Kannan Thirunavukkarasu

The osteoblast-specific transcription factor Cbfa1 contributes to the expression of osteoprotegerin, a potent inhibitor of osteoclast differentiation and function.

Bone formation and resorption are tightly coupled under normal conditions, and the interaction of osteoclast precursors with cells of the osteoblast lineage is a prerequisite for osteoclast formation. Cbfa1 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. At present, it is not known whether Cbfa1 regulates any of the osteoblast-derived factors involved in the bone resorption pathway. Osteoprotegerin (OPG) is an osteoblast-secreted glycoprotein that functions as a potent inhibitor of osteoclast differentiation and bone resorption. Cloning and computer analysis of a 5.9-kilobase human OPG promoter sequence revealed the presence of 12 putative Cbfa1 binding elements (osteoblast-specific element 2 (OSE2)), suggesting a possible regulation of OPG by Cbfa1. We cloned the promoter upstream of the β-galactosidase reporter gene (pOPG5.9βgal) and evaluated whether Cbfa1 could regulate its expression in transient transfection assays. The 5.9-kilobase promoter directed increased levels of reporter gene expression, reminiscent of OPG protein levels in osteoblastic cell lines (BALC and U2OS) as compared with the nonosteoblastic cell line COS1. Cotransfection of a Cbfa1 expression construct along with pOPG5.9βgal reporter construct led to 39-, 7-, and 16-fold increases in β-galactosidase activity in COS1, BALC, and U2OS cells, respectively. Removal of all the putative OSE2 elements led to an almost complete loss of transactivation. Mutational analysis demonstrated that the proximal OSE2 element contributes to a majority of the effects of Cbfa1, and Cbfa1 bound to the proximal element in a sequence-specific manner. Further, overexpression of Cbfa1 led to a 54% increase in OPG protein levels in U2OS cells. These results indicate that Cbfa1 regulates the expression of OPG, thereby further contributing to a molecular link between bone formation and resorption.

Analysis of regulator of G‐protein signaling‐2 (RGS‐2) expression and function in osteoblastic cells

Regulator of G‐protein signaling‐2 (RGS‐2) belongs to a novel family of GTPase‐activating proteins that rapidly turn‐off G‐protein coupled receptor signaling. RGS proteins contain a characteristic RGS domain by which they interact with the α‐subunit of G‐proteins and drive them into their inactive GDP‐bound forms. Previously, we have reported that RGS‐2 mRNA is rapidly and transiently increased by PTH in rat bone and in osteoblast cultures in vitro. In this study, we further explored the molecular basis for the regulation of RGS‐2 by cloning and functionally characterizing the RGS‐2 gene promoter. We cloned 2.3‐ and 2.8‐kb fragments of the 5′‐flanking regions of the rat and mouse RGS‐2 genes, respectively, and generated a stable clone of UMR106 osteoblastic cells containing the rat RGS‐2 promoter driving the β‐gal reporter gene (p2.3RGS‐2‐β‐gal). Treatment of the stable clone with PTH resulted in a maximal 2.2‐ to 3.6‐fold increase in promoter activity at 8 h, reminiscent of the early response observed with endogenous RGS‐2 mRNA regulation. Further, PTH (1–38), (1–31), PTHrP (1–34), and forskolin, which elevate cAMP levels, stimulated the promoter, while PTH (3–34) and (7–34), which do not readily stimulate cAMP accumulation, and PMA that directly activates protein kinase C, had no effect on promoter activity. Taken together, these results implicate the involvement of the Gαs‐adenylate cyclase‐protein kinase A pathway in stimulating RGS‐2 expression. Maintenance of a hyperphosphorylated state via the inhibition of type 2A protein phosphatases by okadaic acid, resulted in a strong dose‐dependent increase in transcriptional activity of the RGS‐2 promoter as well as that of the endogenous RGS‐2 gene. Furthermore, overexpression of the osteoblast‐specific transcription factor Runx2 also led to a stimulation of RGS‐2 promoter activity. Functional analysis using RGS‐2 overexpression suggests the potential negative regulatory effects of RGS‐2 on PTH‐ and forskolin‐induced cAMP production in osteoblastic cells. In summary, our data suggest that PTH treatment results in a direct transcriptional stimulation of RGS‐2 that in turn may play a role in modulating the duration/intensity of PTH receptor signaling. J. Cell. Biochem. 85: 837–850, 2002.

Development of a novel clinical biomarker assay to detect and quantify aggrecanase-generated aggrecan fragments in human synovial fluid, serum and urine

OBJECTIVE Proteolytic degradation of aggrecan in articular cartilage is a hallmark feature of osteoarthritis (OA). The present study was aimed at developing a sensitive enzyme linked immunosorbent assay (ELISA) for the detection of aggrecanase-cleaved fragments of aggrecan in human serum and urine to facilitate the clinical development of aggrecanase inhibitors for OA. METHODS The BC3 monoclonal antibody that detects the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized alpha-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial fluid, serum and urine were detected and quantified using this ELISA. RESULTS The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARGS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARGS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARGS containing fragments in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity. CONCLUSIONS We have developed a novel ELISA using an optimized ARGS antibody and have demonstrated for the first time, an ELISA-based measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA.

Identification of signal transduction pathways and promoter sequences that mediate parathyroid hormone 1–38 inhibition of osteoprotegerin gene expression

Osteoprotegerin (OPG), a secreted member of the tumor necrosis receptor superfamily, is a potent inhibitor of osteoclast formation and bone resorption. Parathyroid hormone (PTH), a potent inducer of osteoclast formation, suppresses OPG mRNA expression in vitro and in vivo. To determine the molecular basis of this inhibition, we analyzed the effects of PTH on the human OPG promoter (−5917 to +19) fused with β‐galactosidase reporter gene in stable and transient transfections into rat osteoblast‐like UMR106 cells. The effect of PTH on OPG promoter expression was biphasic and concentration‐dependent. PTH (1–100 nM) induced the transcriptional activity of the OPG promoter (1.7‐fold) at 8 h followed by a gradual decrease with maximal inhibition (6.6‐fold) at 24–48 h. To ascertain the signal transduction pathways mediating PTH (1–38) effects on OPG gene expression, we compared the effects of PTH with PTH analogs, parathyroid hormone‐related protein 1–34 (PTHrP 1–34), forskolin, 3‐isobutyl‐1‐methylxanthine (IBMX), dibutyryl cAMP, phorbol‐12‐myristate‐13‐acetate (PMA), thapsigargin and calcium ionophore A23187. PTH 1–31 and PTHrP 1–34, which stimulate the cAMP/PKA pathway, and other activators of cAMP/PKA, forskolin, IBMX, N6, O2′‐dibityryl adenosine 3′,5′‐cyclic monophosphate (dibutyryl cAMP), all elicited a similar biphasic response on OPG promoter expression. PTH analogs PTH 3–34 and PTH 7–34, that do not stimulate cAMP production, had no effect on OPG expression. In contrast, phorbol‐12‐myristate‐13‐acetate (PMA), an activator of PKC, stimulated OPG promoter expression, while thapsigargin and calcium ionophore A23187, which increase intracellular Ca2+, showed a dose‐dependent inhibition of OPG promoter expression. To delineate the promoter sequences that mediate the inhibitory effects of PTH on OPG transcription, we analyzed systematic deletions of the OPG promoter for responsiveness in transient transfection assays. The major inhibitory effects of PTH were localized to 391 bp (−372 to +19) of the proximal promoter. Deletions of the promoter region led to a complete loss of responsiveness. Taken together, these results demonstrate that the inhibitory effects of PTH on OPG are mediated at the transcriptional level through cis elements in the proximal promoter. The similar biphasic response of OPG to PTH, PTH 1–31, PTHrP 1–34, forskolin, IBMX and dibutyryl cAMP suggests that PTH regulates OPG transcription via activation of the cAMP/PKA signal transduction pathway. J. Cell. Biochem. 84: 1–11, 2002.

A short-term pharmacodynamic model for monitoring aggrecanase activity: injection of monosodium iodoacetate (MIA) in rats and assessment of aggrecan neoepitope release in synovial fluid using novel ELISAs.

OBJECTIVE To develop a short-term in vivo model in rats, with an enzyme-linked immunosorbent assay (ELISA) readout for specific aggrecanase-cleaved aggrecan fragments, to facilitate testing of aggrecanase inhibitors. METHODS Monosodium iodoacetate (MIA), a metabolic inhibitor, was injected into the right knee joint of male Lewis rats and the release of aggrecanase-cleaved fragments of aggrecan containing the NITEGE or ARGN neoepitope was measured in the synovial fluid at 7 days post MIA injection using novel ELISAs. The ELISAs utilize a commercial antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan, in combination with either an alpha-NITEGE antibody (NITEGE ELISA) or an alpha-ARGS/BC3 antibody (ARGS ELISA), to detect aggrecanase-cleavage of aggrecan within the interglobular domain (IGD). Aggrecan fragments present in in vitro digests, in cytokine-treated cartilage explant culture supernatants and in rat synovial fluid lavage samples were detected and quantified using the two ELISAs. Small molecule inhibitors of aggrecanase activity were dosed orally on days 3-7 to determine their ability to inhibit MIA-induced generation of the NITEGE and ARGN neoepitopes measured in the rat synovial fluid. RESULTS The NITEGE assay was shown to specifically detect the N-terminal fragment of aggrecan comprising the G1 domain and the NITEGE neoepitope sequence. This assay can readily measure aggrecanase-cleaved bovine, human and rat aggrecan without the need for deglycosylation. The ARGS assay specifically detects C-terminal fragments of aggrecan comprising the ARGS/ARGN neoepitope and the G2 domain. Keratan sulfate (KS) residues of aggrecan interfere with this ELISA, and hence this assay works well with native rat articular cartilage aggrecan (that lacks KS residues) and with deglycosylated bovine and human aggrecan. Injection of MIA into the rat knee joints resulted in a time-dependent increase in the release of aggrecanase-cleaved aggrecan fragments into the synovial fluid and treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of the generation of these neoepitopes. CONCLUSIONS We have established a short-term in vivo model in rats that involves measurement of synovial fluid biomarkers that are dependent on aggrecanase activity in the joint. The short duration of the model combined with the mechanistic biomarker readout makes it very useful for the initial in vivo screening of aggrecanase inhibitors prior to testing them in time and resource-intensive disease models of osteoarthritis (OA).

Novel Autotaxin Inhibitors for the Treatment of Osteoarthritis Pain: Lead Optimization via Structure-Based Drug Design

In an effort to develop a novel therapeutic agent aimed at addressing the unmet need of patients with osteoarthritis pain, we set out to develop an inhibitor for autotaxin with excellent potency and physical properties to allow for the clinical investigation of autotaxin-induced nociceptive and neuropathic pain. An initial hit identification campaign led to an aminopyrimidine series with an autotaxin IC50 of 500 nM. X-ray crystallography enabled the optimization to a lead compound that demonstrated favorable potency (IC50 = 2 nM), PK properties, and a robust PK/PD relationship.

Identification of potent and selective hydantoin inhibitors of aggrecanase-1 and aggrecanase-2 that are efficacious in both chemical and surgical models of osteoarthritis.

A disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and ADAMTS-5 are zinc metalloproteases commonly referred to as aggrecanase-1 and aggrecanase-2, respectively. These enzymes are involved in the degradation of aggrecan, a key component of cartilage. Inhibitors of these enzymes could be potential osteoarthritis (OA) therapies. A series of hydantoin inhibitors of ADAMTS-4 and ADAMTS-5 were identified from a screening campaign and optimized through structure-based drug design to give hydantoin 13. Hydantoin 13 had excellent selectivity over other zinc metalloproteases such as TACE, MMP2, MMP3, MMP13, and MMP14. The compound also produced efficacy in both a chemically induced and surgical model of OA in rats.

Pharmacological Characterization of a Potent Inhibitor of Autotaxin in Animal Models of Inflammatory Bowel Disease and Multiple Sclerosis.

Autotaxin is a secreted enzyme that catalyzes the conversion of lysophosphatidyl choline into the bioactive lipid mediator lysophosphatidic acid (LPA). It is the primary enzyme responsible for LPA production in plasma. It is upregulated in inflammatory conditions and inhibition of autotaxin may have anti-inflammatory activity in a variety of inflammatory diseases. To determine the role of autotaxin and LPA in the pathophysiology of inflammatory disease states, we used a potent and orally bioavailable inhibitor of autotaxin that we have recently identified, and characterized it in mouse models of inflammation, inflammatory bowel disease (IBD), multiple sclerosis (MS), and visceral pain. Compound-1, a potent inhibitor of autotaxin with an IC50 of ∼2 nM, has good oral pharmacokinetic properties in mice and results in a substantial inhibition of plasma LPA that correlates with drug exposure levels. Treatment with the inhibitor resulted in significant anti-inflammatory and analgesic effects in the carrageenan-induced paw inflammation and acetic acid-induced visceral pain tests, respectively. Compound-1 also significantly inhibited disease activity score in the dextran sodium sulfate–induced model of IBD, and in the experimental autoimmune encephalomyelitis model of MS. In conclusion, the present study demonstrates the anti-inflammatory and analgesic properties of a novel inhibitor of autotaxin that may serve as a therapeutic option for IBD, MS, and pain associated with inflammatory states.

Intraarticular Matrix Metalloproteinases and Aggrecan Degradation Are Elevated After Articular Fracture

BackgroundPosttraumatic osteoarthritis (OA) is a variant of OA that can develop after articular injury. Although the mechanism(s) of posttraumatic OA are uncertain, the presence and impact of postinjury proteolytic enzymes on articular cartilage remain unknown. To our knowledge, there are no studies that evaluate the presence of matrix metalloproteinases (MMPs) or aggrecan degradation after articular fracture.Questions/purposes(1) Are MMP concentrations and aggrecan degradation elevated after intraarticular fracture? (2) Are MMP concentrations and aggrecan degradation greater in high-energy injuries compared with low-energy injuries? (3) Do the concentrations of these biomarkers remain elevated at a secondary aspiration?MethodsBetween December 2011 and June 2013, we prospectively enrolled patients older than 18 years of age with acute tibial plateau fracture. Exclusion criteria included age older than 60 years, preexisting knee OA, injury greater than 24 hours before evaluation, contralateral knee injury, history of autoimmune disease, open fracture, and non-English-speaking patients. During the enrollment period, we enrolled 45 of the 91 (49%) tibial plateau fractures treated at our facility. Knee synovial fluid aspirations were obtained from both the injured and uninjured knees; two patients received aspirations in the emergency department and the remaining patients received aspirations in the operating room. Twenty patients who underwent spanning external fixator followed by definitive fixation were aspirated during both surgical procedures. MMP-1, -2, -3, -7, -9, -10, -12, and -13 concentrations were quantified using multiplex assays. Aggrecan degradation was quantified using sandwich enzyme-linked immunosorbent assay.ResultsThere were higher concentrations of MMP-1 (3.89 ng/mL [95% confidence interval {CI}, 2.37–6.37] versus 0.37 ng/mL [95% CI, 0.23–0.61], p < 0.001), MMP-3 (457.35 ng/mL [95% CI, 274.5–762.01] versus 129.17 ng/mL [95% CI, 77.01–216.66], p < 0.001), MMP-9 (6.52 ng/mL [95% CI, 3.86–11.03] versus 0.96 ng/mL [95% CI, 0.56–1.64], p < 0.001), MMP-10 (0.52 ng/mL [95% CI, 0.40–0.69] versus 0.23 ng/mL [95% CI, 0.17–0.30], p < 0.001), and MMP-12 (0.18 ng/mL [95% CI, 0.14–0.23] versus 0.10 ng/mL [95% CI, 0.0.081–0.14], p = 0.005) in injured knees compared with uninjured knees. There was not a detectable difference in MMP concentrations or aggrecan degradation between high- and low-energy injuries. MMP-1 (53.25 versus 3.89 ng/mL, p < 0.001), MMP-2 (76.04 versus 0.37 ng/mL, p < 0.001), MMP-3 (1250.62 versus 457.35 ng/mL, p = 0.002), MMP-12 (1.37 versus 0.18, p < 0.001), MMP-13 (0.98 versus 0.032 ng/mL, p < 0.001), and aggrecan degradation (0.58 versus 0.053, p < 0.001) were increased at the second procedure (mean, 9.5 days; range, 3–21 days) as compared with the initial procedure.ConclusionsBecause MMPs and aggrecan degradation are elevated after articular fracture, future studies are necessary to evaluate the impact of elevated MMPs and aggrecan degradation on human articular cartilage.Clinical RelevanceIf further clinical followup can demonstrate a relationship between posttraumatic OA and elevated MMPs and aggrecan degradation, they may provide potential for therapeutic targets to prevent or delay the destruction of the joint. Additionally, these markers may offer prognostic information for patients.

Use of Osmotic Pumps to Establish the Pharmacokinetic–Pharmacodynamic Relationship and Define Desirable Human Performance Characteristics for Aggrecanase Inhibitors

The development of reliable relationships between in vivo target engagement, pharmacodynamic activity, and efficacy in chronic disease models is beneficial for enabling hypothesis-driven drug discovery and facilitating the development of patient-focused candidate selection criteria. Toward those ends, osmotic infusion pumps can be useful for overcoming limitations in the PK properties of proof-of-concept (POC) compounds to accelerate the development of such relationships. In this report, we describe the application of this strategy to the development of hydantoin-derived aggrecanase inhibitors (eg, 3) for the treatment of osteoarthiritis (OA). Potent, selective inhibitors were efficacious in both chemical and surgical models of OA when exposures were sustained in excess of 10 times the plasma IC50. The use of these data for establishing patient-focused candidate selection criteria is exemplified with the characterization of compound 8, which is projected to sustain the desired level of target engagement at a dose of 45 mg qd.