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Objective Osteoarthritis (OA) is a chronic and slowly progressive disease for which biomarkers may be able to provide a more rapid indication of therapeutic responses to therapy than is currently available; this could accelerate and facilitate OA drug discovery and development programs. The goal of this document is to provide a summary and guide to the application of in vitro (biochemical and other soluble) biomarkers in the development of drugs for OA and to outline and stimulate a research agenda that will further this goal. Methods The Biomarkers Working Group representing experts in the field of OA biomarker research from both academia and industry developed this consensus document between 2007–2009 at the behest of the Osteoarthritis Research Society International (OARSI FDA initiative). Results This document summarizes definitions and classification systems for biomarkers, the current outcome measures used in OA clinical trials, applications and potential utility of biomarkers for development of OA therapeutics, the current state of qualification of OA-related biomarkers, pathways for biomarker qualification, critical needs to advance the use of biomarkers for drug development, recommendations regarding practices and clinical trials, and a research agenda to advance the science of OA-related biomarkers. Conclusions Although many OA-related biomarkers are currently available they exist in various states of qualification and validation. The biomarkers that are likely to have the earliest beneficial impact on clinical trials fall into two general categories, those that will allow targeting of subjects most likely to either respond and/or progress (prognostic value) within a reasonable and manageable time frame for a clinical study (for instance within one to two years for an OA trial), and those that provide early feedback for preclinical decision-making and for trial organizers that a drug is having the desired biochemical effect. As in vitro biomarkers are increasingly investigated in the context of specific drug treatments, advances in the field can be expected that will lead to rapid expansion of the list of available biomarkers with increasing understanding of the molecular processes that they represent.

A question remaining to be answered about RecA protein function concerns the role of ATP hydrolysis during the DNA-strand-exchange reaction. In this paper we describe the formation ofjoint molecules in the absence of ATP hydrolysis, using adenosine 5'-[r-thio]triphosphate (ATP[yS]) as nucleotide cofactor. Upon the addition of double-stranded DNA, the ATP[ySJ-RecA protein-single-stranded DNA presynaptic complexes can form homologously paired molecules that are stable after deproteinization. Formation of these joint molecules requires both homology and a free homologous end, suggesting that they are plectonemic in nature. This reaction is very sensitive to magnesium ion concentration, with a maximum rate and extent observed at 4-5 mM magnesium acetate. Under these conditions, the average length of heteroduplex DNA within the joint molecules is 2.4-3.4 kilobase pairs. Thus, RecA protein can form extensive regions of heteroduplex DNA in the presence of ATP [yS], suggesting that homologous pairing and the exchange of the DNA molecules can occur without ATP hydrolysis. A model for the RecA protein-catalyzed DNA-strand-exchange reaction that incorporates these results and its relevance to the mechanisms ofeukaryotic recombinases are presented.The RecA protein-catalyzed DNA-strand-exchange reaction has been divided into at least three distinct phases (1, 2). In the first phase of this reaction, RecA protein binds to single-stranded DNA (ssDNA) in the presence of ATP (presynapsis). During the second phase, synapsis, the ssDNA and double-stranded DNA (dsDNA) (4)]. Since plectonemic, as well as paranemic, joint molecules can be formed in the presence of ATP, it was suggested that conversion of paranemic to plectonemic joint molecules requires ATP hydrolysis (4). In related studies, addition ofATP[yS] to an ongoing DNA-strand-exchange reaction was shown to immediately stop formation of heteroduplex DNA (3). The data were interpreted to suggest that ATP hydrolysis was also required for the formation of extensive regions of heteroduplex DNA (after joint molecules are formed). Thus, homologous pairing and the formation of protein-stabilized paranemic joint molecules are thought to occur in the absence of ATP hydrolysis, whereas the formation of plectonemic joint molecules and extensive regions of heteroduplex DNA requires hydrolysis of ATP. We now report the catalysis ofDNA strand exchange in the presence of ATP [yS]. Our data demonstrate that the joint molecules formed in the presence ofATP [yS] are stable in the absence of RecA protein and are, therefore, plectonemic in nature. The average length of heteroduplex DNA in these joint molecules can be as much as 3.4 kilobase pairs (kb) with no detectable hydrolysis of ATP [yS]. The relevance of these results to the role of ATP hydrolysis in the mechanism of the DNA strand exchange catalyzed by the Escherichia coli RecA protein and by other recombinases is discussed. MATERIALS AND METHODSReagents. All chemicals used were reagent grade and solutions were made in gla...

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