A pathway for repair of NAD(P)H in plants.

Unwanted enzyme side reactions and spontaneous decomposition of metabolites can lead to a build-up of compounds that compete with natural enzyme substrates and must be dealt with for efficient metabolism. It has recently been realized that there are enzymes that process such compounds, formulating the concept of metabolite repair. NADH and NADPH are vital cellular redox cofactors but can form non-functional hydrates (named NAD(P)HX) spontaneously or enzymatically that compete with enzymes dependent on NAD(P)H, impairing normal enzyme function. Here we report on the functional characterization of components of a potential NAD(P)H repair pathway in plants comprising a stereospecific dehydratase (NNRD) and an epimerase (NNRE), the latter being fused to a vitamin B6 salvage enzyme. Through the use of the recombinant proteins, we show that the ATP-dependent NNRD and NNRE act concomitantly to restore NAD(P)HX to NAD(P)H. NNRD behaves as a tetramer and NNRE as a dimer, but the proteins do not physically interact. In vivo fluorescence analysis demonstrates that the proteins are localized to mitochondria and/or plastids, implicating these as the key organelles where this repair is required. Expression analysis indicates that whereas NNRE is present ubiquitously, NNRD is restricted to seeds but appears to be dispensable during the normal Arabidopsis life cycle.

Targeting the Wnt signalling pathway in cancer: prospects and perils.

The Wnt pathway, involved in cancer development and progression, has for a long time been said to be undruggable, owing to its complexity and involvement in stem cell biology. This mindset has shifted in the last few years as new research and insights into the pathway mechanisms specific to tumour cells become apparent, leading to the development of multiple compounds targeting the pathway. In this review, we introduce the Wnt pathway and its connections to cancer biology and therapy resistance. We further dive into the details of drugs that have entered clinical trials, examining their successes and side effects. We show that these drugs all have one thing in common: in order to be successful, the drugs must target tumour specific activated sub-branches of the pathway, either at the receptor level or at the nuclear transcription level.

A high-throughput assay pipeline for specific targeting of frizzled GPCRs in cancer.

Frizzleds (FZDs) are a family of GPCRs controlling key events in all branches of the developmental Wnt signaling pathway. In this capacity these receptors are mostly active prenatally and have only a limited set of functions in the human adult. Numerous cancer types and subtypes were shown to be dependent on aberrant Wnt signaling and FZDs in particular. Taken together with their GPCR properties, this makes them an attractive drug target for the development of highly specific and efficient targeted therapies against cancer. Despite that, there are few chemical or other agents described targeting FZDs, and an even smaller number bears any clinical relevance. This sparsity dictates the necessity for broader efforts in order to advance in the Wnt pathway-targeting drug discovery. The current work describes the concepts and methodology of an inexpensive high-throughput screening followed by the pipeline of secondary assays in order to identify anti-FZD agents, which will efficiently deactivate Wnt signaling in cancer cells. Specifically, we describe a process and criteria for the selection, generation and statistical validation of a stable cancer cell line based on the well-described luciferase readout (TopFlash) which is then converted into a disease-representative high-throughput-ready screening system. We also provide information on the follow-up test sequence and the post-screening criteria to select FZD-targeting compounds among the hits and to validate them as such. We expect that use of this pipeline will boost the research on clinically valuable Wnt-targeting anti-cancer compounds.

A Second WNT for Old Drugs: Drug Repositioning against WNT-Dependent Cancers.

Aberrant WNT signaling underlies cancerous transformation and growth in many tissues, such as the colon, breast, liver, and others. Downregulation of the WNT pathway is a desired mode of development of targeted therapies against these cancers. Despite the urgent need, no WNT signaling-directed drugs currently exist, and only very few candidates have reached early phase clinical trials. Among different strategies to develop WNT-targeting anti-cancer therapies, repositioning of existing drugs previously approved for other diseases is a promising approach. Nonsteroidal anti-inflammatory drugs like aspirin, the anti-leprotic clofazimine, and the anti-trypanosomal suramin are among examples of drugs having recently revealed WNT-targeting activities. In total, 16 human-use drug compounds have been found to be working through the WNT pathway and show promise for their prospective repositioning against various cancers. Advances, hurdles, and prospects of developing these molecules as potential drugs against WNT-dependent cancers, as well as approaches for discovering new ones for repositioning, are the foci of the current review.