KESTREL and KITE Phase 3 Studies: 100-Week Results With Brolucizumab in Patients With Diabetic Macular Edema.

PURPOSE: To report the 100-week outcomes from the KESTREL and KITE trials. DESIGN: Two phase 3, double-masked, active-controlled, randomized trials. METHODS: Patients with diabetic macular edema (DME) were randomized 1:1:1 to brolucizumab 3 mg/6 mg (BRO3/BRO6) or aflibercept 2 mg (AFL) in KESTREL (N = 566) or 1:1 to BRO6 or AFL in KITE (N = 360). BRO3/BRO6 arms received 5 loading doses every 6 weeks (q6w) followed by q12w dosing, with an option to adjust to q8w at predefined disease activity assessment visits. In KITE, at week 72, based on the disease stability assessment, treatment intervals could be extended by 4 weeks in the BRO6 arm. AFL arms received 5 monthly loading doses followed by fixed q8w dosing. RESULTS: At week 100, change from baseline in BCVA (letters) was +8.8 for BRO6 and +10.6 for AFL in KESTREL; and +10.9 for BRO6 and +8.4 for AFL in KITE. In both studies, fewer BRO6 subjects had intraretinal fluid and/or subretinal fluid than AFL subjects. Results were achieved with 32.9% (KESTREL) and 47.5% (KITE) of BRO6 subjects maintained on q12w and q12w/q16w dosing, respectively. Intraocular inflammation rates for BRO6 vs AFL were 4.2% vs 1.1% (KESTREL) and 2.2% vs 1.7% (KITE), of which retinal vasculitis rates were 0.5% vs 0% in KESTREL, with no cases in KITE. Retinal vascular occlusion rates were 1.6% vs 0.5% (KESTREL) and 0.6% in both treatment arms in KITE. CONCLUSIONS: Results show the long-term efficacy and durability of brolucizumab in improving visual and anatomical outcomes in DME; the overall safety profile of brolucizumab remained unchanged through year 2.

KESTREL and KITE: 52-Week Results From Two Phase III Pivotal Trials of Brolucizumab for Diabetic Macular Edema.

PURPOSE: To compare the efficacy and safety of brolucizumab with aflibercept in patients with diabetic macular edema (DME). DESIGN: Double-masked, 100-week, multicenter, active-controlled, randomized trials. METHODS: Subjects were randomized 1:1:1 to brolucizumab 3 mg/6 mg or aflibercept 2 mg in KESTREL (n = 566) or 1:1 to brolucizumab 6 mg or aflibercept 2 mg in KITE (n = 360). Brolucizumab groups received 5 loading doses every 6 weeks (q6w) followed by 12-week (q12w) dosing, with optional adjustment to every 8 weeks (q8w) if disease activity was identified at predefined assessment visits; aflibercept groups received 5 doses every 4 weeks (q4w) followed by fixed q8w dosing. The primary endpoint was best-corrected visual acuity (BCVA) change from baseline at Week 52; secondary endpoints included the proportion of subjects maintained on q12w dosing, change in Diabetic Retinopathy Severity Scale score, and anatomical and safety outcomes. RESULTS: At Week 52, brolucizumab 6 mg was noninferior (NI margin 4 letters) to aflibercept in mean change in BCVA from baseline (KESTREL: +9.2 letters vs +10.5 letters; KITE: +10.6 letters vs +9.4 letters; P < .001), more subjects achieved central subfield thickness (CSFT) <280 µm, and fewer had persisting subretinal and/or intraretinal fluid vs aflibercept, with more than half of brolucizumab 6 mg subjects maintained on q12w dosing after loading. In KITE, brolucizumab 6 mg showed superior improvements in change of CSFT from baseline over Week 40 to Week 52 vs aflibercept (P = .001). The incidence of ocular serious adverse events was 3.7% (brolucizumab 3 mg), 1.1% (brolucizumab 6 mg), and 2.1% (aflibercept) in KESTREL; and 2.2% (brolucizumab 6 mg) and 1.7% (aflibercept) in KITE. CONCLUSION: Brolucizumab 6 mg showed robust visual gains and anatomical improvements with an overall favorable benefit/risk profile in patients with DME.

The neurotoxic secreted phospholipase A2 from the Vipera a. ammodytes venom targets cytochrome c oxidase in neuronal mitochondria.

The ß-neurotoxic secreted phospholipases A2 (sPLA2s) block neuro-muscular transmission by poisoning nerve terminals. Damage inflicted by such sPLA2s (ß-ntx) on neuronal mitochondria is characteristic, very similar to that induced by structurally homologous endogenous group IIA sPLA2 when its activity is elevated, as, for example, in the early phase of Alzheimer's disease. Using ammodytoxin (Atx), the ß-ntx from the venom of the nose-horned viper (Vipera a. ammodytes), the sPLA2 receptor R25 has been detected in neuronal mitochondria. This receptor has been purified from porcine cerebral cortex mitochondria by a new Atx-affinity-based chromatographic procedure. Mass spectrometry analysis revealed R25 to be the subunit II of cytochrome c oxidase (CCOX), an essential constituent of the respiratory chain complex. CCOX was confirmed as being the first intracellular membrane receptor for sPLA2 by alternative Atx-affinity-labellings of purified CCOX, supported also by the encounter of Atx and CCOX in PC12 cells. This discovery suggests the explanation of the mechanism by which ß-ntx hinders production of ATP in poisoned nerve endings. It also provides a new insight into the potential function and dysfunction of endogenous GIIA sPLA2 in mitochondria.

The First Intrinsic Tenase Complex Inhibitor with Serine Protease Structure Offers a New Perspective in Anticoagulant Therapy.

Components of the intrinsic blood coagulation pathway, among them factor VIIIa (FVIIIa), have been recognized as suitable therapeutic targets to treat venous thromboembolism, pathological process behind two very serious cardiovascular diseases, deep vein thrombosis and pulmonary embolism. Here, we describe a unique glycoprotein from the nose-horned viper (Vipera ammodytes ammodytes [Vaa]) venom, Vaa serine proteinase homolog 1 (VaaSPH-1), structurally a serine protease but without an enzymatic activity and expressing potent anticoagulant action in human blood. We demonstrated that one of its targets in the blood coagulation system is FVIIIa of the intrinsic tenase complex, where it antagonizes the binding of FIXa. Anticoagulants with such characteristics are intensively sought, as they would be much safer for medical application as the contemporary drugs, which frequently induce excessive bleeding and other complications. VaaSPH-1 is unlikely to be orally available for chronic usage as it has molecular mass of 35 kDa. However, it represents a very promising template to design low molecular mass FVIIIa-directed anticoagulant substances, based on structural features of the interaction surface between VaaSPH-1 and FVIIIa. To this end, we constructed a three-dimensional model of VaaSPH-1 bound to FVIIIa. The model exposes the 157-loop and the preceding α-helix as the most appropriate structural elements of VaaSPH-1 to be considered as a guideline to synthesize small FVIIIa-binding molecules, potential new generation of anticoagulants.

Clues for Polygenic Inheritance of Pituitary Stalk Interruption Syndrome From Exome Sequencing in 20 Patients.

Context: Pituitary stalk interruption syndrome (PSIS) consists of a small/absent anterior pituitary lobe, an interrupted/absent pituitary stalk, and an ectopic posterior pituitary lobe. Mendelian forms of PSIS are detected infrequently (<5%), and a polygenic etiology has been suggested. GLI2 variants have been reported at a relatively high frequency in PSIS. Objective: To provide further evidence for a non-Mendelian, polygenic etiology of PSIS. Methods: Exome sequencing (trio approach) in 20 patients with isolated PSIS. In addition to searching for (potentially) pathogenic de novo and biallelic variants, a targeted search was performed in a panel of genes associated with midline brain development (223 genes). For GLI2 variants, both (potentially) pathogenic and relatively rare variants (<5% in the general population) were studied. The frequency of GLI2 variants was compared with that of a reference population. Results: We found four additional candidate genes for isolated PSIS (DCHS1, ROBO2, CCDC88C, and KIF14) and one for syndromic PSIS (KAT6A). Eleven GLI2 variants were present in six patients. A higher frequency of a combination of two GLI2 variants (M1352V + D1520N) was found in the study group compared with a reference population (10% vs 0.68%). (Potentially) pathogenic variants were identified in genes associated with midline brain anomalies, including holoprosencephaly, hypogonadotropic hypogonadism, and absent corpus callosum and in genes involved in ciliopathies. Conclusion: Combinations of variants in genes associated with midline brain anomalies are frequently present in PSIS and sustain the hypothesis of a polygenic cause of PSIS.

De Novo Mutations in SLC25A24 Cause a Disorder Characterized by Early Aging, Bone Dysplasia, Characteristic Face, and Early Demise.

A series of simplex cases have been reported under various diagnoses sharing early aging, especially evident in congenitally decreased subcutaneous fat tissue and sparse hair, bone dysplasia of the skull and fingers, a distinctive facial gestalt, and prenatal and postnatal growth retardation. For historical reasons, we suggest naming the entity Fontaine syndrome. Exome sequencing of four unrelated affected individuals showed that all carried the de novo missense variant c.649C>T (p.Arg217Cys) or c.650G>A (p.Arg217His) in SLC25A24, a solute carrier 25 family member coding for calcium-binding mitochondrial carrier protein (SCaMC-1, also known as SLC25A24). SLC25A24 allows an electro-neutral and reversible exchange of ATP-Mg and phosphate between the cytosol and mitochondria, which is required for maintaining optimal adenine nucleotide levels in the mitochondrial matrix. Molecular dynamic simulation studies predict that p.Arg217Cys and p.Arg217His narrow the substrate cavity of the protein and disrupt transporter dynamics. SLC25A24-mutant fibroblasts and cells expressing p.Arg217Cys or p.Arg217His variants showed altered mitochondrial morphology, a decreased proliferation rate, increased mitochondrial membrane potential, and decreased ATP-linked mitochondrial oxygen consumption. The results suggest that the SLC25A24 mutations lead to impaired mitochondrial ATP synthesis and cause hyperpolarization and increased proton leak in association with an impaired energy metabolism. Our findings identify SLC25A24 mutations affecting codon 217 as the underlying genetic cause of human progeroid Fontaine syndrome.

On the role of protein disulfide isomerase in the retrograde cell transport of secreted phospholipases A2.

Following the finding that ammodytoxin (Atx), a neurotoxic secreted phospholipase A2 (sPLA2) in snake venom, binds specifically to protein disulfide isomerase (PDI) in vitro we show that these proteins also interact in living rat PC12 cells that are able to internalize this group IIA (GIIA) sPLA2. Atx and PDI co-localize in both differentiated and non-differentiated PC12 cells, as shown by fluorescence microscopy. Based on a model of the complex between Atx and yeast PDI (yPDI), a three-dimensional model of the complex between Atx and human PDI (hPDI) was constructed. The Atx binding site on hPDI is situated between domains b and b'. Atx interacts hPDI with an extensive area on its interfacial binding surface. The mammalian GIB, GIIA, GV and GX sPLA2s have the same fold as Atx. The first three sPLA2s have been detected intracellularly but not the last one. The models of their complexes with hPDI were constructed by replacement of Atx with the respective mammalian sPLA2 in the Atx-hPDI complex and molecular docking of the structures. According to the generated models, mammalian GIB, GIIA and GV sPLA2s form complexes with hPDI very similar to that with Atx. The contact area between GX sPLA2 and hPDI is however different from that of the other sPLA2s. Heterologous competition of Atx binding to hPDI with GV and GX sPLA2s confirmed the model-based expectation that GV sPLA2 was a more effective inhibitor than GX sPLA2, thus validating our model. The results suggest a role of hPDI in the (patho)physiology of some snake venom and mammalian sPLA2s by assisting the retrograde transport of these molecules from the cell surface. The sPLA2-hPDI model constitutes a valuable tool to facilitate further insights into this process and into the (patho)physiology of sPLA2s in relation to their action intracellularly.

Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography.

In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far-ammodytoxins (Atxs)-are contributing to the venom's toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.

Hemorrhagin VaH4, a covalent heterodimeric P-III metalloproteinase from Vipera ammodytes ammodytes with a potential antitumour activity.

In the envenomation caused by a bite of Vipera ammodytes ammodytes, the most venomous snake in Europe, hemorrhage is usually the most severe consequence in man. Identifying and understanding the hemorrhagic components of its venom is therefore particularly important in optimizing medical treatment of patients. We describe a novel high molecular mass hemorrhagin, VaH4. The isolated molecule is a covalent dimer of two homologous subunits, VaH4-A and VaH4-B. Complete structural characterization of A and partial characterization of B revealed that both belong to the P-III class of snake venom metalloproteinases (SVMPs), comprising a metalloproteinase, a disintegrin-like domain and a cysteine-rich domain. However, neither VaH4-A nor VaH4-B possess the Cys174 involved in the inter-subunit disulphide bond of P-III SVMPs. A three-dimensional model of the VaH4 dimer suggests that Cys132 serves this function. This implies that dimers in the P-III class of SVMPs can be formed either between their Cys132 or Cys174 residues. The proteolytic activity and stability of VaH4 depend on Zn²âº and Ca²âº ions and the presence of glycosaminoglycans, which indicates physiological interaction of VaH4 with the latter element of the extracellular matrix (ECM). The molecular mass of VaH4, determined by MALDI/TOF mass spectrometry, is 110.2 kDa. N-deglycosylation reduced the mass of each monomer by 8.7 kDa. The two possible N-glycosylation sites in VaH4-A are located at completely different positions from those in homodimeric P-IIIc VaH3 from the same venom, however, without any evident functional implications. The hemorrhagic activity of this slightly acidic SVMP is ascribed to its hydrolysis of components of the ECM, particularly fibronectin and nidogen, and of some blood coagulation proteins, in particular the α-chain of fibrinogen. VaH4 is also significant medically as we found it cytotoxic against cancer cells and due to its substantial sequence similarity to ADAM/ADAMTS family of physiologically very important human proteins of therapeutic potential.

VaH3, one of the principal hemorrhagins in Vipera ammodytes ammodytes venom, is a homodimeric P-IIIc metalloproteinase.

Hemorrhage is the most potent manifestation of envenomation by Vipera ammodytes ammodytes (V. a. ammodytes) venom in man. A detailed description of the venom components contributing to this effect is thus medically very important. We have characterized a novel component, termed here VaH3, as a potently hemorrhagic snake venom metalloproteinase (SVMP). Its proteolytic activity and overall stability depend on the presence of Zn(2+) and Ca(2+) ions. The molecular mass of this slightly acidic molecule, determined by MALDI/TOF analysis, is 104 kDa. Chemical reduction and S-carbamoylmethylation result in a single monomer of 53.7 kDa. N-deglycosylation decreased this mass by 4.6 kDa. The complete amino acid sequence of VaH3 was determined by protein and cDNA sequencing, showing that each of the identical glycoprotein subunits comprise a metalloproteinase, a disintegrin-like domain and a cysteine-rich domain, VaH3 belongs to the P-IIIc class of SVMPs. It shows strong sequence similarity to vascular endothelial cell apoptosis-inducing reprolysins. Anti-ammodytagin antibodies strongly cross-reacted with VaH3 and completely neutralized its hemorrhagic activity in rat, despite the fact that the two hemorrhagic P-III SVMPs from V. a. ammodytes venom do not share a very high degree of amino acid sequence identity. In spite of its narrow proteolytic specificity, VaH3 rapidly cleaved some basal membrane and extracellular matrix proteins, such as collagen IV, fibronectin and nidogen. Moreover, it also hydrolyzed plasma proteins involved in blood coagulation. It is an effective α-fibrinogenase that cleaves prothrombin and factor X without activating them. The degradation of these proteins likely contributes to the hemorrhagic activity of VaH3. A three-dimensional model of VaH3 was built to help explain structure-function relationships in ADAM/ADAMTS, a family of proteins having significant therapeutic potential and substantial sequence similarity to VaH3.

Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR.

Here we present Cys-Ph-TAHA, a new nonadentate lanthanide tag for the paramagnetic labelling of proteins. The tag can be easily synthesized and is stereochemically homogenous over a wide range of temperatures, yielding NMR spectra with a single set of peaks. Bound to ubiquitin, it induced large residual dipolar couplings and pseudocontact shifts that could be measured easily and agreed very well with the protein structure. We show that Cys-Ph-TAHA can be used to label large proteins that are biochemically challenging such as the Lac repressor in a 90 kDa ternary complex with DNA and inducer.

Conformational flexibility and allosteric regulation of cathepsin K.

The human cysteine peptidase cathepsin K is a key enzyme in bone homoeostasis and other physiological functions. In the present study we investigate the mechanism of cathepsin K action at physiological plasma pH and its regulation by modifiers that bind outside of the active site. We show that at physiological plasma pH the enzyme fluctuates between multiple conformations that are differently susceptible to macromolecular inhibitors and can be manipulated by varying the ionic strength of the medium. The behaviour of the enzyme in vitro can be described by the presence of two discrete conformations with distinctive kinetic properties and different susceptibility to inhibition by the substrate benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin. We identify and characterize sulfated glycosaminoglycans as natural allosteric modifiers of cathepsin K that exploit the conformational flexibility of the enzyme to regulate its activity and stability against autoproteolysis. All sulfated glycosaminoglycans act as non-essential activators in assays using low-molecular-mass substrates. Chondroitin sulfate and dermatan sulfate bind at one site on the enzyme, whereas heparin binds at an additional site and has a strongly stabilizing effect that is unique among human glycosaminoglycans. All glycosaminoglycans stimulate the elastinolytic activity of cathepsin K at physiological plasma pH, but only heparin also increases the collagenolytic activity of the enzyme under these conditions. Altogether these results provide novel insight into the mechanism of cathepsin K function at the molecular level and its regulation in the extracellular space.

Recombinant human SMOCs produced by in vitro refolding: calcium-binding properties and interactions with serum proteins.

Secreted modular calcium-binding (SMOC) proteins are little known members of the BM-40 family of matricellular proteins. SMOC-1 is localized in basement membranes, while SMOC-2 exhibits pro-angiogenic properties and stimulates cell cycle progression via integrin-linked kinase. In this work we have expressed recombinant human SMOCs in inclusion bodies in Escherichia coli. Soluble proteins were prepared by in vitro refolding with a final yield of approximately 3mg of purified SMOCs per liter of bacterial culture. The folding state of the products and their ability to reversibly bind calcium ions were verified by CD spectroscopy. The EF hands of the refolded SMOCs were both functional, one had high affinity for calcium ions (K(d) values in the 0.7-1 microM range), while the other had lower affinity (K(d) values 20-25 microM). The proteins were also examined for their ability to bind blood serum proteins. Three of the bands specifically retained on SMOC-Sepharose were identified as C-reactive protein, an acute phase protein from the pentraxin family, the basement membrane and elastic fiber-associated fibulin-1, and vitronectin, which is involved in cell adhesion, migration and proliferation and binds numerous extracellular and membrane proteins, including integrins. The interactions were additionally confirmed in solution using purified individual proteins by the method of biotin label transfer from one interacting partner to the other. Their identification is among the first pieces of information about the action of the SMOCs on molecular level and opens new possibilities for future research aimed towards elucidating the physiological roles of these versatile proteins.

A presynaptically toxic secreted phospholipase A2 is internalized into motoneuron-like cells where it is rapidly translocated into the cytosol.

The molecular mechanism of the presynaptic toxicity of secreted phospholipase A2 (sPLA2) neurotoxins, including that of ammodytoxin A (AtxA), has not been resolved. Here we report the action of AtxA on mouse motoneuron-like cells, on which it induced characteristic neurotoxic effects on synaptic vesicles and on the reorganization of F-actin. AtxA also released fatty acids from the plasmalemma. Its significantly less neurotoxic V31W mutant showed similar effects on cells but with a much higher rate of hydrolysis than the wild-type, indicating that high enzymatic activity alone is not sufficient for the observed effects. The neurotoxic action was observed by confocal microscopy of a fluorescently labelled AtxA and by electron microscopy of a nanogold-labelled toxin. The Atx-binding proteins were tagged by a photo-cross-linking reagent conjugated to the toxin. AtxA was taken up rapidly by the cells, where it interacted within minutes with calmodulin and 14-3-3 proteins in the cytosol. These data demonstrate, for the first time, the translocation of an sPLA2 from the extracellular space into the cytosol of a cell. Such an event may thus be important in explaining the action of a range of homologous endogenous sPLA2 enzymes in mammals whose roles in various cellular processes are not yet completely understood.

Generation of ammodytoxin-anti-cathepsin B immuno-conjugate as a model for delivery of secretory phospholipase A2 into cancer cells.

Ammodytoxin C (AtxC) is a toxic secreted phospholipase A2 (sPLA2) from the venom of Vipera ammodytes snake. To evaluate its potential to kill cancer cells, the toxin was cross-linked to the monoclonal antibody against cathepsin B which endocytoses upon binding to cathepsin B, an antigen overexpressed on the plasma membrane of cancer cells. A photo-reactive derivative of AtxC, possessing the same biological activity as the native toxin, was reacted with antibodies to form a covalent immuno-conjugate. In conditions of the cytosolic redox potential, AtxC was gradually released from the conjugate due to reduction of the disulfide bond in the spacer arm of the cross-linker. The phospholipase activity of the preparation reached maximum in 10min and then decreased gradually. As demonstrated by fluorescence microscopy, the immuno-conjugate targeted Caco-2 colon adenocarcinoma cells but was very slowly internalized, the likely reason of only slight cytotoxicity being observed. Despite the lack of a clear cytotoxic effect of AtxC-antibody conjugate on Caco-2 cells, we demonstrated in this work a new methodology for the targeted delivery of (toxic) sPLA2 into cells, promising in research or therapy.