A variety of plasma-derived (pd) and recombinant (r) factor VIII (FVIII) concentrates are used to prevent and treat bleeding in severe hemophilia A patients. A significant side effect of FVIII replacement is the development of FVIII neutralizing antibodies (inhibitors) in up to 30% of patients receiving FVIII concentrates. The FVIII protein content (FVIII:Ag) per unit of FVIII:C in FVIII concentrates, and how effectively the FVIII:Ag in FVIII concentrates binds to von Willebrand factor (VWF) may provide information relevant for the survival of FVIII:C in vivo and for estimating the risk for inhibitor development. The FVIII:Ag content of nine r-FVIII and nine pd-FVIII concentrates were quantified in this study using two enzyme-linked immunosorbent assay (ELISA) platforms. The two ELISA platforms were based on the use of a monoclonal anti-(FVIII light chain)-IgG and polyclonal anti-FVIII antibodies as capture antibodies and both ELISAs were equally able to detect > or =0.005 IU of FVIII:Ag. Measured in international units, the r-FVIII concentrates contained significantly higher FVIII:Ag per unit of FVIII:C than the pd-FVIII concentrates. The VWF-binding profiles of the r-FVIII and pd-FVIII concentrates were also determined by gel filtration chromatography. Unlike the plasma-derived products, the r-FVIII concentrates invariably contained a fraction of FVIII:Ag molecules (approximately 20%) which was unable to associate with VWF. Given that VWF regulates both factor VIII proteolysis and survival of FVIII:Ag in vivo, the fraction of FVIII:Ag unable to bind to VWF may have a reduced survival and be more susceptible to proteolytic degradation in vivo. The extent to which the fractions of FVIII:Ag in concentrates able and unable to bind to VWF contribute to inhibitor development in severe FVIII-deficient patients is unknown.

The highly-selective blood-brain barrier (BBB) prevents neurotoxic substances in blood from crossing into the extracellular fluid of the central nervous system (CNS). As such, the BBB has a close relationship with CNS disease development and treatment, so predicting whether a substance crosses the BBB is a key task in lead discovery for CNS drugs. Machine learning (ML) is a promising strategy for predicting the BBB permeability, but existing studies have been limited by small datasets with limited chemical diversity. To mitigate this issue, we present a large benchmark dataset, B3DB, complied from 50 published resources and categorized based on experimental uncertainty. A subset of the molecules in B3DB has numerical log BB values (1058 compounds), while the whole dataset has categorical (BBB+ or BBB−) BBB permeability labels (7807). The dataset is freely available at https://github.com/theochem/B3DB and 10.6084/m9.figshare.15634230.v3 (version 3). We also provide some physicochemical properties of the molecules. By analyzing these properties, we can demonstrate some physiochemical similarities and differences between BBB+ and BBB− compounds.

DNA topoisomerases are essential enzymes involved in all the DNA processes and among them, type IA topoisomerases emerged as a key actor in the maintenance of genome stability. The hyperthermophilic archaeon, Sulfolobus solfataricus, contains three topoisomerases IA including one classical named TopA. SsoTopA is very efficient at unlinking DNA catenanes, grouping SsoTopA into the topoisomerase III family. SsoTopA is active over a wide range of temperatures and at temperatures of up to 85°C it produces highly unwound DNA. At higher temperatures, SsoTopA unlinks the two DNA strands. Thus depending on the temperature, SsoTopA is able to either prevent or favor DNA melting. While canonical topoisomerases III require a single-stranded DNA region or a nick in one of the circles to decatenate them, we show for the first time that a type I topoisomerase, SsoTopA, is able to efficiently unlink covalently closed catenanes, with no additional partners. By using single molecule experiments we demonstrate that SsoTopA requires the presence of a short single-stranded DNA region to be efficient. The unexpected decatenation property of SsoTopA probably comes from its high ability to capture this unwound region. This points out a possible role of TopA in S. solfataricus as a decatenase in Sulfolobus.

Exatecan and deruxtecan are antineoplastic camptothecin derivatives in development as tumor-targeted-delivery warheads in various formulations including peptides, liposomes, polyethylene glycol (PEG) nanoparticles, and antibody-drug conjugates (ADCs). Here, we report the molecular pharmacology of exatecan compared to the clinically approved topoisomerase I (TOP1) inhibitors and preclinical models for validating biomarkers and the combination of exatecan with ATR inhibitors. Modeling exatecan binding at the interface of a TOP1 cleavage complex suggests two novel molecular interactions with the flanking DNA base and the TOP1 residue N352, in addition to the three known interactions of camptothecins with the TOP1 residues R364, D533 and N722. Accordingly, exatecan showed much stronger TOP1 trapping, higher DNA damage and apoptotic cell death than the classical TOP1 inhibitors used clinically. We demonstrate the value of SLFN11 expression and homologous recombination (HR)-deficiency (HRD) as predictive biomarkers of response to exatecan. We also show that exatecan kills cancer cells synergistically with the clinical ATR inhibitor ceralasertib (AZD6738). To establish the translational potential of this combination, we tested CBX-12, a clinically developed pH-sensitive peptide-exatecan conjugate that selectively targets cancer cells and is currently in clinical trials. The combination of CBX-12 with ceralasertib significantly suppressed tumor growth in mouse xenografts. Collectively, our results demonstrate the potency of exatecan as a TOP1 inhibitor and its clinical potential in combination with ATR inhibitors, using SLFN11 and HRD as predictive biomarkers.

Hirudin and hirulog-1 [D-Phe-Pro-Arg-Pro-[Gly]4-desulphohirudin-(54-65)] abrogate the enzyme activities of alpha-thrombin by binding the enzyme simultaneously at its catalytic centre and fibrin(ogen)-recognition exosite. In contrast, hirugen [hirudin-(54-65)] binds alpha-thrombin solely at the fibrin(ogen)-recognition exosite, and competitively inhibits fibrinopeptide A release. To investigate the extent to which the fibrin(ogen)-recognition exosite is involved when alpha-thrombin catalyses the amplification reactions of coagulation, we compared the abilities of hirudin, hirulog-1 and hirugen to inhibit simultaneously Factor X, Factor V and prothrombin activation. Whereas 0.1 microM-hirudin and 0.1 microM-hirulog-1 (i.e. less than 10% of the concentration of prothrombin in plasma) inhibited Factor X, Factor V and prothrombin activation, 10 microM was the minimum concentration of hirugen to achieve a similar anticoagulant action. Concentrations of hirudin and hirulog-1 equimolar to and 5 times greater than those of alpha-thrombin respectively abrogated Factor V activation by exogenous alpha-thrombin. In contrast, a 500-fold molar excess of hirugen could not. The inability of hirugen to inhibit the activation of the three clotting factors effectively suggests that the fibrin(ogen)-recognition exosite does not play a mandatory role when thrombin activates Factor V.

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The historical paradigm of the deep ocean as a biological 'desert' has shifted to one of a 'rainforest' owing to the isolation of many novel microbes and their associated bioactive compounds. To explore the potential of the bioactive compounds in our marine microbial natural product library, we screened it for the selective cytotoxicity of six different cancer cell lines to human normal lung fibroblast cell line HLF. The crude extract from a marine-derived fungal strain showed notable selectivity against cancer cell lines. For a bioactivity-guided fractionation and purification, a novel cyclopentenone, (-)-(4R *, 5S *)-3-ethyl-4,5-dihydroxycyclopent-2-enone (1, trichoderone), and a known compound with new activity, cholesta-7,22- diene-3 beta,5 alpha,6 beta-triol (2), were identified from a marine Trichoderma sp. that was isolated from the deep sea sediment of the South China Sea. Their structures were determined by NMR and MS data analyses. Trichoderone (1) displayed potent cytotoxicity against a panel of six cancer cell lines, whereas it did not show much cytotoxicity against normal human lung fibroblast cell line HLF even at a concentration of 7.02 mM. The selectivity index (SI) value for 1 was greater than 100. To the best of our knowledge, both compounds were isolated from marine fungi for the first time. They also exhibited bioactivities against HIV protease and Taq DNA polymerase. Optimization of the compounds would shed new light on treating cancer and infectious diseases.

The validity of the hypothesis that Factor Xa activates Factor V in heparinized plasma was examined by establishing the temporal relationships between Factor V proteolysis and prothrombin consumption in plasma. Factor V was cleaved into Factor Va heavy chain (approx. 110 kDa) and an intermediate (approx. 230 kDa) 30 s after CaCl2 was added to contact-activated plasma (CAP). The larger fragment was converted into Factor V activation peptide (approx. 150 kDa) and Factor Va light chain (approx. 80 kDa) 15 s later. Heparin (approx. 0.05 microM) delayed Factor V proteolysis in CAP by at least 30 s. On supplementing CAP with 1 nM-Factor Xa or 1 nM-thrombin, Factor V was activated 15 s later. Heparin prolonged by 15 s and 45 s the time required to demonstrate Factor V activation in CAP supplemented with Factor Xa and thrombin respectively. Factor V was activated 20 s after tissue factor and CaCl2 were added to plasma, both in the absence and in the presence of approx. 0.05 microM-heparin. In contrast, hirudin and D-Phe-Pro-Arg-CH2Cl (two thrombin inhibitors more effective than heparin) delayed Factor V activation in this plasma by at least 30 s. The fragments of Factor V obtained in heparinized CAP suggest thrombin escapes inhibition and contributes to Factor V activation in that plasma. Production of Factor Va heavy chain and the 230 kDa Factor V fragment invariably preceded efficient prothrombin activation. These observations suggest that heparin, hirudin and D-Phe-Pro-Arg-CH2Cl delay Factor V activation by inhibiting thrombin. The availability of Factor Xa markedly moderates the ability of heparin to inhibit Factor V activation.