terminal of glycoproteins and gangliosides are used as one of the receptors of many types of viruses. Therefore, it is very interesting to determine whether sialic acid is a potential receptor of SARS-CoV-2. To address this question, we took N-Acetylneuraminic acid (Neu5Ac), a type of predominant sialic acid found in human cells, as the molecular probe to computationally search the surface of the spike protein to locate the potential binding sites of Neu5Ac. SPR analysis and mass spectrum analysis confirmed the interaction between Neu5Ac and spike protein. This study shows that sialic acids can moderately interact with the spike protein of SARS-CoV-2 by binding between the two RBDs of the spike protein, indicating it could be a potential secondary or auxiliary receptor of SARS-CoV-2.

A Novel Intronic Pathogenic Variant in STAR With a Dominant Negative Mechanism Causes Attenuated Lipoid Congenital Adrenal Hyperplasia.

Lipoid congenital adrenal hyperplasia (LCAH) is typically inherited as an autosomal recessive condition. There are 3 reports of individuals with a dominantly acting heterozygous variant leading to a clinically significant phenotype. We report a 46,XY child with a novel heterozygous intronic variant in STAR resulting in LCAH with an attenuated genital phenotype. The patient presented with neonatal hypoglycemia and had descended testes with a fused scrotum and small phallus. Evaluation revealed primary adrenal insufficiency with deficiencies of cortisol, aldosterone, and androgens. He was found to have a de novo heterozygous novel variant in STAR: c.65-2A>C. We report a case of a novel variant and review of other dominant mutations at the same position in the literature. Clinicians should be aware of the possibility of attenuated genital phenotypes of LCAH and the contribution of de novo variants in STAR at c.65-2 to the pathogenesis of that phenotype.

Clinical Utility of Genetic Testing in the Precision Diagnosis and Management of Pediatric Patients with Kidney and Urinary Tract Diseases.

Background: As genetic testing increasingly integrates into the practice of nephrology, our understanding of the basis of many kidney disorders has exponentially increased. Given this, we recently initiated a Renal Genetics Clinic (RGC) at our large, urban children's hospital for patients with kidney disorders. Methods: Genetic testing was performed in Clinical Laboratory Improvement Amendments-certified laboratories using single gene testing, multigene panels, chromosomal microarray, or exome sequencing. Results: A total of 192 patients were evaluated in this clinic, with cystic kidney disease (49/192) being the most common reason for referral, followed by congenital anomalies of the kidney and urinary tract (41/192) and hematuria (38/192). Genetic testing was performed for 158 patients, with an overall diagnostic yield of 81 out of 158 (51%). In the 16 out of 81 (20%) of patients who reached a genetic diagnosis, medical or surgical treatment of the patients were affected, and previous clinical diagnoses were changed to more accurate genetic diagnoses in 12 of 81 (15%) patients. Conclusions: Our genetic testing provided an accurate diagnosis for children and, in some cases, led to further diagnoses in seemingly asymptomatic family members and changes to overall medical management. Genetic testing, as facilitated by such a specialized clinical setting, thus appears to have clear utility in the diagnosis and counseling of patients with a wide range of kidney manifestations.

A TNXB splice donor site variant as a cause of hypermobility type Ehlers-Danlos syndrome in patients with congenital adrenal hyperplasia.

BACKGROUND: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is an autosomal recessive disease of steroidogenesis that affects 1 in 15,000. Approximately, 10% of the CAH population also suffer from CAH-X, a connective tissue dysplasia consistent with hypermobility type Ehlers-Danlos syndrome (EDS). Most patients with CAH-X carry a contiguous gene deletion involving CYP21A2 encoding 21-hydroxylase and TNXB encoding tenascin-X (TNX), but some are of unknown etiology. METHODS: We conducted clinical evaluation and medical history review of EDS-related manifestations in subjects from two unrelated CAH families who carry a heterozygous TNXB c.12463+2T>C variant that alters the splice donor site of intron 42. A next generation sequencing (NGS) based EDS panel composed of 45 genes was performed for index patients from each family. TNX expression in patient skin biopsy tissues and dermal fibroblasts was assessed by qRT-PCR and Sanger sequencing. RESULTS: All three evaluated CAH patients carrying the TNXB splice site variant had moderate EDS manifestations. An NGS panel excluded involvement of other known EDS-related variants. RNA assay on skin biopsies and dermal fibroblasts did not detect splicing errors in TNX mRNA; however, the removal of intron 42 was less efficient in the allele harboring the splice site variant as evidenced by the existence of a premature TNX RNA form, leading to an allele specific decrease in TNX mRNA. CONCLUSIONS: Carrying a TNXB c.12463+2T>C variant at the intron 42 splice donor site causes an allele specific decrease in TNX expression, which can be associated with moderate EDS in CAH patients.

Natural product piperine alleviates experimental allergic encephalomyelitis in mice by targeting dihydroorotate dehydrogenase.

Multiple sclerosis (MS) is the most popular chronic and debilitating inflammatory disease of the central nervous system (CNS) that remains incurable. Dihydroorotate dehydrogenase (DHODH) is critical to the activity of T lymphocytes and represents a potential therapeutic target for MS. Here we identify piperine, a bioactive constituent of black pepper, as a potent inhibitor of DHODH with an IC50 value of 0.88 µM. Isothermal titration calorimetry and thermofluor assay demonstrate the directly interaction between piperine and DHODH. The co-complex crystal structure of DHODH and piperine at 1.98 Å resolution further reveal that Tyr356 residue of DHODH is crucial for piperine binding. Importantly, we show that piperine can inhibit T cell overactivation in a DHODH-dependent manner in concanavalin A-triggered T-cell assay and mixed lymphocyte reaction assay. Finally, piperine exhibits strong preventive and therapeutic effect in the MOG-induced experimental allergic encephalomyelitis (EAE), a useful model for studying potential treatments for MS, by restricting inflammatory cells infiltration into the CNS and preventing myelin destruction and blood-brain barrier (BBB) disruption. Taken together, these findings highlight DHODH as a therapeutic target for autoimmune disease of the nervous system, and demonstrate a novel role for piperine in the treatment of MS.

SS31 attenuates oxidative stress and neuronal apoptosis in early brain injury following subarachnoid hemorrhage possibly by the mitochondrial pathway.

BACKGROUND: SS31 has been shown to have neuroprotective effects in a number of neurological degenerative diseases. However, the mechanisms and its role of neuroprotection after subarachnoid hemorrhage (SAH) remain unexplored. The aim of the present study is to evaluate the neuroprotective effects of SS31 on early brain injury (EBI) induced by SAH in rats and the potential mechanisms of the protective effects of SS31. METHODS: Sprague-Dawley rats were randomly divided into four groups: Sham, SAH, SAH + vehicle, and SAH + SS31 groups. The SAH-induced prechiasmatic cistern rat model was established in this study. Neurological scores were evaluated at 24 h and 72 h after SAH. The brain edema, blood-brain barrier (BBB) permeability, neuronal apoptosis, malondialdehyde (MDA), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities, as well as the expression of mitochondrial and cytosolic cytochrome C (Cyt C), and Bax were analyzed at 24 h after SAH. RESULTS: When compared with the vehicle-treated group, treatment with SS31 significantly reduced MDA levels and restored the activities of GPx and SOD in the temporal cortex following SAH when compared with the vehicle-treated group. In addition, the levels of mitochondrial Cyt C and Bax respectively increased and decreased by SS31 treatment. Moreover, SS31 treatment ameliorated brain edema and Evans blue dye extravasation, improved neurological deficits, and decreased neuronal apoptosis at 24 h after SAH. CONCLUSION: Our data provides initial evidence that SS31 could alleviate EBI after SAH through its antioxidant property and ability in inhibiting neuronal apoptosis, likely by modulating the mitochondrial apoptotic pathway.

Structure-Aided Identification and Optimization of Tetrahydro-isoquinolines as Novel PDE4 Inhibitors Leading to Discovery of an Effective Antipsoriasis Agent.

Psoriasis is a common, chronic inflammatory disease characterized by abnormal skin plaques, and the effectiveness of phosphodiesterase 4 (PDE4) inhibitor to lessen the symptoms of psoriasis has been proved. Aiming to find a novel PDE4 inhibitor acting as an effective, safe, and convenient therapeutic agent, we constructed a library consisting of berberine analogues, and compound 2 with a tetrahydroisoquinoline scaffold was identified as a novel and potent hit. The structure-aided and cell-based structure-activity relationship studies on a series of tetrahydro-isoquinolines lead to efficient discovery of a qualified lead compound (16) with the high potency and selectivity, well-characterized binding mechanism, high cell permeability, good safety and pharmacokinetic profile, and impressive in vivo efficacy on antipsoriasis, in particular with a topical application. Thus, our study presents a prime example for efficient discovery of novel, potent lead compounds derived from natural products using a combination of medicinal chemistry, biochemical, biophysical, and pharmacological approaches.

Pharmacological inhibition of dihydroorotate dehydrogenase induces apoptosis and differentiation in acute myeloid leukemia cells.

Acute myeloid leukemia is a disorder characterized by abnormal differentiation of myeloid cells and a clonal proliferation derived from primitive hematopoietic stem cells. Interventions that overcome myeloid differentiation have been shown to be a promising therapeutic strategy for acute myeloid leukemia. In this study, we demonstrate that CRISPR/Cas9-mediated knockout of dihydroorotate dehydrogenase leads to apoptosis and normal differentiation of acute myeloid leukemia cells, indicating that dihydroorotate dehydrogenase is a potential differentiation regulator and a therapeutic target in acute myeloid leukemia. By screening a library of natural products, we identified a novel dihydroorotate dehydrogenase inhibitor, isobavachalcone, derived from the traditional Chinese medicine Psoralea corylifolia Using enzymatic analysis, thermal shift assay, pull down, nuclear magnetic resonance, and isothermal titration calorimetry experiments, we demonstrate that isobavachalcone inhibits human dihydroorotate dehydrogenase directly, and triggers apoptosis and differentiation of acute myeloid leukemia cells. Oral administration of isobavachalcone suppresses subcutaneous HL60 xenograft tumor growth without obvious toxicity. Importantly, our results suggest that a combination of isobavachalcone and adriamycin prolonged survival in an intravenous HL60 leukemia model. In summary, this study demonstrates that isobavachalcone triggers apoptosis and differentiation of acute myeloid leukemia cells via pharmacological inhibition of human dihydroorotate dehydrogenase, offering a potential therapeutic strategy for acute myeloid leukemia.

Iron dysregulates APP processing accompanying with sAPPα cellular retention and ß-secretase inhibition in rat cortical neurons.

Amyloid precursor protein (APP) and iron both play pivotal roles in the central nervous system, but whether and how iron influences the processing of endogenous APP in neurons remain unclear. Here, we investigated the regulatory effects and underlying mechanisms of iron on non-amyloidogenic and amyloidogenic processing of APP in rat primary cortical neurons. Treatment of the neurons with ferric ammonium citrate (FAC, 100 µmol/L) markedly facilitated the non-amyloidogenic processing of APP, as evidenced by a robust increase in α-secretase-derived carboxy-terminal fragment α (CTFα). Furthermore, the distribution of sAPPα was altered after iron treatment, and sAPPα remained in the cellular lysates instead of being secreted into the extracellular milieu. Moreover, the levels of APP amyloidogenic products, including sAPPß and Aß were both decreased. We further revealed that FAC did not alter the expression of ß-secretase, but significantly suppressed its enzymatic activity in iron-treated neurons. In a cell-free ß-secretase activity assay, FAC dose-dependently inhibited the activity of purified ß-secretase with an IC50 value of 21.67 µmol/L. Our data provide the first evidence that iron overload alters the neuronal sAPPα distribution and directly inhibits ß-secretase activity. These findings shed light on the regulatory mechanism of bio-metals on APP processing.

Revisiting the prevalence of nonclassic congenital adrenal hyperplasia in US Ashkenazi Jews and Caucasians.

PurposeNonclassic 21-hydroxylase deficiency, a mild form of congenital adrenal hyperplasia (CAH), is estimated to be the most common autosomal recessive condition, with an especially high prevalence in Ashkenazi Jews (3.7% affected, 30.9% carriers), based on a 1985 HLA-B linkage study of affected families. Affected individuals, especially women, may suffer from hyperandrogenism and infertility. State-of-the-art genetic studies have not been done to confirm these remarkable rates.MethodsCYP21A2 genotyping was performed in 200 unrelated healthy Ashkenazi Jewish subjects and 200 random US Caucasians who did not self-identify as a specific ethnicity using multiplex minisequencing, real-time polymerase chain reaction and junction site analysis.ResultsNonclassic CAH carriership was found similarly in 15% (95% confidence interval (CI): 10.4-20.7) of Ashkenazi Jews and 9.5% (95% CI: 5.8-14.4) of Caucasians (P=0.13). The proportion of Ashkenazi Jewish nonclassic CAH carriers (0.15 versus 0.309, P<0.0001) and disease affected (0.005 versus 0.037, P=0.009) was not as high as previously reported. The estimated prevalence of nonclassic CAH in Caucasians was 1 in 200 (0.5%, 95% CI: 0.01-2.8).ConclusionNonclassic CAH is a common condition, regardless of ethnicity, and should be considered with preconception and infertility counseling.

Genetics of Congenital Adrenal Hyperplasia.

Congenital adrenal hyperplasia (CAH) refers to a group of autosomal recessive disorders due to single-gene defects in the various enzymes required for cortisol biosynthesis. CAH represents a continuous phenotypic spectrum with more than 95% of all cases caused by 21-hydroxylase deficiency. Genotyping is an important tool in confirming the diagnosis or carrier state, provides prognostic information on disease severity, and is essential for genetic counseling. In this article, the authors provide an in-depth discussion on the genetics of CAH, including genetic diagnosis, molecular analysis, genotype-phenotype relationships, and counseling of patients and their families.

Ehlers-Danlos Syndrome Caused by Biallelic TNXB Variants in Patients with Congenital Adrenal Hyperplasia.

Some variants that cause autosomal-recessive congenital adrenal hyperplasia (CAH) also cause hypermobility type Ehlers-Danlos syndrome (EDS) due to the monoallelic presence of a chimera disrupting two flanking genes: CYP21A2, encoding 21-hydroxylase, necessary for cortisol and aldosterone biosynthesis, and TNXB, encoding tenascin-X, an extracellular matrix protein. Two types of CAH tenascin-X (CAH-X) chimeras have been described with a total deletion of CYP21A2 and characteristic TNXB variants. CAH-X CH-1 has a TNXB exon 35 120-bp deletion resulting in haploinsufficiency, and CAH-X CH-2 has a TNXB exon 40 c.12174C>G (p.Cys4058Trp) variant resulting in a dominant-negative effect. We present here three patients with biallelic CAH-X and identify a novel dominant-negative chimera termed CAH-X CH-3. Compared with monoallelic CAH-X, biallelic CAH-X results in a more severe phenotype with skin features characteristic of classical EDS. We present evidence for disrupted tenascin-X function and computational data linking the type of TNXB variant to disease severity.

Structural and Thermodynamic Characterization of Protein-Ligand Interactions Formed between Lipoprotein-Associated Phospholipase A2 and Inhibitors.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) represents a promising therapeutic target for atherosclerosis and Alzheimer's disease. Here we reported the first crystal structures of Lp-PLA2 bound with reversible inhibitors and the thermodynamic characterization of complexes. High rigidity of Lp-PLA2 structure and similar binding modes of inhibitors with completely different scaffolds are revealed. It not only provides the molecular basis for inhibitory activity but also sheds light on the essential features of Lp-PLA2 recognition with reversible inhibitors.

Structure-Based Discovery of PDEs Inhibitors.

Phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP and cGMP, thereby regulating the cyclic nucleotide signalling pathways and biological responses. PDEs inhibitors can be used clinically for treatment of several diseases including central nervous system disorders, erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, and inflammatory diseases such as chronic obstructive pulmonary disease. However, the unfavourable risk-benefit ratio and side-effect profiles of non-selective PDEs inhibitors have impeded their therapeutic success and therefore spurred the pharmaceutical industry to develop family-selective PDE inhibitors. Given the recent remarkable advances in structure-based drug design, this review will summarize developments and achievements in structure-based search, design and optimization of PDEs inhibitors, and highlight the challenges that need to be addressed.

Broadening the Spectrum of Ehlers Danlos Syndrome in Patients With Congenital Adrenal Hyperplasia.

CONTEXT: The contiguous gene deletion syndrome (CAH-X) was described in a subset (7%) of congenital adrenal hyperplasia (CAH) patients with a TNXA/TNXB chimera, resulting in deletions of CYP21A2, encoding 21-hydroxylase necessary for cortisol biosynthesis, and TNXB, encoding the extracellular matrix glycoprotein tenascin-X (TNX). This TNXA/TNXB chimera is characterized by a 120-bp deletion in exon 35 and results in TNXB haploinsufficiency, disrupted TGF-ß signaling, and an Ehlers Danlos syndrome phenotype. OBJECTIVE: The objective of the study was to determine the genetic status of TNXB and resulting protein defects in CAH patients with a CAH-X phenotype but not the previously described TNXA/TNXB chimera. Design, Settings, Participants, and Intervention: A total of 246 unrelated CAH patients were screened for TNXB defects. Genetic defects were investigated by Southern blotting, multiplex ligation-dependent probe amplification, Sanger, and next-generation sequencing. Dermal fibroblasts and tissue were used for immunoblotting, immunohistochemical, and coimmunoprecipitation experiments. MAIN OUTCOME MEASURES: The genetic and protein status of tenascin-X in phenotypic CAH-X patients was measured. RESULTS: Seven families harbor a novel TNXB missense variant c.12174C>G (p.C4058W) and a clinical phenotype consistent with hypermobility-type Ehlers Danlos syndrome. Fourteen CAH probands carry previously described TNXA/TNXB chimeras, and seven unrelated patients carry the novel TNXB variant, resulting in a CAH-X prevalence of 8.5%. This highly conserved pseudogene-derived variant in the TNX fibrinogen-like domain is predicted to be deleterious and disulfide bonded, results in reduced dermal elastin and fibrillin-1 staining and altered TGF-ß1 binding, and represents a novel TNXA/TNXB chimera. Tenascin-X protein expression was normal in dermal fibroblasts, suggesting a dominant-negative effect. CONCLUSIONS: CAH-X syndrome is commonly found in CAH due to 21-hydroxylase deficiency and may result from various etiological mechanisms.

A dynamic view of ATP-coupled functioning cycle of Hsp90 N-terminal domain.

Heat-shock protein 90 (Hsp90) is one of the most important chaperones involved in multiple cellular processes. The chaperoning function of Hsp90 is intimately coupled to the ATPase activity presented by its N-terminal domain. However, the molecular mechanism for the ATP-dependent working cycle of Hsp90 is still not fully understood. In this study, we use NMR techniques to investigate the structural characteristics and dynamic behaviors of Hsp90 N-terminal domain in its free and AMPPCP (ATP analogue) or ADP-bound states. We demonstrated that although AMPPCP and ADP bind to almost the same region of Hsp90, significantly different effects on the dynamics behaviors of the key structural elements were observed. AMPPCP binding favors the formation of the active homodimer of Hsp90 by enhancing the slow-motion featured conformational exchanges of those residues (A117-A141) within the lid segment (A111-G135) and around region, while ADP binding keeps Hsp90 staying at the inactive state by increasing the conformational rigidity of the lid segment and around region. Based on our findings, a dynamic working model for the ATP-dependent functioning cycle of Hsp90 was proposed.

Design, synthesis and biological evaluation of tasiamide B derivatives as BACE1 inhibitors.

Nineteen new derivatives based on the structure of marine natural product tasiamide B were designed, synthesized, and evaluated for their inhibitory activity against BACE1, a potential therapeutic target for Alzheimer's disease. The hydrophobic substituents Val at P3 position, Leu at P1' position, Ala at P2' position, and Phe at P3' position were found to significantly affect the inhibition. Free carboxylic acid at C-terminus was also found to be important to the activity. In addition, the structure-activity relationships (SARs) were supported by molecular docking simulation.

Discovery of potent N-(isoxazol-5-yl)amides as HSP90 inhibitors.

HSP90 is ubiquitously overexpressed in a broad spectrum of human cancers and has been recognized as an attractive target for cancer treatment. Here, we described the fragment screening, synthesis and structure-activity relationship studies of small molecule inhibitors with 4,5-diarylisoxazole scaffold targeting HSP90. Among them, the compound N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((4-morpholinopiperidin-1-yl)methyl)phenyl)isoxazol-5-yl)cyclopropanecarboxamide (108) showed high affinity for binding to HSP90 (FP binding assay, IC50 = 0.030 µM) and inhibited the proliferation of various human cancer cell lines with averaging GI50 about 88 nM. Compound 108 exhibited its functional inhibition of HSP90 by depleting key signaling pathways and concomitantly elevating of HSP70 and HSP27 in U-87MG cells. Further in vivo studies showed that compound 108 strongly suppressed the tumor growth of human glioblastoma xenograft model U-87MG with T/C = 18.35% at 50 mg/kg q3w/2.5w. Moreover, compound 108 also exhibited good pharmacokinetic properties. Together, our study implicates that compound 108 is a promising candidate of HSP90 inhibitor and is currently advanced to preclinical study.

Thermodynamic and structural characterization of halogen bonding in protein-ligand interactions: a case study of PDE5 and its inhibitors.

The significance of halogen bonding in protein-ligand interactions has been recognized recently. We present here the first comprehensive thermodynamic and structural characterization of halogen bonding in PDE5-inhibitor interactions. ITC studies reveal that binding strength of the halogen bonding between chlorine, bromine, and iodine of inhibitor and the protein is -1.57, -3.09, and -5.59 kJ/mol, respectively. The halogens interact with the designed residue Y612 and an unexpected buried water molecule.

Identification of a new series of potent diphenol HSP90 inhibitors by fragment merging and structure-based optimization.

Heat shock protein 90 (HSP90) is a molecular chaperone to fold and maintain the proper conformation of many signaling proteins, especially some oncogenic proteins and mutated unstable proteins. Inhibition of HSP90 was recognized as an effective approach to simultaneously suppress several aberrant signaling pathways, and therefore it was considered as a novel target for cancer therapy. Here, by integrating several techniques including the fragment-based drug discovery method, fragment merging, computer aided inhibitor optimization, and structure-based drug design, we were able to identify a series of HSP90 inhibitors. Among them, inhibitors 13, 32, 36 and 40 can inhibit HSP90 with IC50 about 20-40 nM, which is at least 200-fold more potent than initial fragments in the protein binding assay. These new HSP90 inhibitors not only explore interactions with an under-studied subpocket, also offer new chemotypes for the development of novel HSP90 inhibitors as anticancer drugs.