Hans-Jürgen Federsel

Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.

The bacterial ω‐transaminase from Chromobacterium violaceum (Cv‐ωTA, EC2.6.1.18) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5′‐phosphate (PLP). Here, we present four crystal structures of Cv‐ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular mass of ∼ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo‐Cv‐ωTA structure reveals unique ‘relaxed’ conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered ‘roof ’ over the PLP‐binding site, whereas in the other apo form and the holo form the ‘roof’ is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the ‘roof’ structure may be associated with substrate binding in Cv‐ωTA and some other transaminases.

Key Amino Acid Residues for Reversed or Improved Enantiospecificity of an ω‐Transaminase

Transaminases inherently possess high enantiospecificity and are valuable tools for stereoselective synthesis of chiral amines in high yield from a ketone and a simple amino donor such as 2‐propylamine. Most known ω‐transaminases are (S)‐selective and there is, therefore, a need of (R)‐selective enzymes. We report the successful rational design of an (S)‐selective ω‐transaminase for reversed and improved enantioselectivity. Previously, engineering performed on this enzyme group was mainly based on directed evolution, with few exceptions. One reason for this is the current lack of 3D structures. We have explored the ω‐transaminase from Chromobacterium violaceum and have used a homology modeling/rational design approach to create enzyme variants for which the activity was increased and the enantioselectivity reversed. This work led to the identification of key amino acid residues that control the activity and enantiomeric preference. To increase the enantiospecificity of the C. violaceum ω‐transaminase, a possible single point mutation (W60C) in the active site was identified by homology modeling. By site‐directed mutagenesis this enzyme variant was created and it displayed an E value improved up to 15‐fold. In addition, to reverse the enantiomeric preference of the enzyme, two other point mutations (F88A/A231F) were identified. This double mutation created an enzyme variant, which displayed substrate dependent reversed enantiomeric preference with an E value shifted from 3.9 (S) to 63 (R) for 2‐aminotetralin.

Logistics of process R&D: transforming laboratory methods to manufacturing scale

In the past, process R&D — which is responsible for producing candidate drugs in the required quantity and of the requisite quality — has had a low profile, and many people outside the field remain unaware of the challenges involved. However, in recent years, the increasing pressure to achieve shorter times to market, the demand for considerable quantities of candidate drugs early in development, and the higher structural complexity — and therefore greater cost — of the target compounds, have increased awareness of the importance of process R&D. Here, I discuss the role of process R&D, using a range of real-life examples, with the aim of facilitating integration with other parts of the drug discovery pipeline.

Process R&D under the magnifying glass: Organization, business model, challenges, and scientific context☆

Initially, the aim is to provide the big picture illustrating the as is situation in the pharmaceutical industry: a lack of productivity resulting in too few products reaching the market; a loss of billions in revenue over the next few years as some of the major megabrands go off patent; a spiraling cost for developing new drugs and taking them through clinical and safety studies. Following on, a look deeper into the organization will offer an insight into the state-of-the-art in a technical function accountable for chemical Process R&D (with a remit to develop scalable, robust, and cost efficient processes for small molecules). The vast majority of compounds already launched in the form of drug products on the market or still being pursued through the phases of discovery and development, fall within the category of small molecules (as opposed to biopharmaceuticals, e.g., proteins, monoclonal antibodies). This typically means molecular weights of <1000Da and puts organic synthesis in the widest sense of the word at the forefront of technologies needed to support R&D programs in the pharma industry. Understandably, the demands on Medicinal Chemistry are quite different to what applies in a Process R&D (PR&D) organization. In the former, making large numbers of potentially interesting molecules, many of which are discarded after testing, is a key driver and for this virtually any synthetic methodology will suffice. For PR&D, however, homing in on selected compounds there is an expectation that the best synthetic routes will be delivered that meet a number of tough criteria, for instance from an environmental and safety point of view, allowing operation on large scale, offering cost competitiveness, avoiding patent infringements, showing sustainability for long-term production, etc. The intention is to focus on issues to be addressed during this transition by providing examples of changes that had to be put in place in order to make the supply of larger amounts of material feasible. At the end some forward looking conclusions will be shared.

A guide to drug discovery: Logistics of process R&D: transforming laboratory methods to manufacturing scale

In the past, process R&D — which is responsible for producing candidate drugs in the required quantity and of the requisite quality — has had a low profile, and many people outside the field remain unaware of the challenges involved. However, in recent years, the increasing pressure to achieve shorter times to market, the demand for considerable quantities of candidate drugs early in development, and the higher structural complexity — and therefore greater cost — of the target compounds, have increased awareness of the importance of process R&D. Here, I discuss the role of process R&D, using a range of real-life examples, with the aim of facilitating integration with other parts of the drug discovery pipeline.

Building bridges from process R&D: from a customer–supplier relationship to full partnership

A new and forward-looking way of running process R&D is introduced that integrates this core business in an efficient manner into the network of activities in different disciplines, which constitute the arena for the development of pharmaceutical products. The interfaces with surrounding areas are discussed in addition to the novel organizational principles implemented in process R&D and the workflow emanating from this. Furthermore, the Tollgate model used to keep track of the progress in a project and the pre-study concept are presented in detail. Finally, the main differences between operating modes in the past and in the future are highlighted.

Start small, think big — the art of process R&D

Process R&D is becoming increasingly crucial to the overall efficiency of drug development.

CHAPTER 13:EziG: A Universal Platform for Enzyme Immobilisation

EnginZyme, a Swedish biocatalysis company founded in 2014, aims to reduce the environmental impact of the chemical industry by developing products that make it easier and more cost efficient to use enzymes. Currently, the company is addressing the issues of high enzyme cost and process implementation by marketing EziG™, a universal enzyme immobilisation material. EziG eliminates several common issues with enzyme immobilisation and is workable for all enzyme types. This designed enzyme carrier can be used for direct extraction of enzyme from crude preparations, and high loadings of active enzyme are achieved. As an alternative to costly immobilisation testing towards an uncertain end, assured biocatalyst reusability gives benefits beyond the direct economic gain of lower biocatalyst cost, including focus changes in enzyme engineering and simpler work-up procedures. EziG can be used as a standard heterogeneous catalyst, as well as in flow chemistry with packed bed reactors. The importance of this technology in growing the field remains to be evaluated. However, the issues that are now resolved have been identified as key barriers to increased industrial implementation. EziG may play an important role in the ongoing greening of the chemical production industry.

Logistics of process R&D: transforming laboratory methods to manufacturing scale: A guide to drug discovery

In the past, process R&D — which is responsible for producing candidate drugs in the required quantity and of the requisite quality — has had a low profile, and many people outside the field remain unaware of the challenges involved. However, in recent years, the increasing pressure to achieve shorter times to market, the demand for considerable quantities of candidate drugs early in development, and the higher structural complexity — and therefore greater cost — of the target compounds, have increased awareness of the importance of process R&D. Here, I discuss the role of process R&D, using a range of real-life examples, with the aim of facilitating integration with other parts of the drug discovery pipeline.