Application of biomarkers in the development of drugs intended for the treatment of osteoarthritis.

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 and 2009 at the behest of the Osteoarthritis Research Society International Federal Drug Administration initiative (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 1-2 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.

Mice overexpressing chemokine ligand 2 (CCL2) in astrocytes display enhanced nociceptive responses.

Recent findings demonstrate that chemokines, and more specifically CC chemokine ligand 2 (CCL2 or monocyte chemoattractant protein-1), play a major role in pain processing. In the present study, we assess nociceptive responses of mice that overexpressed CCL2 under control of glial fibrillary acidic protein promoter (CCL2 tg). In models of acute nociception CCL2 tg mice demonstrated significantly enhanced nociceptive behavior relative to wild-type controls in responses to both thermal (hot plate) and chemical (formalin test) stimulus modalities. There were no differences in mechanical allodynia in the partial sciatic nerve ligation model, in terms of either magnitude or duration of the allodynic response; however, both groups responded to the maximal extent measurable. In a model of inflammatory pain, elicited by intraplantar administration of complete Freund's adjuvant (CFA), CCL2 tg mice displayed both greater edema and thermal hyperalgesia compared with control mice. In control mice, edema and hyperalgesia returned to baseline values 5-7 days post CFA. However, in CCL2 tg mice, thermal hyperalgesia was significantly different from baseline up to 3 weeks post CFA. Parallel to these enhanced behavioral responses CCL2 serum levels were significantly greater in CCL2 overexpressing mice and remained elevated 7 days post CFA. Consequently, proinflammatory cytokine mRNA expression (IL-1beta, IL-6, and TNFalpha) levels were greater in skin, dorsal root ganglia (DRG), and spinal cord, whereas the anti-inflammatory cytokine (IL-10) level was lower in skin and DRG in CCL2 overexpressing mice than in control mice. Taken together with data from CCR2-deficient mice, these present data confirm a key role of CCL2/CCR2 axis in pain pathways and suggest that inhibiting this axis may result in novel pain therapies.

Analysis of a mutation in phosphodiesterase type 4 that alters both inhibitor activity and nucleotide selectivity.

Cyclic nucleotide phosphodiesterase type 4 (PDE4) is a cAMP-specific phosphodiesterase that is found as four distinct genes in the mammalian genome (PDE4A, 4B, 4C, and 4D). Mutation analysis was done to identify the amino acids involved in activity and inhibitor selectivity. Mutations at Asp333 were made in HSPDE4D3 based on mutations that affect rolipram sensitivity in RNPDE4B1. The PDE4D3 Asp-Asn mutant was resistant to inhibition by rolipram as well as several other PDE4 inhibitors tested. These results suggest that this residue is near the inhibitor binding pocket in PDE4D3. Sequence comparison of PDE4 with cGMP-specific PDE proteins shows a conserved aspartic acid at position 333 in PDE4D3 and a conserved asparagine at this position in PDE enzymes that hydrolyze cGMP. Therefore, cGMP hydrolysis by PDE4D3 Asp-Asn was measured. PDE4D3 Asp-Asn hydrolyzes cGMP with kinetic constants similar to those observed for this protein with cAMP (K(m) approximately 20 microM, V(max) approximately 2 micromol AMP/min/mg recombinant protein). Under identical conditions, the K(m) value for cAMP hydrolysis by wild-type PDE4D3 is 3 microM and the V(max) value is 1 micromol AMP/min/mg recombinant protein. In addition, the PDE4D3 Asp-Ala mutant protein could hydrolyze cGMP. Finally, the analogous mutation in HSPDE4B1 (Asp413Asn) also allows hydrolysis of cGMP. These results show that this aspartic acid residue is important in inhibitor binding and nucleotide discrimination and suggest this residue is in the active site of PDE4.

The structure of the nuclear factor-kappaB protein-DNA complex varies with DNA-binding site sequence.

Transcriptional regulation of many immune responsive genes is under the control of the transcription factor NF-kappaB. This factor is found in cells as a dimer which can contain any two members of the Rel family of proteins (p50, p65, p52, c-Rel, and RelB). The different dimers show distinct preferences for DNA-binding site sequences. To understand the relationship between the DNA binding properties of the dimer forms and transcriptional activation, the physical properties of the complexes of p50 and p65 with DNA have been analyzed. Comparison of apparent DNA binding affinity showed differences in selectivity of DNA-binding site sequence. The ionic strength dependence of apparent binding affinity has shown that the number of ionic interactions in the protein-DNA complex depends on the DNA-binding site sequence and the dimer form, which are consistent with changes in the structure of the protein-DNA complex. Using a fluorescent technique to measure DNA structure changes, protein binding does not appear to alter the structure of the DNA-binding site within the limits of detection. These results are consistent with a change in protein structure that may result in activation differences due to alternative interactions with other transcription proteins.

V-D-J rearrangements at the T cell receptor delta locus in mouse thymocytes of the alpha beta lineage.

The T cell receptor (TCR) delta locus lies within the TCR alpha locus and is excised from the chromosome by V alpha-J alpha rearrangement. We show here that delta sequences persist in a large fraction of the DNA from mature CD4+CD8- alpha beta+ mouse thymocytes. Virtually all delta loci in these cells are rearranged and present in extrachromosomal DNA. In immature alpha beta lineage thymocytes (CD3-/loCD4+CD8+) and in CD4+CD8- alpha beta+ thymocytes expressing a transgene-encoded alpha beta receptor, rearranged delta genes are present both in chromosomal and extrachromosomal DNA. Thus, contrary to earlier proposals, commitment to the alpha beta lineage does not require recombinational silencing of the delta locus or its deletion by a site-specific mechanism prior to V alpha-J alpha rearrangement.

V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes.

Lymphoid cells from scid mice initiate V(D)J recombination normally but have a severely reduced ability to join coding segments. Thymocytes from scid mice contain broken DNA molecules at the TCR delta locus that have coding ends, as well as molecules with signal ends, whereas in normal mice we previously detected only signal ends. Remarkably, these coding (but not signal) ends are sealed into hairpin structures. The formation of hairpins at coding ends may be a universal, early step in V(D)J recombination; this would provide a simple explanation for the origin of P nucleotides in coding joints. These findings may shed light on the mechanism of cleavage and suggest a possible role for the scid factor.

The physical and enzymatic properties of Escherichia coli recA protein display anion-specific inhibition.

The enzymatic activities of Escherichia coli recA protein are sensitive to ionic composition. Here we report that sodium glutamate (NaGlu) is much less inhibitory to the DNA strand exchange, DNA-dependent ATPase, and DNA binding activities of the recA protein than is NaCl. Both joint molecule formation and complete exchange of DNA strands occur (albeit at reduced rates) at NaGlu concentrations as high as 0.5 M whereas concentrations of NaCl greater than 0.2 M are sufficient for complete inhibition. The single-stranded DNA (ssDNA)-dependent ATPase activity is even less sensitive to inhibition by NaGlu; ATP hydrolysis stimulated by M13 ssDNA is unaffected by 0.5 M NaGlu and is further stimulated by E. coli ssDNA binding protein approximately 2-fold. Finally, NaGlu has essentially no effect on the stability of recA protein-epsilon M13 DNA complexes, with concentrations of NaGlu as high as 1.5 M failing to dissociate the complexes. Surprisingly, NaGlu also has little effect on the concentration of NaCl required to disrupt the recA protein-epsilon M13 DNA complex, demonstrating that destabilization is dependent on both the concentration and type of anionic rather than cationic species. Quantitative analysis of DNA binding isotherms establishes that the intrinsic binding affinity of recA protein is affected by the anionic species present and that the cooperativity parameter is relatively unaffected. Consequently, the sensitivity of recA protein-ssDNA complexes to disruption by NaCl does not result from the competitive effects associated with cation displacement from the ssDNA upon protein binding but rather results from anion displacement upon complex formation. The magnitude of this anion-specific effect on ssDNA binding is large relative to that of other nucleic acid binding proteins.

V(D)J recombination in mouse thymocytes: double-strand breaks near T cell receptor delta rearrangement signals.

In the murine T cell receptor delta locus, V(D)J recombination events frequently involve the D2 and J1 elements. Here we report the presence of double-strand breaks at recombination signals flanking D2 in approximately 2% of thymus DNA. An excised linear species containing the sequences between D2 and J1 and a circular product of the joining of D2 and J1 recombination signals were also found. Although broken molecules with signal ends were detected, no species with coding ends could be identified. Observation of these broken molecules in thymus, but not in liver or spleen, provides the first direct evidence for an association between specific cleavage of chromosomal DNA and recombination in mammalian cells, and supports a breakage-reunion model of V(D)J recombination.

V(D)J recombination activity in lymphoid cell lines is increased by agents that elevate cAMP.

V(D)J [variable--(diversity)--joining] recombination is regulated developmentally, being restricted to cells of the early B- and T-lymphocyte lineages. In this report we show that recombination activity can also be regulated in response to chemical effectors. Compounds that increase intracellular cAMP increase V(D)J recombination of extrachromosomal substrates in pre-B-cell lines as much as 10-fold. In contrast, V(D)J recombination is reduced 5- to 8-fold in response to phorbol 12-myristate 13-acetate or to the calcium ionophore A23187. The effect of cAMP agonists on recombination appears to reflect an increase in cellular recombination activity, as indicated by the caffeine-induced rise in the level of mRNA from the recombination-activating genes RAG1 and RAG2. Our data demonstrate that intracellular second messengers modulate recombination activity in lymphoid cell lines, implying that recombination activity can be regulated by these signals in developing B and T lymphocytes.

Biochemical properties of the Escherichia coli recA430 protein. Analysis of a mutation that affects the interaction of the ATP-recA protein complex with single-stranded DNA.

The biochemical properties of the recA430 protein have been examined and compared to those of wild-type recA protein. We find that, while the recA430 protein possesses ssDNA-dependent rATP activity, this activity is inhibited by the Escherichia coli single-stranded DNA binding protein (SSB protein) under many conditions that enhance wild-type recA protein rATPase hydrolysis. Stimulation of rATPase activity by SSB protein is observed only at high concentrations of both rATP (greater than 1 mM) and recA430 protein (greater than 5 microM). In contrast, stimulation of ssDNA-dependent dATPase activity by SSB protein is less sensitive to protein and nucleotide concentration. Consistent with the nucleotide hydrolysis data, recA430 protein can carry out DNA strand exchange in the presence of either rATP or dATP. However, in the presence of rATP, both the rate and the extent of DNA strand exchange by recA430 protein are greatly reduced compared to wild-type recA protein and are sensitive to recA430 protein concentration. This reduction is presumably due to the inability of recA430 protein to compete with SSB protein for ssDNA binding sites under these conditions. The cleavage of lexA repressor protein by recA430 protein is also sensitive to the nucleotide cofactor present and is completely inhibited by SSB protein when rATP is the cofactor but not when dATP is used. Finally, the steady-state affinity and the rate of association of the recA430 protein-ssDNA complex are reduced, suggesting that the mutation affects the interaction of the ATP-bound form of recA protein with ssDNA. This alteration is the likely molecular defect responsible for inhibition of recA430 protein rATP-dependent function by SSB protein. The biochemical properties observed in the presence of dATP and SSB protein, i.e. the reduced levels of both DNA strand exchange activity and cleavage of lexA repressor protein, are consistent with the phenotypic behavior of recA430 mutations.

Stable DNA heteroduplex formation catalyzed by the Escherichia coli RecA protein in the absence of ATP hydrolysis.

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 of joint molecules in the absence of ATP hydrolysis, using adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) as nucleotide cofactor. Upon the addition of double-stranded DNA, the ATP[gamma S]-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[gamma S], 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 of eukaryotic recombinases are presented.

Enhancement of Escherichia coli RecA protein enzymatic function by dATP.

The Escherichia coli recA protein has been shown to hydrolyze several nucleoside triphosphates in the presence of ssDNA. The substitution of dATP for rATP has significant effects on various recA protein biochemical properties. In the presence of dATP, recA protein can invade more secondary structure in native ssDNA than it can in the presence of rATP. The dATP-recA protein complex can compete more effectively with the E. coli ssDNA binding protein (SSB) for ssDNA binding sites compared with the rATP-recA protein complex. Finally, the rate of dATP hydrolysis stimulated by dsDNA is greater than the rate of rATP hydrolysis. These effects, in turn, are observed as alterations in the recA protein catalyzed DNA strand exchange reaction. In the absence of SSB protein, the rate of joint molecule and product formation in the DNA strand exchange reaction is greater in the presence of dATP than in the presence of rATP. The rate of product formation in the dATP-dependent reaction is also faster than the rATP-dependent reaction when SSB protein is added to the ssDNA before recA protein; the rate of rATP-dependent product formation is inhibited 10-fold under these conditions. This nucleotide, dATP, was previously shown to induce an apparent affinity of recA protein for ssDNA which is higher than any other NTP. These results suggest that the observed enhancement of enzymatic activity may be related to the steady-state properties of the high-affinity ssDNA binding state of recA protein. In addition, the data suggest that recA protein functions in NTP hydrolysis as a dimer of protein filaments and that the binding of ssDNA to only one of the recA filaments is sufficient to activate all recA protein molecules in the dimeric filament. The implications of this finding to the enzymatic function of recA protein are discussed.

Properties of the high-affinity single-stranded DNA binding state of the Escherichia coli recA protein.

The properties of the high-affinity single-stranded DNA (ssDNA) binding state of Escherichia coli recA protein have been studied. We find that all of the nucleoside triphosphates that are hydrolyzed by recA protein induce a high-affinity ssDNA binding state. The effect of ATP binding to recA protein was partially separated from the ATP hydrolytic event by substituting calcium chloride for magnesium chloride in the binding buffer. Under these conditions, the rate of ATP hydrolysis is greatly inhibited. ATP increases the affinity of recA protein for ssDNA in a concentration-dependent manner in the presence of both calcium and magnesium chloride with apparent Kd values of 375 and 500 microM ATP, respectively. Under nonhydrolytic conditions, the molar ratio of ATP to ADP has an effect on the recA protein ssDNA binding affinity. Over an ATP/ADP molar ratio of 2-3, the affinity of recA protein for ssDNA shifts cooperatively from a low-to a high-affinity state.

Transfer of recA protein from one polynucleotide to another. Kinetic evidence for a ternary intermediate during the transfer reaction.

We have analyzed the transfer kinetics of recA protein from one polynucleotide to another by monitoring the change in fluorescence of a modified single-stranded M13 DNA, referred to as etheno M13 DNA, that accompanies recA protein dissociation. The observed rate of transfer is dependent on the concentration of competitor polynucleotide, polythymidylic acid (poly(dT]; increasing the poly(dT) concentration increases the rate of transfer. These data are inconsistent with the rate-limiting step in the transfer mechanism occurring by a simple dissociation process. Under certain conditions, the apparent rate constant displays plateau behavior at high poly(dT) concentrations. This result is indicative of transfer occurring through a ternary intermediate including etheno M13 DNA and poly(dT). The transfer reaction was found to occur through two kinetically distinct species of transferring recA protein X DNA complexes. The relative amounts of these two species was affected by both the MgCl2 and protein concentration, suggesting that the two kinetic components reflect different aggregation states of the recA protein X DNA complex. Because etheno M13 DNA and poly(dT) contain no complementary sequences, we have concluded that recA protein has the intrinsic ability to form a kinetic ternary intermediate with two separate single-stranded DNA molecules in the absence of homology.

Transfer of recA protein from one polynucleotide to another. Effect of ATP and determination of the processivity of ATP hydrolysis during transfer.

The transfer of recA protein from a fluorescently modified single-stranded DNA, containing 1,N6-ethenoadenosine and 3,N4-ethenocytosine, to polydeoxythymidylic acid (poly(dT)) was shown to occur by a complex mechanism in both the absence and presence of ADP (Menetski, J. P., and Kowalczykowski, S. C. (1987) J. Biol. Chem. 262, 2085-2092). A part of the mechanism involves the formation of a kinetic ternary intermediate. Since the binding and hydrolysis of ATP by recA protein is involved in many of the recA protein in vitro activities, we have analyzed the effect of ATP on the transfer reaction. In the presence of ATP, the transfer reaction is dependent on the concentration of the competitor single-stranded DNA, poly(dT). This result suggests that transfer does not occur by a simple dissociation mechanism. The reaction occurs via two kinetically distinct species of protein X DNA complexes with properties that are similar to those characterized for the transfer reaction in the absence of ATP. There is a complicated effect of nucleotide concentration on the rate of transfer. At low concentrations of ATP (less than 50 microM), increasing nucleotide concentration increases the rate of transfer; this is similar to the effect of ADP. However, at high concentrations of ATP (greater than 50 microM), increasing ATP concentration decreases the rate of transfer. Finally, the processivity of ATP hydrolysis during transfer was found to increase with increases in ATP concentration. Less than one ATP molecule was hydrolyzed per transfer event at low ATP concentrations (less than 20 microM) while greater than 50 molecules were hydrolyzed at high ATP concentration (greater than 250 microM). These data suggest that the rate of transfer is not directly coupled to the rate of hydrolysis.

Interaction of recA protein with single-stranded DNA. Quantitative aspects of binding affinity modulation by nucleotide cofactors.

We have investigated quantitative molecular aspects of the interaction of recA protein with single-stranded DNA, by using a fluorescent modified-DNA referred to as etheno-M13 DNA. In addition, the effects of the nucleotide cofactors ATP and ADP, and the analogues ATP-gamma-S, AMP-P-C-P, and AMP-P-N-P on this interaction have been studied. It is shown that ATP, AMP-P-N-P and, in particular, ATP-gamma-S significantly increase the affinity of recA protein for single-stranded DNA, whereas ADP and, to a lesser degree, AMP-P-C-P decrease the affinity. Binding to etheno-M13 single-stranded DNA is co-operative, with the value of the co-operativity parameter, omega, being approximately 50 under all conditions measured. The effect that ADP has on recA protein-DNA affinity is to lower the intrinsic binding constant, but it has no effect on the co-operativity of binding. In addition, the stability of the recA protein-DNA complex is very salt dependent (d log K/d log [NaC1] approximately -10) and it is the intrinsic binding affinity rather than the co-operativity of binding that is affected; thus, under all conditions observed, recA protein binds single-stranded DNA co-operatively with a value of omega = 50 +/- 10. The binding affinity is also influenced by the type of anion present, being approximately 10,000-fold higher when acetate ion is present instead of chloride ion. These data have been interpreted to suggest that recA protein forms up to five ionic interactions when it binds to single-stranded DNA and that five to six anions are displaced upon binding. The modulation of recA protein-DNA complex stability by nucleotide cofactors suggests that these cofactors play a role in the cycling of recA protein on and off single-stranded DNA, with ATP being required for DNA binding under physiological conditions and ADP serving as a "release" factor. These results are discussed in terms of a model for the role of ATP hydrolysis in a recA protein-single stranded DNA binding cycle.