Identification, binding, and structural characterization of single domain anti-PD-L1 antibodies inhibitory of immune regulatory proteins PD-1 and CD80.

Programmed death-ligand 1 (PD-L1) is a key immune regulatory protein that interacts with programmed cell death protein 1 (PD-1), leading to T-cell suppression. Whilst this interaction is key in self-tolerance, cancer cells evade the immune system by overexpressing PD-L1. Inhibition of the PD-1/PD-L1 pathway with standard monoclonal antibodies has proven a highly effective cancer treatment; however, single domain antibodies (VHH) may offer numerous potential benefits. Here, we report the identification and characterization of a diverse panel of 16 novel VHHs specific to PD-L1. The panel of VHHs demonstrate affinities of 0.7 nM to 5.1 µM and were able to completely inhibit PD-1 binding to PD-L1. The binding site for each VHH on PD-L1 was determined using NMR chemical shift perturbation mapping and revealed a common binding surface encompassing the PD-1-binding site. Additionally, we solved crystal structures of two representative VHHs in complex with PD-L1, which revealed unique binding modes. Similar NMR experiments were used to identify the binding site of CD80 on PD-L1, which is another immune response regulatory element and interacts with PD-L1 localized on the same cell surface. CD80 and PD-1 were revealed to share a highly overlapping binding site on PD-L1, with the panel of VHHs identified expected to inhibit CD80 binding. Comparison of the CD80 and PD-1 binding sites on PD-L1 enabled the identification of a potential antibody binding region able to confer specificity for the inhibition of PD-1 binding only, which may offer therapeutic benefits to counteract cancer cell evasion of the immune system.

Evaluating the druggability of TrmD, a potential antibacterial target, through design and microbiological profiling of a series of potent TrmD inhibitors.

The post-transcriptional modifier tRNA-(N1G37) methyltransferase (TrmD) has been proposed to be essential for growth in many Gram-negative and Gram-positive pathogens, however previously reported inhibitors show only weak antibacterial activity. In this work, optimisation of fragment hits resulted in compounds with low nanomolar TrmD inhibition incorporating features designed to enhance bacterial permeability and covering a range of physicochemical space. The resulting lack of significant antibacterial activity suggests that whilst TrmD is highly ligandable, its essentiality and druggability are called into question.

Allosteric activation of MALT1 by its ubiquitin-binding Ig3 domain.

The catalytic activity of the protease MALT1 is required for adaptive immune responses and regulatory T (Treg)-cell development, while dysregulated MALT1 activity can lead to lymphoma. MALT1 activation requires its monoubiquitination on lysine 644 (K644) within the Ig3 domain, localized adjacent to the protease domain. The molecular requirements for MALT1 monoubiquitination and the mechanism by which monoubiquitination activates MALT1 had remained elusive. Here, we show that the Ig3 domain interacts directly with ubiquitin and that an intact Ig3-ubiquitin interaction surface is required for the conjugation of ubiquitin to K644. Moreover, by generating constitutively active MALT1 mutants that overcome the need for monoubiquitination, we reveal an allosteric communication between the ubiquitination site K644, the Ig3-protease interaction surface, and the active site of the protease domain. Finally, we show that MALT1 mutants that alter the Ig3-ubiquitin interface impact the biological response of T cells. Thus, ubiquitin binding by the Ig3 domain promotes MALT1 activation by an allosteric mechanism that is essential for its biological function.

Accuracy of sonographic estimation of weight in fetuses with abdominal wall defects.

BACKGROUND: Accurate estimation of fetal weight is essential in guiding management of fetuses with abdominal wall defects (AWDs), as growth restriction is an important predictor of perinatal morbidity and mortality. Several sonographic formulae are available involving multiple biometric parameters, but abdominal circumference measurements may underestimate weight in fetuses with AWDs. No formula has yet shown superior accuracy. AIMS: The objectives of this study were to evaluate, in fetuses with gastroschisis and omphalocoele, the accuracy of a sonographic estimated fetal weight (EFW) formula proposed by Siemer and colleagues, specifically for use in fetuses with AWDs compared to the commonly used Hadlock IV formula in estimating fetal weight, and detecting small for gestational age (SGA) fetuses. MATERIALS AND METHODS: A retrospective cohort of 113 fetuses with AWDs was identified from an Australian teaching hospital over 13 years. Pregnancy data and sonographic fetal biometry parameters were obtained. The accuracy of each formula in predicting birthweight was compared using Bland-Altman limits of agreement, and the intraclass correlation coefficient between EFW and actual birthweight. Performance of each formula in detecting SGA fetuses was determined. RESULTS: The Siemer and Hadlock formulae have similar accuracies for predicting birthweight in fetuses with AWDs. The Hadlock formula has a higher detection rate for SGA < 10th centile and < 3rd centile compared to the Seimer formula (84% vs 68% and 83% vs 67% respectively), albeit with a higher false-positive rate. CONCLUSION: There is no clear clinical advantage in using the Siemer formula, which is specifically designed for fetuses with AWDs, over the Hadlock formula to estimate weight in fetuses with AWDs.

Potent bicyclic inhibitors of malarial cGMP-dependent protein kinase: approaches to combining improvements in cell potency, selectivity and structural novelty.

Focussed studies on imidazopyridine inhibitors of Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG) have significantly advanced the series towards desirable in vitro property space. LLE-based approaches towards combining improvements in cell potency, key physicochemical parameters and structural novelty are described, and a structure-based design hypothesis relating to substituent regiochemistry has directed efforts towards key examples with well-balanced potency, ADME and kinase selectivity profiles.

Potent inhibitors of malarial P. Falciparum protein kinase G: Improving the cell activity of a series of imidazopyridines.

Development of a class of bicyclic inhibitors of the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG), starting from known compounds with activity against a related parasite PKG orthologue, is reported. Examination of key sub-structural elements led to new compounds with good levels of inhibitory activity against the recombinant kinase and in vitro activity against the parasite. Key examples were shown to possess encouraging in vitro ADME properties, and computational analysis provided valuable insight into the origins of the observed activity profiles.

Receptor binding by a ferret-transmissible H5 avian influenza virus.

Cell-surface-receptor binding by influenza viruses is a key determinant of their transmissibility, both from avian and animal species to humans as well as from human to human. Highly pathogenic avian H5N1 viruses that are a threat to public health have been observed to acquire affinity for human receptors, and transmissible-mutant-selection experiments have identified a virus that is transmissible in ferrets, the generally accepted experimental model for influenza in humans. Here, our quantitative biophysical measurements of the receptor-binding properties of haemagglutinin (HA) from the transmissible mutant indicate a small increase in affinity for human receptor and a marked decrease in affinity for avian receptor. From analysis of virus and HA binding data we have derived an algorithm that predicts virus avidity from the affinity of individual HA-receptor interactions. It reveals that the transmissible-mutant virus has a 200-fold preference for binding human over avian receptors. The crystal structure of the transmissible-mutant HA in complex with receptor analogues shows that it has acquired the ability to bind human receptor in the same folded-back conformation as seen for HA from the 1918, 1957 (ref. 4), 1968 (ref. 5) and 2009 (ref. 6) pandemic viruses. This binding mode is substantially different from that by which non-transmissible wild-type H5 virus HA binds human receptor. The structure of the complex also explains how the change in preference from avian to human receptors arises from the Gln226Leu substitution, which facilitates binding to human receptor but restricts binding to avian receptor. Both features probably contribute to the acquisition of transmissibility by this mutant virus.

Normal sonographic renal length measurements in an Australian pediatric population.

BACKGROUND: Reference charts depicting normal growth are important for the sonographic assessment of the pediatric kidney. Limited charts are available for clinical use in an Australian population. OBJECTIVE: To retrospectively collate sonographic renal length measurements in a cohort of low-risk Australian children aged newborn to 16 years to produce a reference table and comparison with other published charts. MATERIALS AND METHODS: We identified consecutive pediatric patients who were at low risk for renal disease and had renal lengths measured. After exclusions, we included 941 renal lengths (male 490, female 451). We used linear regression to estimate the relationship of renal length with age, gender and side. We calculated percentile values of renal length according to age categories. RESULTS: No statistically significant differences in mean renal length were observed between males and females, or for left and right kidneys. We tabulated reference data and provide them in a reference chart (1-, 2.5-, 5-, 10-, 50-, 90-, 97.5- and 99-percentiles). CONCLUSION: We calculated new reference ranges for pediatric renal length using a larger cohort than previously published, from a population with diverse ethnicity.

Knowledge of Safety, Training, and Practice of Neonatal Cranial Ultrasound: A Survey of Operators.

OBJECTIVES: Ultrasound can lead to thermal and mechanical effects in interrogated tissues. This possibility suggests a potential risk during neonatal cranial ultrasound examinations. The aim of this study was to explore safety knowledge and training of neonatal cranial ultrasound among Australian operators who routinely perform these scans. METHODS: An online survey was administered on biosafety and training in neonatal cranial ultrasound, targeting all relevant professionals who can perform neonatal cranial ultrasound examinations in Australia: namely, radiologists, neonatologists, sonographers, and pediatricians. The survey was conducted between November 2013 and May 2014. RESULTS: A total of 282 responses were received. Twenty of 208 (10%) answered all ultrasound biosafety questions correctly, and 49 of 169 (29%) correctly defined the thermal index. Two-thirds (134 of 214 [63%]) of respondents failed to recognize that reducing the overall scanning time is the most effective method of reducing the total power exposure. Only 13% (31 of 237) indicated that a predetermined fixed period of training or that a specified minimum number of supervised scans was used during training. The reported number of supervised scans during training was highly variable. Almost half of the participants (82 of 181 [45%]) stated that they had received supervision for 10 to 50 scans (median, 20 scans). CONCLUSIONS: There is a need to educate operators on biosafety issues and approaches to minimize power outputs and reduce the overall duration of cranial ultrasound scans. Development of standardized training requirements may be warranted.

Structural studies of postentry restriction factors reveal antiparallel dimers that enable avid binding to the HIV-1 capsid lattice.

Restriction factors (RFs) form important components of host defenses to retroviral infection. The Fv1, Trim5α, and TrimCyp RFs contain N-terminal dimerization and C-terminal specificity domains that target assembled retroviral capsid (CA) proteins enclosing the viral core. However, the molecular detail of the interaction between RFs and their CA targets is unknown. Therefore, we have determined the crystal structure of the B-box and coiled-coil (BCC) region from Trim5α and used small-angle X-ray scattering to examine the solution structure of Trim5α BCC, the dimerization domain of Fv1 (Fv1Ntd), and the hybrid restriction factor Fv1Cyp comprising Fv1NtD fused to the HIV-1 binding protein Cyclophilin A (CypA). These data reveal that coiled-coil regions of Fv1 and Trim5α form extended antiparallel dimers. In Fv1Cyp, two CypA moieties are located at opposing ends, creating a molecule with a dumbbell appearance. In Trim5α, the B-boxes are located at either end of the coiled-coil, held in place by interactions with a helical motif from the L2 region of the opposing monomer. A comparative analysis of Fv1Cyp and CypA binding to a preformed HIV-1 CA lattice reveals how RF dimerization enhances the affinity of interaction through avidity effects. We conclude that the antiparallel organization of the NtD regions of Fv1 and Trim5α dimers correctly positions C-terminal specificity and N-terminal effector domains and facilitates stable binding to adjacent CA hexamers in viral cores.

Routine Screening for Callosal Dysgenesis in the Second Trimester Is Achievable With Intensive Training.

OBJECTIVES: The purpose of this study was to determine whether routine direct visualization of the corpus callosum is achievable during second-trimester sonography when performed by a large group of sonographers in a general second-trimester sonographic screening program. The secondary aim was to determine the time taken to obtain a sagittal corpus callosum image. METHODS: We conducted a retrospective cohort study of visualization of the corpus callosum before and after intensive training. Images from 150 consecutive second-trimester scans were reviewed before and after training to evaluate the image quality of the corpus callosum. RESULTS: A total of 300 cases were evaluated before and after training. There was a significant increase in the rate of complete visualization of the corpus callosum after intensive training (P < .0001). Before training 35 of 150 cases (23%) had complete visualization of the corpus callosum versus 107 of 150 (71%) after training. The mean time to perform the corpus callosum views was 53.4 seconds before training compared to 56.2 seconds after training. CONCLUSIONS: Assessing the corpus callosum in the sagittal view is difficult and requires appropriate training and patience; however, this view is feasible without adding substantial time to the examination and provides additional information during a routine second-trimester morphologic scan.

Catechols and 3-hydroxypyridones as inhibitors of the DNA repair complex ERCC1-XPF.

Catechol-based inhibitors of ERCC1-XPF endonuclease activity were identified from a high-throughput screen. Exploration of the structure-activity relationships within this series yielded compound 13, which displayed an ERCC1-XPF IC50 of 0.6 µM, high selectivity against FEN-1 and DNase I and activity in nucleotide excision repair, cisplatin enhancement and γH2AX assays in A375 melanoma cells. Screening of fragments as potential alternatives to the catechol group revealed that 3-hydroxypyridones are able to inhibit ERCC1-XPF with high ligand efficiency, and elaboration of the hit gave compounds 36 and 37 which showed promising ERCC1-XPF IC50 values of <10 µM.

N-Hydroxyimides and hydroxypyrimidinones as inhibitors of the DNA repair complex ERCC1-XPF.

A high throughput screen allowed the identification of N-hydroxyimide inhibitors of ERCC1-XPF endonuclease activity with micromolar potency, but they showed undesirable selectivity profiles against FEN-1. A scaffold hop to a hydroxypyrimidinone template gave compounds with similar potency but allowed selectivity to be switched in favour of ERCC1-XPF over FEN-1. Further exploration of the structure-activity relationships around this chemotype gave sub-micromolar inhibitors with >10-fold selectivity for ERCC1-XPF over FEN-1.

Evolution of the receptor binding properties of the influenza A(H3N2) hemagglutinin.

The hemagglutinin (HA) of influenza A(H3N2) virus responsible for the 1968 influenza pandemic derived from an avian virus. On introduction into humans, its receptor binding properties had changed from a preference for avian receptors (α2,3-linked sialic acid) to a preference for human receptors (α2,6-linked sialic acid). By 2001, the avidity of human H3 viruses for avian receptors had declined, and since then the affinity for human receptors has also decreased significantly. These changes in receptor binding, which correlate with increased difficulties in virus propagation in vitro and in antigenic analysis, have been assessed by virus hemagglutination of erythrocytes from different species and quantified by measuring virus binding to receptor analogs using surface biolayer interferometry. Crystal structures of HA-receptor analog complexes formed with HAs from viruses isolated in 2004 and 2005 reveal significant differences in the conformation of the 220-loop of HA1, relative to the 1968 structure, resulting in altered interactions between the HA and the receptor analog that explain the changes in receptor affinity. Site-specific mutagenesis shows the HA1 Asp-225→Asn substitution to be the key determinant of the decreased receptor binding in viruses circulating since 2005. Our results indicate that the evolution of human influenza A(H3N2) viruses since 1968 has produced a virus with a low propensity to bind human receptor analogs, and this loss of avidity correlates with the marked reduction in A(H3N2) virus disease impact in the last 10 y.

A phospha-oseltamivir-biotin conjugate as a strong and selective adhesive for the influenza virus.

We present the synthesis and application of a molecule containing both the powerful influenza neuraminidase (NA) inhibitor phospha-oseltamivir and d-biotin, connected via an undecaethylene glycol spacer. It inhibits influenza virus neuraminidase (from the H3N2 X31 virus) in the same range as oseltamivir, with a slow off-rate, and produces a stable NA-coated surface when loaded onto streptavidin-coated biosensors. Purified X31 virus binds to these loaded biosensors with an apparent dissociation constant in the low picomolar range and binding of antibodies to the immobilized virus could be readily detected. The compound is thus a potential candidate for the selective immobilization of influenza virus in influenza diagnosis, vaccine choice, development or testing.

Sonographic risk stratification of FDG-avid thyroid nodules using the Thyroid Imaging Reporting and Data System.

INTRODUCTION: The increasing usage of positron emission tomography/computed tomography (PET/CT) for detection and monitoring of malignancy has led to an increase in incidental detection of thyroid nodules. Nodules that demonstrate increased avidity for 2-[18F]-fluoro-2-deoxy-d-glucose (FDG) have been shown to carry a high incidence of malignancy and warrant further investigation. At present, there has been limited research on the risk stratification of FDG-avid thyroid incidentalomas. Thus, this study aims to evaluate the efficacy of the ACR TIRADS classification in the risk stratification of such nodules. METHODS: Data were collected retrospectively for FDG-avid thyroid incidentalomas over a 10-year period. Nodules were characterised using the TIRADS classification and, subsequently, underwent fine-needle aspirate cytology. Cytological findings were classified using the Bethesda reporting system. Non-diagnostic samples (Bethesda class I) were excluded. The remaining samples were divided into two groups: benign (Bethesda class II) or suspicious for malignancy/malignant (Bethesda class III or above). RESULTS: Thirty-six percent of low-risk nodules and 45% of high-risk nodules were malignant, respectively (P = 0.516). The sensitivity and specificity of TIRADS for detection of malignant nodules were 56% and 54%, respectively. There were no malignant TIRADS 1 or 2 nodules. The absence of any suspicious sonographic features had a 1.0 negative predictive value. CONCLUSIONS: FDG-avid nodules classified as TIRADS 1 or 2 or have no suspicious ultrasound features have a 0% incidence of malignancy and thus may not require further assessment with fine-needle aspirate cytology (FNA) when detected incidentally. FDG-avid nodules that are TIRADS 3 or above should undergo FNA regardless of size due to the high risk of malignancy and poor sensitivity of the TIRADS classification system.

A review of outcomes of an intracavernosal injection therapy programme.

UNLABELLED: Study Type--Therapy (outcomes research) Level of Evidence 2b. What's known on the subject? and What does the study add? Intracavernosal injection (ICI) therapy is an important treatment option for erectile dysfunction. However, high discontinuation rates have been reported for ICI therapy, and a risk of priapism has long been a concern. There has never been a large sample study performed with multivariate analysis to characterise outcomes of ICI therapy. The present paper reviews ICI therapy outcomes in a very large population of men at a tertiary care Sexual Medicine Clinic over 5 years. Multivariate analysis was used to further characterise these outcomes. The present study shows that for a large percentage of our sample of patients, ICI therapy is a successful treatment strategy. And, while discontinuation rates are still high, many of those not continuing ICI therapy achieved success with phosphodiesterase inhibitors. Also, the incidence of priapism was less in the present study than previously reported. OBJECTIVES: • To review the outcomes, adverse events and discontinuation rates of intracavernosal injection (ICI) therapy in men with erectile dysfunction (ED) in a sexual medicine practice over a 5-year period at a tertiary referral centre. • Since 1983, ICI has become a staple therapeutic option and high success rates have been reported. However, priapism is a significant concern and discontinuation rates are estimated to be >50%. PATIENTS AND METHODS: • Men presenting with ED who were enrolled in our ICI programme between September 2002 and August 2006 were followed at least annually. • Patient demographic information, agents used, erectile function outcomes and adverse events were recorded. • Failure was defined as the inability to have penetrative sex. Discontinuation was defined as patient declaration of such, failure to attend an annual follow-up visit or failure to call for a repeat prescription. • Multivariable analysis was used to define predictors of failure to respond to ICI therapy, as well as predictors of discontinuation within 36 months of starting ICI in those patients responding. RESULTS: • In all, 1412 patients had complete data and constituted the study population. Most patients were using Trimix and 89% of Trimix users were capable of having sexual intercourse. • Response rates were lower in pelvic radiation and diabetic patients. • However, the discontinuation rate was significant; it was lower in men who had not undergone radical prostatectomy (RP). Of note, many RP patients discontinued ICI because of recovery of natural or phosphodiesterase type 5 inhibitor-assisted erections. CONCLUSIONS: • ICI therapy is associated with very high success rates even in men with high comorbidity profiles; however, the discontinuation rates, even in men who had not undergone RP, by the end of the third year are significant. • Of note, the recorded priapism rate was extremely low (0.5%).

Structures of receptor complexes formed by hemagglutinins from the Asian Influenza pandemic of 1957.

The viruses that caused the three influenza pandemics of the twentieth century in 1918, 1957, and 1968 had distinct hemagglutinin receptor binding glycoproteins that had evolved the capacity to recognize human cell receptors. We have determined the structure of the H2 hemagglutinin from the second pandemic, the "Asian Influenza" of 1957. We compare it with the 1918 "Spanish Influenza" hemagglutinin, H1, and the 1968 "Hong Kong Influenza" hemagglutinin, H3, and show that despite its close overall structural similarity to H1, and its more distant relationship to H3, the H2 receptor binding site is closely related to that of H3 hemagglutinin. By analyzing hemagglutinins of potential H2 avian precursors of the pandemic virus, we show that the human receptor can be bound by avian hemagglutinins that lack the human-specific mutations of H2 and H3 pandemic viruses, Gln-226Leu, and Gly-228Ser. We show how Gln-226 in the avian H2 receptor binding site, together with Asn-186, form hydrogen bond networks through bound water molecules to mediate binding to human receptor. We show that the human receptor adopts a very similar conformation in both human and avian hemagglutinin-receptor complexes. We also show that Leu-226 in the receptor binding site of human virus hemagglutinins creates a hydrophobic environment near the Sia-1-Gal-2 glycosidic linkage that favors binding of the human receptor and is unfavorable for avian receptor binding. We consider the significance for the development of pandemics, of the existence of avian viruses that can bind to both avian and human receptors.

Establishing an Ultrasound Screening Protocol for Chronic Liver Disease with a Handheld Device: A Pilot Project in Southern Ethiopia.

Chronic liver disease (CLD) poses significant challenges in the developing world. The prevalence of this problem and the health burden on local health services are not well understood. The diagnosis and monitoring of CLD are difficult in these settings because of limited access to expensive imaging with limited mobility and/or liver biopsy. The aim of this project was to develop and implement an efficient evidence-based robust ultrasound protocol for the assessment of chronic liver disease using a hand-held ultrasound device that could be effectively used in the developing world. A protocol was established using scoring systems that have established accuracy for the diagnosis of hepatic fibrosis/cirrhosis and hepatic steatosis. Included in the protocol was the identification of hepatic masses, portal venous enlargement, hepatic size and splenic size. Hepatic steatosis was common, identified in 46 of 94 participants (49%). Hepatic fibrosis was observed in only 13 of 94 participants (14%). A significant limitation of the methodology was the inability to validate the results with biopsy or other forms of cross-sectional imaging. The protocol was successfully implemented in a community in a rural setting in South Ethiopia with a mean examination time of around 6 min. It is feasible to use handheld ultrasound for the screening of CLD in remote settings. This project provides an evidence-based framework for further studies in this area.