Inhibitory antibodies designed for matrix metalloproteinase modulation

The family of matrix metalloproteinases (MMPs) consists of a set of biological targets that are involved in a multitude of severe pathogenic events such as different forms of cancers or arthritis. Modulation of the target class with small molecule drugs has not led to the anticipated success until present, as all clinical trials failed due to unacceptable side effects or a lack of therapeutic outcome. Monoclonal antibodies offer a tremendous therapeutic potential given their high target selectivity and good pharmacokinetic profiles. For the treatment of a variety of diseases there are already antibody therapies available and the number is increasing. Recently, several antibodies were developed for the selective inhibition of single MMPs that showed high potency and were therefore investigated in in vivo studies with promising results. In this review, we highlight the progress that has been achieved toward the design of inhibitory antibodies that successfully modulate MMP-9 and MMP-14

Scientific results of the Bryotrop expedition to Zaire and Rwanda : 3., photosynthetic gas exchange of bryophytes from different forest types in eastern Central Africa.

During the BRYOTROP-Expedition to Zaire and Rwanda bryophytes were collected from a rainforest habitat at 800 m a.s.l. and from bamboo forest and tree-heath environments between 2200 and 3200 m. The microclimates influencing the mosses are different at the altitudinally separated locations. Conditions are rather constant with 24 °C, 100 % rel. hum. and PAR below 100 μmol photons m-2 sec-1 at the lowland station, rather versatile in the mountains with six times higher daily sums of PAR, temperatures between 10 and 25 °C and relative humidities between 60 and 1oo %. In the bamboo forest epiphytic mosses dry out during the day to less than 70 % of their water content, but regain saturation from the vapor-saturated air during night. Bryophyte photosynthesis and respiration were studied by Warburg manometry with moisture saturated samples. Temperature curves of gas exchange peaked between 22 and 30 °C. Optima of the lowland species were somewhat higher than those from samples collected at the mountain sites. Habitat separation of characteristics of photosynthesis was more pronounced with respect to light responses. Saturation gas exchange rates were reached by all species still below 400 μmol photons m-2 sec-1. But the slopes of the curves in the low-light range were distinctly steeper, and the light compensation points smaller in the lowland than in the highland species (compensation points of the former: 3 - 12 μmol photons m-2 sec-1, of the latter: 8 - 20 μmol photons m-2 sec-1). It is emphasized that bryophytes in the rainforest understory experience extremely high ambient C02 concentrations near the floor. This, their low light requirements for photosynthesis, and the permanently optimal temperature and humidity conditions for maximal carbon gain enable them to live successfully, but with less biomass development in this dark and damp environment. By contrast, bryophytes from the bamboo forest and tree-heath environments can utilize light conditions combined with variable temperatures and humidities similarly as species from extratropical vegetation types

Targeted fluoro positioning for the discovery of a potent and highly selective matrix metalloproteinase inhibitor

The incorporation of fluorine atoms into functional molecules is of wide interest in synthetic organic chemistry as well as cognate disciplines. In particular, in medicinal chemistry, there is a strong desire to positively influence the physicochemical molecular properties of drug compounds by introducing fluorine into biologically active molecules. Here, we present targeted fluoro positioning as the key design principle of converting a weak matrix metalloproteinase-13 (MMP-13 ) inhibitor into a very potent (IC50 = 6nM) and highly selective (selectivity factors of > 1000 over MMP-1, 2, 3, 7, 8, 9, 10, 12, 14) inhibitor with excellent plasma and microsomal stability, and no binding to the hERG channel (hERG: human ether-a-go-go related gene)

Multigrid methods for anisotropic BTTB systems

AbstractMultigrid methods are highly efficient solution techniques for large sparse multilevel Toeplitz systems which are positive definite and ill-conditioned. In this paper, we develop multigrid methods which are especially designed for anisotropic two-level Toeplitz (BTTB) matrices. First, a method is described for systems with anisotropy along coordinate axes as a suitable combination of semicoarsening and full coarsening steps. Although the basic idea is known from the solution of partial differential equations, we present it here in a more formal way using generating functions and their level curves. This enables us not only to prove the optimal convergence of the two-grid method, but also to carry over the results to systems with anisotropy in other directions. We introduce new coordinates in order to describe these more complicated systems in terms of generating functions. This enables us to solve them with the same efficiency. For the two-level method, we present a convergence proof in this more general case

Molecular recognition of the catalytic zinc(II) ion in MMP-13 : structure-based evolution of an allosteric inhibitor to dual binding mode inhibitors with improved lipophilic ligand efficiencies

Matrix metalloproteinases (MMPs) are a class of zinc dependent endopeptidases which play a crucial role in a multitude of severe diseases such as cancer and osteoarthritis. We employed MMP-13 as the target enzyme for the structure-based design and synthesis of inhibitors able to recognize the catalytic zinc ion in addition to an allosteric binding site in order to increase the affinity of the ligand. Guided by molecular modeling, we optimized an initial allosteric inhibitor by addition of linker fragments and weak zinc binders for recognition of the catalytic center. Furthermore we improved the lipophilic ligand efficiency (LLE) of the initial inhibitor by adding appropriate zinc binding fragments to lower the clogP values of the inhibitors, while maintaining their potency. All synthesized inhibitors showed elevated affinity compared to the initial hit, also most of the novel inhibitors displayed better LLE. Derivatives with carboxylic acids as the zinc binding fragments turned out to be the most potent inhibitors (compound 3 (ZHAWOC5077): IC50 = 134 nM) whereas acyl sulfonamides showed the best lipophilic ligand efficiencies (compound 18 (ZHAWOC5135): LLE = 2.91)

Strategic targeting of multiple water-mediated interactions : a concise and rational structure-based design approach to potent and selective MMP-13 inhibitors

Water in the architecture of life: Potent and selective matrix metalloproteinase-13 (MMP-13) inhibitors were rationally designed by targeting multiple water-mediated interactions between the target protein and small-molecule inhibitors. This structure-based design concept offers tremendous opportunities for the discovery of unique small molecules with tailored biological activity

Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering

Industrial plant biotechnology applications include the production of sustainable fuels, complex metabolites and recombinant proteins, but process development can be impaired by a lack of reliable and scalable screening methods. Here, we describe a rapid and versatile expression system which involves the infusion of Agrobacterium tumefaciens into three‐dimensional, porous plant cell aggregates deprived of cultivation medium, which we have termed plant cell packs (PCPs). This approach is compatible with different plant species such as Nicotiana tabacum BY2, Nicotiana benthamiana or Daucus carota and 10‐times more effective than transient expression in liquid plant cell culture. We found that the expression of several proteins was similar in PCPs and intact plants, for example, 47 and 55 mg/kg for antibody 2G12 expressed in BY2 PCPs and N. tabacum plants respectively. Additionally, the expression of specific enzymes can either increase the content of natural plant metabolites or be used to synthesize novel small molecules in the PCPs. The PCP method is currently scalable from a microtiter plate format suitable for high‐throughput screening to 150‐mL columns suitable for initial product preparation. It therefore combined the speed of transient expression in plants with the throughput of microbial screening systems. Plant cell packs therefore provide a convenient new platform for synthetic biology approaches, metabolic engineering and conventional recombinant protein expression techniques that require the multiplex analysis of several dozen up to hundreds of constructs for efficient product and process development

Multiple pathways of plasmid DNA transfer in Helicobacter pylori

Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species

Fossil water lily fruits with seeds Nymphaea subgenus Lotos, from the Oligocene of Armissan/Narbonne (France)

Based on specimens from a historical collection from the classical Oligocene locality Armissan (Southern France) fossil fruits of Nymphaeaceae are described that can be affiliated to the Nymphaeaceae genus Nymphaea subgenus Lotos with confidence. Such findings were first described in 1865 by Saporta from Armissan as an extinct genus, Anoectomeria. However, in the absence of preserved seeds, and due to a misinterpretation of the organisation of the fruits, the fossils could only be affiliated to Nymphaeaceae in general. The identification of the historical specimens with preserved seeds allows a new interpretation of such fossils and, hence their identification as fruits of the subgenus Lotos. Especially the characteristical mode of fruit dehiscence, which leads to typical stigmatic discs in extant as well as in fossil specimens, confirms this affiliation. In this study, the flowers/fruits described by Saporta (rhizome, leaves and isolated seeds were also included in his taxa) are revised to Nymphaea (subgenus Lotos) brongniartii. Comparable Paleogene fossils from other localities are also discussed. Based on N. (L.) brongniartii the minimal geological age of the subgenus Lotos can be determined as Late Oligocene. By interpretation of the comparable Paleogene fossils as belonging to subgenus Lotos the minimal geological age of this taxon would even be Middle Eocene. The Late Oligocene minimal geological age of Lotos is in agreement with phylogenetic trees based on molecular data and allows a better calibration of molecular phylogenetic trees of Nymphaeaceae.Nach Exemplaren einer historischen Aufsammlung von der klassischen Oligozän-Fundstelle Armissan (Südfrankreich) werden fossile Nymphaeaceen-Früchte mit Samen beschrieben, die eindeutig der rezenten Nymphaeaceen Gattung Nymphaea Untergattung Lotos zuzuordnen sind. Derartige Funde wurden zuerst 1865 von Saporta ebenfalls von Armissan als Anoectomeria brongniartii, A. nana und A. media beschrieben, und konnten aber wegen des Fehlens von Samen und der Fehldeutung des Fruchtaufbaus nur dieser Kunstgattung und allgemein den Nymphaeaceae zugeordnet werden. Die Auffindung der historischen Exemplare, die anders als die von Saporta beschriebenen, Samen aufweisen und die damit mögliche Neuinterpretation dieser fossilen Früchte erlaubt eine eindeutige Zuordnung zur Untergattung Lotos. Besonders die sehr charakteristische Fruchtdehiszenz, die rezent wie fossil zu isolierten Scheiben aus verwachsenen Griffelnarben führt, erlaubt nur diese Zuordnung. Die Blüten bzw. Früchte der Saporta-Taxa (Rhizome, Blätter und isolierte Samen wurden von ihm auch jeweils in die Taxa mit einbezogen) werden hier zu Nymphaea (subgenus Lotos) brongniartii revidiert. Vergleichbare paläogene Funde von anderen Lokalitäten werden diskutiert. Mit Nymphaea (Lotos) brongniartii kann das minimale geologische Alter der Untergattung Lotos als Oberoligozän bestimmt werden. Mit der Zuordnung vergleichbarer paläogener Funde zu Lotos wäre das minimale geologische Alter sogar Mitteleozän. Das Oberoligozäne minimale geologische Alter von Lotos ist mit den aus molekularen Daten erstellten Stammbäumen vereinbar und ermöglicht eine bessere Kalibrierung molekularer Stammbäume der Nymphaeaceae