Identification of Novel Zika Virus Inhibitors: A Screening using Thiosemicarbazones and Thiazoles Templates.

BACKGROUND: Zika virus (ZIKV) remains an important cause of congenital infection, fetal microcephaly, and Guillain-Barré syndrome in the population. In 2016, WHO declared a cluster of microcephaly cases and other neurological disorders reported as a global public health emergency in Brazil. There is still no specific treatment for Zika virus fever, only palliative care. Therefore, there is a need for new therapies against this disease. According to the literature, thiosemicarbazone, phthalimide and thiazole are privileged structures with several biological activities, including antiviral activity against various viruses. OBJECTIVE: Based on this, this work presents an antiviral screening using previously synthesized compounds derived from thiosemicarbazone, phthalimide, and thiazole as new hits active against ZIKV. METHODS: After synthesis and characterization, all compounds were submitted to Cytotoxicity by MTT and Antiviral activity against ZIKV assays. RESULTS: Compounds 63, 64, 65, and 73 exhibited major reductions in the ZIKV title from this evaluation. Compounds 63 (99.74%), 64 (99.77%), 65 (99.92%), and 73 (99.21%) showed a higher inhibition than the standard 6MMPr (98.74%) at the CC20 dose. These results revealed new chemical entities with anti-ZIKV activity. CONCLUSION: These derivatives are promising candidates for further assays. In addition, the current approach brings a new privileged scaffolding, which may drive future drug discovery for ZIKV.

Endocrinological features of a patient with 14q microdeletion and Dubowitz phenotype.

BACKGROUND: Dubowitz syndrome (DS) is a complex and rare condition characterized by postnatal growth retardation, microcephaly, short stature, mild developmental delay, facial dysmorphism, skin eruption and bone marrow failure. Though approximately 200 cases have been described so far, no specific genetic analysis, laboratory tests or radiological exams are available to confirm the diagnosis which is still based on clinical and facial features. Although short stature is a major feature of the syndrome, no endocrine alterations have been reported so far and scant data are available about the efficacy and safety of GH treatment in these patients. METHODS: A 13-year-old male patient was referred to our attention for short stature. Endocrinological evaluation including GH axis, adrenal and gonadal functions were assessed. aCGH was performed. RESULTS: 14q terminal microdeletion associated with Dubowitz phenotype was found. Endocrinological investigations revealed the presence of hypopituitarism which showed a satisfactory response to short-term growth hormone therapy. The subject also started glucocorticoid replacement therapy. Disorders in pubertal progression and gonadal function were noted. CONCLUSIONS: Dubowitz syndrome (DS) includes different clinical findings variably occurring. Subjects with a Dubowitz phenotype should be carefully monitored for endocrinological anomalies. The prompt recognition of potential life-threatening endocrinological condition for example adrenal insufficiency is mandatory in order to start an adequate and early treatment.

Immunologic phenotype of a child with the MEHMO syndrome.

MEHMO syndrome is a rare X-linked syndrome characterized by Mental retardation, Epilepsy, Hypogenitalism, Microcephaly, and Obesity associated with the defect of protein synthesis caused by the EIF2S3 gene mutations. We hypothesized that the defect in protein synthesis could have an impact on the immune system. We describe immunologic phenotype and possible treatment outcomes in patient with MEHMO syndrome carrying a frame-shift mutation (I465fs) in the EIF2S3 gene. The proband (currently 9-year-old boy) had normal IgG and IgM levels, but had frequent respiratory and urinary tract infections. On subcutaneous immunoglobulin therapy achieving supra-physiological IgG levels the frequency of infections significantly decreased in Poisson regression by 54.5 % (CI 33.2-89.7, p=0.017). The MEHMO patient had had frequent acute infections despite normal IgG and IgM serum levels and responded well to the immunoglobulin treatment.

Pre- and postnatal growth failure with microcephaly due to two novel heterozygous IGF1R mutations and response to growth hormone treatment.

AIM: To explore the phenotype and response to growth hormone in patients with heterozygous mutations in the insulin-like growth factor I receptor gene (IGF1R). METHODS: Children with short stature, microcephaly, born SGA combined with biochemical sign of IGF-I insensitivity were analysed for IGF1R mutations or deletions using Sanger sequencing and Multiple ligation-dependent probe amplification analysis. RESULTS: In two families, a novel heterozygous non-synonymous missense IGF1R variant was identified. In family 1, c.3364G > T, p.(Gly1122Cys) was found in the proband and co-segregated perfectly with the phenotype in three generations. In family 2, a de novo variant c.3530G > A, p.(Arg1177His) was detected. Both variants were rare, not present in the GnomAD database. Three individuals carrying IGF1R mutations have received rhGH treatment. The average gain in height SDS during treatment was 0.42 (range: 0.26-0.60) and 0.64 (range: 0.32-0.86) after 1 and 2 years of treatment, respectively. CONCLUSION: Our study presents two heterozygous IGF1R mutations causing pre- and postnatal growth failure and microcephaly and also indicates that individuals with heterozygous IGF1R mutations can respond to rhGH treatment. The findings highlight that sequencing of the IGF1R should be considered in children with microcephaly and short stature due to pre- and postnatal growth failure.

Zika Virus Dependence on Host Hsp70 Provides a Protective Strategy against Infection and Disease.

The spread of mosquito-borne Zika virus (ZIKV), which causes neurological disorders and microcephaly, highlights the need for countermeasures against sudden viral epidemics. Here, we tested the concept that drugs targeting host proteostasis provide effective antivirals. We show that different cytosolic Hsp70 isoforms are recruited to ZIKV-induced compartments and are required for virus replication at pre- and post-entry steps. Drugs targeting Hsp70 significantly reduce replication of different ZIKV strains in human and mosquito cells, including human neural stem cells and a placental trophoblast cell line, at doses without appreciable toxicity to the host cell. By targeting several ZIKV functions, including entry, establishment of active replication complexes, and capsid assembly, Hsp70 inhibitors are refractory to the emergence of drug-resistant virus. Importantly, these drugs protected mouse models from ZIKV infection, reducing viremia, mortality, and disease symptoms. Hsp70 inhibitors are thus attractive candidates for ZIKV therapeutics with the added benefit of a broad spectrum of action.

Amyloid clearance defect in ApoE4 astrocytes is reversed by epigenetic correction of endosomal pH.

Endosomes have emerged as a central hub and pathogenic driver of Alzheimer's disease (AD). The earliest brain cytopathology in neurodegeneration, occurring decades before amyloid plaques and cognitive decline, is an expansion in the size and number of endosomal compartments. The strongest genetic risk factor for sporadic AD is the ε4 allele of Apolipoprotein E (ApoE4). Previous studies have shown that ApoE4 potentiates presymptomatic endosomal dysfunction and defective endocytic clearance of amyloid beta (Aß), although how these two pathways are linked at a cellular and mechanistic level has been unclear. Here, we show that aberrant endosomal acidification in ApoE4 astrocytes traps the low-density lipoprotein receptor-related protein (LRP1) within intracellular compartments, leading to loss of surface expression and Aß clearance. Pathological endosome acidification is caused by ε4 risk allele-selective down-regulation of the Na+/H+ exchanger isoform NHE6, which functions as a critical leak pathway for endosomal protons. In vivo, the NHE6 knockout (NHE6KO) mouse model showed elevated Aß in the brain, consistent with a causal effect. Increased nuclear translocation of histone deacetylase 4 (HDAC4) in ApoE4 astrocytes, compared with the nonpathogenic ApoE3 allele, suggested a mechanistic basis for transcriptional down-regulation of NHE6. HDAC inhibitors that restored NHE6 expression normalized ApoE4-specific defects in endosomal pH, LRP1 trafficking, and amyloid clearance. Thus, NHE6 is a downstream effector of ApoE4 and emerges as a promising therapeutic target in AD. These observations have prognostic implications for patients who have Christianson syndrome with loss of function mutations in NHE6 and exhibit prominent glial pathology and progressive hallmarks of neurodegeneration.

A Single Injection of Human Neutralizing Antibody Protects against Zika Virus Infection and Microcephaly in Developing Mouse Embryos.

Zika virus (ZIKV) is a mosquito-transmitted flavivirus that is generally benign in humans. However, an emergent strain of ZIKV has become widespread, causing severe pre- and post-natal neurological defects. There is now an urgent need for prophylactic and therapeutic agents. To address this, we investigated six human monoclonal antibodies with ZIKV epitope specificity and neutralizing activity in mouse models of ZIKV infection and microcephaly. A single intraperitoneal injection of these antibodies conveyed distinct levels of adult and in utero protection from ZIKV infection, which closely mirrored their respective in vitro neutralizing activities. One antibody, ZK2B10, showed the most potent neutralization activity, completely protected uninfected mice, and markedly reduced tissue pathology in infected mice. Thus, ZK2B10 is a promising candidate for the development of antibody-based interventions and informs the rational design of ZIKV vaccine.

Seizures as a Complication of Congenital Zika Syndrome in Early Infancy.

Zika virus transmission in Brazil was linked to a large outbreak of microcephaly but less is known about longer term anthropometric and neurological outcomes. We studied a cohort of infants born between October 31, 2015, and January 9, 2016, in a state maternity hospital, followed up for 101 ± 28 days by home visits. Microcephaly (< 2 standard deviations, Intergrowth standard) occurred in 62 of 412 (15%) births. Congenital Zika syndrome (CZS) was diagnosed in 29 patients. Among CZS patients, we observed a significant gain in anthropometric measures (P < 0.001) but no significant gain in percentile for these measures. The main neurological outcome was epilepsy, occurring in 48% of infants at a rate of 15.6 cases per 100 patient-months, frequently requiring multiple anti-seizure medications. The cumulative fatality rate was 7.4% (95% confidence interval: 2.1-23.4%). Health-care professionals should be alerted on the high risk of epilepsy and death associated with CZS in early infancy and the need to actively screen for seizures and initiate timely treatment.

Zika virus outbreak: a review of neurological complications, diagnosis, and treatment options.

Zika virus (ZIKV) is an arbovirus transmitted mainly by mosquitos of Aedes species. The virus has emerged in recent years and spread throughout North and South Americas. The recent outbreak of ZIKV started in Brazil (2015) has resulted in infections surpassing a million mark. Contrary to the previous beliefs that Zika causes mildly symptomatic infections fever, headache, rash, arthralgia, and conjunctivitis, the recent outbreak associated ZIKV to serious neurological complications such as microcephaly, Guillain-Barré syndrome, and eye infections. The recent outbreak has resulted in an astonishing number of microcephaly cases in fetus and infants. Consequently, numerous studies were conducted using in vitro cell and in vivo animal models. These studies showed clear links between ZIKV infections and neurological abnormalities. Diagnosis methods based on nucleic acid and serological detection facilitated rapid and accurate identification of ZIKV infections. New transmission modalities such as sexual and transplacental transmission were uncovered. Given the seriousness of ZIKV infections, WHO declared the development of safe and effective vaccines and new antiviral drugs as an urgent global health priority. Rapid work in this direction has led to the identification of several vaccine and antiviral drug candidates. Here, we review the remarkable progress made in understanding the molecular links between ZIKV infections and neurological irregularities, new diagnosis methods, potential targets for antiviral drugs, and the current state of vaccine development.

Galloway-mowat syndrome - unusual form of nephrotic syndrome in adolescent.

Galloway-Mowat syndrome (GMS), also acknowledged as Microcephaly-Hiatal hernia nephrotic syndrome, is an uncommon genetic disorder inherited as an autosomal recessive trait usually seen before two years of life. It is an exceptional multisystem genetic disorder with a collection of skeletal, neurological, facial, gastrointestinal, growth, and renal abnormalities. This case report describes GMS in a girl, suffering from developmental delay, stunted growth, and various dysmorphic features, in whom nephrotic syndrome became apparent at adolescent age.

Zika virus cell tropism in the developing human brain and inhibition by azithromycin.

The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic.

Two new cases of serine deficiency disorders treated with l-serine.

OBJECTIVE AND PATIENTS: We report on two new cases of serine deficiency due respectively to 3-phosphoglycerate dehydrogenase (PHGDH) deficiency (Patient 1) and phosphoserine aminotransferase (PSAT1) deficiency (Patient 2), presenting with congenital microcephaly (<3rd centile at birth) and encephalopathy with spasticity. Patient 1 had also intractable seizures. A treatment with oral l-serine was started at age 4.5 years and 3 months respectively. RESULTS: Serine levels were low in plasma and CSF relative to the reference population, for which we confirm recently redefined intervals based on a larger number of samples. l-Serine treatment led in patient 1 to a significant reduction of seizures after one week of treatment and decrease of electroencephalographic abnormalities within one year. In patient 2 treatment with l-serine led to an improvement of spasticity. However for both patients, l-serine failed to improve substantially head circumference (HC) and neurocognitive development. In a couple related to patient's 2 family, dosage of serine was performed on fetal cord blood when the fetus presented severe microcephaly, showing reduced serine levels at 30 weeks of pregnancy. CONCLUSIONS: l-Serine treatment in patients with 2 different serine synthesis defects, led to a significant reduction of seizures and an improvement of spasticity, but failed to improve substantially neurocognitive impairment. Therefore, CSF and plasma serine levels should be measured in all cases of severe microcephaly at birth to screen for serine deficiency, as prompt treatment with l-serine may significantly impact the outcome of the disease. Reduced serine levels in fetal cord blood may also be diagnostic as early as 30 weeks of pregnancy.

Effect of recombinant insulin-like growth factor-1 treatment on short-term linear growth in a child with Majewski osteodysplastic primordial dwarfism type II and hepatic insufficiency.

We report the case of a boy affected by severe intrauterine and postnatal growth retardation, microcephaly, facial dysmorphisms and postnecrotic cirrhosis, diagnosed at birth as having Seckel syndrome, and subsequently confirmed as Majewski osteodysplastic primordial dwarfism type II (MOPD II) on the basis of clinical and radiological features of skeletal dysplasia. At our observation (6 years 7 months) he presented height -10.3 standard deviation score (SDS), weight -22.1 SDS, head circumference -8 SDS, delayed bone age of 4 years with respect to chronological age. In consideration of the low levels of insulin-like growth factor-1 (IGF-1) as well as of hepatic insufficiency, we started the treatment with recombinant human IGF-1 (rhIGF-1) at the dose of 0.04 mg/kg in 2 doses/day, with an increase of 0.04 mg/kg after 1 week until the maximum dose of 0.12 mg/kg. We observed an early response to rhIGF-1 treatment, with a shift of height velocity from 1.8 cm/year (-4.6 SDS) at 4 cm/year (-1.9 SDS), and an increase in bone age of 1.5 years during the first 6 months. rhIGF-1 treatment does not seem to be able to replace the physiological action of IGF-1 in patients with MOPD II and hepatic insufficiency, however, it seems to preserve the typical growth pattern of MOPD II patients, avoiding a further widening of the growth deficiency in these subjects.

Idiopathic pulmonary arterial hypertension in a young patient with the Cohen syndrome.

We admitted a 16-year-old boy with the Cohen syndrome to our institution for increasing dyspnoea. Investigations revealed idiopathic pulmonary hypertension. He was commenced on bosentan and oral anticoagulation and was followed up for nearly 7 years, during which he was readmitted for dynamic measurements of pulmonary artery pressure. Despite initial improvement, the right heart pressures increased again and sildenafil was added. His final hospitalisation was due to increasing breathlessness and episodes of syncope. The addition of prostacyclin conferred no reduction in pulmonary artery pressure. The patient suffered a cardiac arrest and remained intubated for 2 weeks, during which Klebsiella pneumonia and superinfection with the H1N1 swine flu virus occurred. The patient died due to multi-organ failure, nearly 7 years after his initial diagnosis. The Cohen syndrome, its phenotype and clinical findings, and the incidence and treatment of pulmonary hypertension are discussed.

An update on serine deficiency disorders.

Serine deficiency disorders are caused by a defect in one of the three synthesising enzymes of the L-serine biosynthesis pathway. Serine deficiency disorders give rise to a neurological phenotype with psychomotor retardation, microcephaly and seizures in newborns and children or progressive polyneuropathy in adult patients. There are three defects that cause serine deficiency of which 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency, the defect affecting the first step in the pathway, has been reported most frequently. The other two disorders in L-serine biosynthesis phosphoserine aminotransferase (PSAT) deficiency and phosphoserine phosphatase (PSP) deficiency have been reported only in a limited number of patients. The biochemical hallmarks of all three disorders are low concentrations of serine in cerebrospinal fluid and plasma. Prompt recognition of affected patients is important, since serine deficiency disorders are treatable causes of neurometabolic disorders. The use of age-related reference values for serine in CSF and plasma can be of great help in establishing a correct diagnosis of serine deficiency, in particular in newborns and young children.

Small molecule inhibition of p38 MAP kinase extends the replicative life span of human ATR-Seckel syndrome fibroblasts.

Ataxia-telangiectasia and rad3 (ATR)-related Seckel syndrome is associated with growth retardation and premature aging features. ATR-Seckel fibroblasts have a reduced replicative capacity in vitro and an aged morphology that is associated with activation of stress-associated p38 mitogen-activated protein kinase and phosphorylated HSP27. These phenotypes are prevented using p38 inhibitors, with replicative capacity restored to the normal range. However, this stressed phenotype is retained in telomerase-immortalized ATR-Seckel fibroblasts, indicating that it is independent of telomere erosion. As with normal fibroblasts, senescence in ATR-Seckel is bypassed by p53 abrogation. Young ATR-Seckel fibroblasts show elevated levels of p21(WAF1), p16(INK4A), phosphorylated actin-binding protein cofilin, and phosphorylated caveolin-1, with small molecule drug inhibition of p38 reducing p16(INK4A) and caveolin-1 phosphorylation. In conclusion, ATR-Seckel fibroblasts undergo accelerated aging via stress-induced premature senescence and p38 activation that may underlie certain clinical features of Seckel syndrome, and our data suggest a novel target for pharmacological intervention in this human syndrome.

Epilepsy in Mowat-Wilson syndrome: delineation of the electroclinical phenotype.

Mowat-Wilson syndrome (MWS) is a genetic disease caused by heterozygous mutations or deletions of the ZEB2 gene and is characterized by distinctive facial features, epilepsy, moderate to severe intellectual disability, corpus callosum abnormalities and other congenital malformations. Epilepsy is considered a main manifestation of the syndrome, with a prevalence of about 70-75%. In order to delineate the electroclinical phenotype of epilepsy in MWS, we investigated epilepsy onset and evolution, including seizure types, EEG features, and response to anti-epileptic therapies in 22 patients with genetically confirmed MWS. Onset of seizures occurred at a median age of 14.5 months (range: 1-108 months). The main seizure types were focal and atypical absence seizures. In all patients the first seizure was a focal seizure, often precipitated by fever. The semiology was variable, including hypomotor, versive, or focal clonic manifestations; frequency ranged from daily to sporadic. Focal seizures were more frequent during drowsiness and sleep. In 13 patients, atypical absence seizures appeared later in the course of the disease, usually after the age of 4 years. Epilepsy was usually quite difficult to treat: seizure freedom was achieved in nine out of the 20 treated patients. At epilepsy onset, the EEGs were normal or showed only mild slowing of background activity. During follow-up, irregular, diffuse frontally dominant and occasionally asymmetric spike and waves discharges were seen in most patients. Sleep markedly activated these abnormalities, resulting in continuous or near-to-continuous spike and wave activity during slow wave sleep. Slowing of background activity and poverty of physiological sleep features were seen in most patients. Our data suggest that a distinct electroclinical phenotype, characterized by focal and atypical absence seizures, often preceded by febrile seizures, and age-dependent EEG changes, can be recognized in most patients with MWS.

Growth in individuals with Majewski osteodysplastic primordial dwarfism type II caused by pericentrin mutations.

Microcephalic primordial dwarfism (MPD) is a class of disorders characterized by intrauterine growth restriction (IUGR), impaired postnatal growth and microcephaly. Majewski osteodysplastic primordial dwarfism type II (MOPD II) is one of the more common conditions within this group. MOPD II is caused by truncating mutations in pericentrin (PCNT) and is inherited in an autosomal recessive manner. Detailed growth curves for length, weight, and OFC are presented here and derived from retrospective data from 26 individuals with MOPD II confirmed by molecular or functional studies. Severe pre- and postnatal growth failure is evident in MOPD II patients. The length, weight, and OFC at term (when corrected for gestational age) were -7.0, -3.9, and -4.6 standard deviation (SD) below the population mean and equivalent to the 50th centile of a 28-29-, 31-32-, and 30-31-week neonate, respectively. While at skeletal maturity, the height, weight, and OFC were -10.3, -14.3, and -8.5 SD below the population mean and equivalent to the size of 3-year 10- to 11-month-old, a 5-year 2- to 3-month-old, and 5- to 6-month-old, respectively. During childhood, MOPD II patients grow with slowed, but fairly constant growth velocities and show no evidence of any pubertal growth spurt. Treatment with human growth hormone (n = 11) did not lead to any significant improvement in final stature. The growth charts presented here will be of assistance with diagnosis and management of MOPD II, and should have particular utility in nutritional management of MOPD II during infancy.

Growth hormone treatment, final height, insulin-like growth factors, ghrelin, and adiponectin in four siblings with Seckel syndrome.

OBJECTIVE: To report on the effect of growth hormone (GH) treatment on final height (FH) and to describe the insulin-like growth factor (IGF) system, ghrelin, and adiponectin (ADPN) in children with Seckel syndrome. SUBJECTS AND RESULTS: Four severely growth-retarded Iraqi siblings (two girls and two boys) with Seckel syndrome were referred at ages 16.5, 14.4, 12.4, and 10.4 years. They were born at term, but their growth was retarded and birth weight ranged between 1 and 1.5 kg. The children were healthy and had a normal response to GH provocative test. Long-term GH treatment of the youngest brother and sister increased the FH by 7.2 and 3.4 cm, respectively, compared with their older brother and sister. At FH, body mass index standard deviation scores (BMISDS) ranged from -3.0 to -3.9. Serum levels of immunoreactive IGF-1, bioactive IGF-1, and IGF-binding protein 3 were all within normal to high range before GH treatment and increased after GH treatment. Fasting plasma ghrelin remained severely reduced. Despite low BMISDS, plasma ADPN was moderately reduced and showed an almost complete absence of the low-molecular-weight subform. CONCLUSION: This is the first report on the effect of GH treatment on FH in children with Seckel syndrome. GH may have increased FH. In addition to growth defects and reduced BMISDS, patients with Seckel syndrome are characterized by low fasting ghrelin levels, low total ADPN, and near deficiency of the low-molecular-weight ADPN subform. The possible significance of the hormonal changes requires further investigations.