Cure Arthritis Naturally

Cure Arthritis Naturally

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The most important function of the innate immune response is to eradicate foreign infectious agents such as microbes and viruses. In such cases, phagocytes and a variety of effector molecules accumulate at the site of infection and induce the secretion of several cytokines and other inflammatory mediators. This class of molecules has effects on subsequent immunological events such as inflammation and lymphoid apoptosis. Cytokines, secreted by macrophages in response to the foreign antigens such as microbes, are often called monokines, since they are produced by monocytes. Monokines include interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), and tumor necrosis factor-a (TNF-a). Most of these molecules are pleiotropic (i.e., affect different biological functions). These effectors contribute to many elements and the inflammatory response at the site of infection.

As early as in 1893, Coley (1) observed beneficial inflammatory effects in terminally ill cancer patients. Subsequent investigations revealed that the curative effects were caused by induced release of TNF-a to the bacterial endotoxin (2). The name tumor necrosis factor was coined by Lloyd Old (2)

* Formerly at University of Pennsylvania, Philadelphia, Pennsylvania.

for its ability to trigger necrosis and involution of transplantable tumor (3). For some time, it was believed that the factor could be a chemotherapeutic agent against cancer. TNF-a was shown to be highly toxic to both humans and animals (3). In an unrelated experiment, cachectin isolated from waste body fluids of animals and humans with chronic diseases was determined to be the same as the necrosis factor. The effect of TNF-a on lipopolysaccharide (LPS)-induced biological functions led to the conclusion that TNF-a is also a strong mediator of shock, disseminated coagulator, metabolic acidosis, and end-organ damage brought about by LPS.

TNF has been the focus of research for several years and remains one of the most active areas of research. Early in 1980, TNF-a was cloned, sequenced, and purified. Subsequently, several biochemical and biological properties of TNF-a have been elucidated. Production of TNF-a has been found to promote chronic inflammatory disease. Overproduction or prolonged production of TNF-a has been observed to modify the anticoagulant properties of endothelial cells and neutrophils and promotes release of other proinflam-matory cytokines such as IL-1 leading to cardiovascular collapse (4). On the other hand, low levels of TNF-a promote bone resorption, fever, and anemia (5). Normal production of TNF-a is thought to be critical for immune regulation against infection (6).

In addition to lysis of tumor and induction of sepsis, TNF-a has been shown to play a role in the development of other diseases. High concentrations of TNF-a in plasma correlate with the development of autoimmune processes (7-13) such as rheumatoid arthritis (RA) and Crohn's disease. Excessive secretion of TNF-a due to infection has also been shown to influence the development of pathophysiological conditions (6), including osteoporosis (14-16), cancer (17,18), and acquired immunodeficiency syndrome (AIDS) (19).

Multiple roles mediated by TNF-a in the development of autoimmune diseases prompted efforts to refocus on inhibition of TNF-a as a viable therapeutic agent for diseases other than cancer and sepsis (20). The molecule has been found to play a critical role in modulating programmed cell death (apoptosis) (7,21,22). Inhibition of TNF-a has proven to be of therapeutic value in some preliminary general studies (23,24). Rheumatoid arthritic symptoms have been improved through the use of anti-TNF-a antibody (13). Also, anti-TNF-a agents may be valuable in the treatment of bone resorption (25,26), obesity due to insulin resistance (27-29), and eye injury (30-32).

We have recently developed a novel anti-TNF-a peptidomimetic (33) based on a detailed structural knowledge of the TNF receptor (55 kD) and its complex with TNF-P (34-36). In this chapter, we will focus on the strategies used in the development of small molecular forms of anti-TNF-a and their status.


An understanding of how the receptors work is essential for effective therapeutic design. The receptors for cytokines differ from growth factor receptors such as epidermal growth factor (EGF), prostaglandin F (PDGF), and ErbB2 receptors, because they lack endodomain with intracellular kinase domains. By association and aggregation with other members of receptors, cytokine receptors mediate a comparable complex pattern in modulating signal transduction and biological functions. Active cytokine receptors increase their phosphorylation by recruiting other components to assemble with the receptors (37). Functional redundancy observed in the cytokines' functions may be attributed to cross reactivity among the members of the superfamily of cytokines receptors (38). Cytokine receptors have been shown to share structural motifs such as, for example, interferon-y receptor, immunoglobulin (Ig) superfamily fold, and chemokines, based on their structure, genetic organization, and cellular source (39). Ligand cytokines are found either in soluble forms or as membrane-associated molecules. Membrane-associated cytokines are often cleaved to perform the same functions as soluble ones and enable the two forms to perform complex biological functions by physical contact between cells and paracrine pathways.

TNF receptors (TNFR) exist in two forms: a 55-kD (TNFR-I) and 75-kD (TNFR-II) receptors. These receptors have been observed both as membrane-bound (TNFR) and soluble forms (sTNFR) and both show an ability to bind TNF-a. However, their biological functions differ. The functional role of TNFR-II is not quite clear, but studies indicate that TNFR-I is more critical for immune protection than TNFR-II (40,41).

Attempts to disable TNF-a functions are targeted at several levels and use different approaches. The methods extend from gene therapy to small molecule antagonists. The processes include (1) blocking the production of TNF-a synthesis through protease inhibitors, (2) interference with molecules involved in the signaling such as kinase inhibitors, (3) gene therapy to modulate nuclear factor-KB (NF-kB) expression, and (4) blocking excessive TNF-a in the plasma binding to its receptor either by antibody, soluble receptor, or small molecule antagonists. No one method has been proven to be uniformly effective. Some approaches in reducing excessive TNF-a have shown promise for therapeutic values in certain type of disease. For example, anti-TNF-a antibody, in general, has been shown to be effective against some arthritic problems (42,43) but not others (20,24). Nevertheless, the curative potential of anti-TNF-a agents may be improved by (1) understanding the role of TNF-a in the development of diseases and by (2) balancing the TNF-a concentra tion by precise intervention. To this purpose, it is important to address the current approaches and evaluate them for their advantages or disadvantages.

Modulation of Signal Transduction

The elucidation of the assembly of the TNF receptor-ligand complex and the associated signal transduction and biological function is an active area of research. Some progress has been made in identifying various molecules involved in the TNF-a-induced apoptosis pathways (22,44-46) and mechanisms of diseases (6,10,24,27,47-49).

TNF-a binding to its receptor leads to the activation of transcription factors such as transcription-activator proteins (AP-1) and NF-kB (50,51). These transcription factors play a role in the programmed cell death pathways (apoptosis). Thus, there has been considerable effort to control the expression of NF-kB for therapeutic use against cancers as well as other inflammatory processes as an alternate strategy to blocking TNF-a binding to its receptor per se. In this regard, molecules involved in signal transduction due to TNF-a are targeted. Protein tyrosine kinase (PTK) inhibitors such as genistein and erbstatin have been shown to block TNF-a-induced activation of NF-kB (52). Proteasome inhibitors may also be able to block the activation of some transcription factors (53-57). Other approaches including gene therapy (58-61) and antisense methods (41,62-64) have been noted to limit activation of transcription factors. These approaches are being actively pursued, and it may be too early to assess their merits.

Blocking Biosynthesis of TNF-a

Membrane-bound TNF-a is proteolytically processed and released as a soluble mature form. TNF-a is processed by proteases, known as matrix metallopro-teinases (MMP) (65-68), members of a family of enzymes mainly involved in degradation of the extracellular matrix. Their biological role and relevance as therapeutic targets have been reviewed by others (68-70).

A relevant role for TNF-converting enzyme (TACE), which is a metallo-proteinase disintegrin, has been identified in mice lacking the TACE gene. These mutant mice exhibited reduced production of TNF-a, suggesting that TACE is critical for releasing membrane-bound TNF-a (71,72). Subsequently, the gene was cloned (72). Natural and synthetic metalloproteinase inhibitors have been identified and some are in early clinical evaluation (70,71).

Small molecular inhibitors such as MMP inhibitors are nonselective agents. It has been shown that certain MMP inhibitors can block TNF-a not only synthesis but also TNFR cleavage (73). Recent crystal structure determination of MMP (74) may help to design more selective inhibitors. Some of the inhibitors are in early clinical trials mostly for cancer (69). Blocking p38 mitogen-activated protein kinase has been shown to reduce the production and accumulation of TNF-a (75,76).

Macromolecular Inhibitors of TNF-a

The classic approaches in the development of antagonists are either ligand mimics or substrates analogs. Antagonists may be discovered using high-throughput screening. Other approaches such as, for example, monoclonal antibody and minibodies are also being developed (13,42,77,78). These approaches require little knowledge of structure and function.

Monoclonal Antibody

Monoclonal antibodies have been proven to be successful in the treatment of several diseases (79). Monoclonal antibodies that bind TNF-a have shown promise in clinical trials (9,13,80,81). Although the monoclonal antibody approach has been promising in treating some cancers, there is no report on treating TNF-a-related diseases. Despite the limited success of monoclonal antibodies as therapeutic agents, it has some drawbacks: cost of production, humaniza-tion, and other related disadvantages associated with macromolecules (82).

Soluble Receptor

Soluble TNF receptor species have been detected in the plasma. It is thought that soluble receptors play a role of controlling the TNF-a activity and are necessary for normal immune regulation (6,48,83). Soluble receptors as therapeutic agents have been used in treating inflammatory diseases (40,47,81). In some cytokine systems, soluble receptors or antibodies do not neutralize the circulating cytokines but merely extend their half-life, thereby potentiating the effect (49).

One of the problems with soluble receptors as therapeutic agents is related to their long half-life; that is, the molecules will not be cleared from the body through the normal secretion process but may be active for a long time before the large molecules are degraded by proteases. This may have unfavorable effects on systemic immune system reactions in fighting infections. Since TNF-a has pleotropic effects in vitro and may even have more unexpected side effects in vivo. Further, these macromolecules may also induce neutralizing antibodies. Fusion proteins containing the constant domain of Ig (with a soluble receptor)

may be particularly immunogenic, because they bind to the Fc receptors of antigen-presenting cells, thereby facilitating uptake and antigen presentation (49).

Disadvantages of Macromolecular Therapeutics

Although the advantage of macromolecules as drugs has been reported and it has been shown that they (1) are highly specific, (2) are selective, (3) often do not require extensive analysis of biodistribution and toxicity, as in the case of small molecules, and (4) have a long half-life, macromolecules also have some drawbacks: (1) commercial-scale production may be either difficult or costly, (2) purity may be difficult to achieve and microheterogeneity may be inevitable, (3) conformational stability may vary with environment of body fluids, and (4) they may be excluded from certain compartments such as the blood-brain barrier (82).

Most of the above disadvantages of macromolecules can be overcome by creating small molecular inhibitors. Small molecules have their own limitations, including their biodistribution and half-life. Often peptides are created first to assess biological effects, but when used as a template lead to further development of viable therapeutic agents (84-86).

Several macromolecular anti-TNF-a agents such as monoclonal antibodies and soluble receptors have already been used in clinical trials. Some successes have been reported in the treatment of certain diseases. In the treatment of sepsis, anti-TNF-a treatments did not reduce the mortality (20), but in Crohn's disease where chronic TNF-a synthesis is thought to occur, treatment by monoclonal antibodies has improved the disease (87). As mentioned, one potential side effect with macromolecular anti-TNF-a agents treatments such as antibodies is that they have a longer half-life, and the prolonged inhibition of a critical inflammatory agent such as TNF-a might compromise the natural immune response. A small molecule inhibitor may therefore be more suitable, not only for the development of viable drugs, but also for more controlled intervention of TNF-a in the plasma.


A knowledge-based approach to drug design has been shown to be effective. The design of a human immunodeficiency virus (HIV) protease inhibitors from the structure and function of the enzyme highlights the progress made in recent years in structural biology. Unlike conventional approaches, rational design offers not only a viable lead, but may also provide the opportunity to decipher the biological role of target molecules. Our approach to the design and development of the antireceptor antagonists stems from a combined structural analysis of relevant molecules, antibodies, ligands, and receptors. An understanding of the properties of molecular recognition at the atomic level has allowed us to engineer molecules that either can mimic the ligands or modulate the receptors' signaling function.

Recent progresses in crystallographic, nuclear magnetic resonance (NMR), and molecular modeling techniques have created a large three-dimensional structure database. These efforts have shown that related macromolecu-lar structures are often highly conserved. Macromolecules, in general, have distinct components: (1) Scaffolds: required for stability of the folding (e.g., the framework regions in an antibody); (2) functional regions: mainly small regions in a molecule which are involved in molecular recognition (e.g., complementary determining region [CDR] loops in an antibody, P-turns, or long flexible loops); and (3) functional surfaces/cavities: folded proteins contain solvent accessible or occupied clefts or cavities which are required for their functions (e.g., active site in an enzyme is a large cavity where substrate binds). When the structure of either target protein or its associating members is known, it may be possible rationally to design agonists or antagonists (88,89).

Structure of Immunoglobulins

The immunoglobulin fold is one of the most common folds in proteins and receptors involved in immunological functions. Macromolecules which contain this fold range from antibody fragments to T-cell receptor complexes. Immunoglobulin domains were first identified in antibodies. The fold contains six to seven P-strands arranged like a barrel with extended CDR loops. The CDR loops are the functional secondary structures that are responsible for molecular recognition.

Molecular and crystallographic analyses of immunoglobulins have revealed that critical ligand-binding surfaces are predominantly the CDR loop projections. Canonical conformations of the CDR of the Vk light chain CDR, and two of three of the heavy chain CDR have been noted. The third CDR of the heavy chain, as a consequence of the complex genetic mechanism which influences its structure, has medium or long loops which have diverse patterns of interactions. In general, the canonical CDR, aside from the CDR3 of the heavy chain, have reverse turn conformations which sometimes have the regular features of P turns. In addition, the two constant domains, C1 and C2, although similarly fashioned, have different roles; the C1 domains are involved in antigen interactions, whereas C2 domains subserve Fc receptor and adhesive structures such as leukocyte function-associated antigen-3 (LFA-3), myclin-associated glycoprotein (MAG), CD2, neural cell adhesion molecule (NCAM), and intercellular adhesion molecule (ICAM) (90,91).

Conformational properties of CDR loops or reverse turns are considered to be important mediators in the biological activity of polypeptides. Turns provide suitable orientations of binding groups essential for bioactivity by stabilizing a folded conformation, and thus may be involved in both binding and recognition (92,93). Moreover, crystallographic studies of antigen-antibody complexes reveal that molecular recognition often requires an interface area of about 600-1000 A2. Yet, most of the intermolecular interactions are mediated by few residues; predominantly from the heavy chain CDR3 region and in some cases from light chains (94). The studies of small naturally secreted peptides, such as somatostatin and enkephalins, have shown that the structural aspects of P turns, namely, optimal disposition of side chains, leads to effective binding to receptors.

Structure of Receptors

Analysis of the evolution of biological molecules has revealed that the structural topology is more conserved across different species than the primary sequence. This is evident from the fact that many proteins of immunological interest and the large number of cell surface receptors and growth factors share the immunoglobulin fold. A common feature of receptor types shows that they often have predominantly immunoglobulin folds or cystine knot repeats. The immunoglobulin fold is characterized by six to eight P strands which are sandwiched against each other. Some of the P strands are stabilized by at least one disulfide bond. On the other hand, the cystine knot is characterized by three or four P strands which are stabilized by three disulfide bonds. Unlike the immunoglobulin fold, which has a unique globular topology, the cystine knot has been shown to adopt different topologies (95,96). In the receptors, cystine knots are arranged in a head-to-tail or elongated fashion in the receptors studied to date. In multidomain macromolecules, the role of individual subdomains can be considered either as framework or scaffolds or as functional units. In immunoglobulin and the cystine knot, P strands stabilized by disulfide bonds can be considered to be scaffolds and loops interleaved between them as functional units.

Further, irrespective of structural topology, the subdomain of all macro-molecules is built by four major secondary structural elements: the alpha helix, P sheet, P turn, and loops. All the secondary structures are well defined and classified except loops. The loop structures are highly variable and often mobile. In many protein-protein and receptor-ligand complexes, the flexible loop structures are often involved in binding to their counterparts, and thus loops have been attributed to their role in molecular recognition and binding (9799). An attempt to classify loops in the protein is still elusive, but in certain domain structures, it is possible to predict and classify small loops owing to the small length of the amino acid sequence involved (99-101). Thus, the functional units in biological molecules appear predominantly as loops or reverse turns between the structural framework regions.

In designing antireceptor small molecules, we have employed a variety of features such as structures of antibodies, receptors, ligands, and biochemical and biological data to design antireceptor antagonists. We have designed small molecule antagonists of CD4 (102,103) and TNF receptors (33). The structure of the CD4 receptor contains an immunoglobulin domain similar to an antibody, and TNF receptors contain ''cystine knot'' repeating domains. In the following sections, the design is illustrated for the TNF receptor. Although these approaches are described in the context of TNF receptors, they can be also used for other related receptors (86).

General Strategy in the Design of Peptidomimetics

Linear peptides possess too much inherent flexibility. Studies have shown that constraining the peptides enhanced their stability and, in some case, their affinity. Achieving structural stability, solubility in physiological relevant solutions, and bioviability are necessary properties for a therapeutic use. We have modified cyclic peptides to increase their stability and bioviability by addition of aromatic residues at the termini.

Placement of Constraining Cystine Residues

Placement of constraining cystine residues in the loop or P turn structure is critical. Constraining cystine residues in a cyclic peptide alters both ring size and the conformation of other residues. For this paired cystine residues are placed systematically at residues (Ca atoms separated at least by 6.2 A) away from the critical residues using the program MODIP (R. Murali, unpublished data). The effect of disulfide closure on the loop structure of a peptide and its loop ring size are evaluated by a conformational search (100) followed by molecular energy minimization and dynamics (INSIGHT, Molecular Simulations, Inc., San Diego, California).

Aromatic Modification of Peptides

Cyclization of peptides confers structural and conformational rigidity which are critical for optimal interaction with macromolecules, but it does little to improve solubility. To increase stability, solubility, and other properties, a variety of strategies (85) have been adopted. For example, mixed anhydride coupling has been used (104), and DeGrado and colleagues have used a semirigid linker m-aminomethyl benzoic acid which links the two ends of the pep-tide in a simple reaction (105,106). These simple chemistries have allowed the development of different forms of constrained readily synthesized small molecules of known structure.

Distribution of hydrophobic residues and to a smaller extent hydrophilic residues has been observed at the protein-protein interfaces and within the antibody-combining site (107-109). A general explanation for such a disproportionate distribution at the interface is attributed to their role in stabilizing interactions. Owing to their large hydrophobicity, especially aromatic residues with planar n charges, they can exclude solvents at the interface and decrease the entropy for a higher binding property (109). Based on these observations, we proposed that bulky aromatics such as phenylalanine and tyrosine, known to decorate by enhancing the binding surface area, would protrude from the antigen-binding surface and promote forming either ordered water binding to main chain residues or exclusion of solvent in the binding site. These bound waters then lead to a network of water chains that become involved in bridging antigen and antibody. This concept provides a basis for more rational structural modeling and has been used to aid our creation of small forms of molecules.

Based on our studies of the dominant features of aromatics in the antigen-binding surface and their ability to lead to propagated water networks that facilitate binding and increase complementarity, we have used aromatic residues in our macrocyclic constructs. In our early studies, we found that addition of aromatic residues Phe or Tyr to the termini of peptides greatly increased the 50% efficiency of cyclization to almost 100% (Berezov and Murali, unpublished results). Furthermore, the aromatically modified cyclic pep-tides behaved far more efficiently than any small compound tested to date, with some possessing nanomolar (nM) affinity. The thermodynamic consequence of the bulky hydrophobic residues on ordering water molecules may be responsible for the improved relative affinity of the compounds. Thus, in our design of peptidomimetics, the peptides are generally flanked by aromatic residues at both N and C termini, but clearly other thermodynamically equivalent modifications could be employed.

Molecular Modeling

There are two major approaches: (1) a de novo folding design using energy minimization and molecular dynamics and (2) comparative modeling followed by energy minimization and molecular dynamics. These two approaches differ only in developing the trial or initial structures. The folding patterns are studied using energy minimization and molecular dynamics. Parameters used in the modeling of peptide mimics have been described earlier by our group (110). A detailed description of various methods used to design peptidomimet-ics is beyond the scope of this chapter. A brief summary of the strategy used by us is discussed.

Initial trial structures were developed using a database consisting of loops from proteins in the Brookhaven protein database (111). Based on the sequence similarity and the loop size, trial structures were selected. Each of the structures was evaluated for the loop size, relative orientation of the side chains, and solvent effects using a combination of energy minimization and molecular dynamics. In the simulation studies, both room temperature (300°K) and high temperature (900°K) are employed. Low-energy conformers are then subjected to further minimization and compared with the native conformation of the template. Each assigned score is based on the similarity (as measured by Ca atoms), relative disposition of critical amino acids with respect to their neighboring residues, predicted solubility, and ability to form oligomers. When required, original amino acid residues in the template are replaced in an iterative manner to conform to the above criteria.

Design of a TNF-a Inhibitor

Structure of the TNF Receptor Complex

Small molecule TNF antagonist molecules are designed from the combined structural knowledge of the TNF receptor complex and anti-TNF antibodies. Several good reviews describing the structural complex of the TNF receptor are reported in the literature (112,113). For readers who are not familiar with the structure of the TNF receptor complex, a brief description is provided below.

Active TNF is a trimeric molecule. Binding of TNF-a to its receptor promotes formation of a trimeric receptor complex. Recent crystallographic work reveals that TNF-a, TNF-P, and TNFR-I exist as trimers. The crystalline structure of the TNF receptor both in complexed and uncomplexed forms provides a general understanding by which these receptors bind to their ligands (34,35) (Fig. 1). TNF receptors are characterized by cysteine residue repeat. This type of repeat has been found in other protein species such as toxins and has come to be known as the cystine knot (95,114). The cystine knot in the TNF receptor family consists of 42 amino acid residues with six cysteine resi-

Figure 1 Complex of TNFR and TNF-a is shown as (a) ribbon and (b) space-filling models. The model is based on the crystal structure of TNFR and TNF-P (36). Crystal structure of TNF-a (34) was superimposed on TNF-P to create the complex. The three major sites considered for peptidomimetic design are shown: domain 1, WP5; domain 2, WP8; and domain 3, WP9.

Figure 1 Complex of TNFR and TNF-a is shown as (a) ribbon and (b) space-filling models. The model is based on the crystal structure of TNFR and TNF-P (36). Crystal structure of TNF-a (34) was superimposed on TNF-P to create the complex. The three major sites considered for peptidomimetic design are shown: domain 1, WP5; domain 2, WP8; and domain 3, WP9.

dues forming three interchain disulfide bonds to create the structural motif. The three-dimensional structure of the TNF receptor reveals four cystine knot repeats with each repeat about 30 A in length and arranged in a head-to-tail fashion exposing the loops on one side of the receptor. These loops appear to be involved either in oligomerization or in ligand binding (34,35). Although there is no direct evidence that the TNF receptors form dimers in solutions, the three-dimensional structure of uncomplexed TNF receptors shows receptors associated as either parallel or anti-parallel dimers. It has been argued that this may represent the oligomerization pattern of TNF receptors (115). Nevertheless, in the parallel dimeric form, the first and last cystine knot domains are involved in dimeric contact (116). In both crystalline structures, the membrane proximal domain is disordered; perhaps due to the lack of the transmembrane that normally holds this domain's structure in a stable form.

In the following sections, the basis and design strategy of an anti-TNF inhibitor is discussed. The design includes consideration of all the three components: (1) antibodies, (2) ligand, TNF-a, and (3) the TNF receptor.

Antibody as Template

Anti-TNF-a antibodies have been shown to block TNF-a and are being evaluated for clinical usefulness against rheumatoid arthritis (117). Often antibodies mirror competing antigens either structurally or sequencewise or both (118). Several antibody epitope-mapping studies suggested that the antibodies might bind to the interface of trimeric TNF-a. It was suggested that one of the mechanisms by which antibodies block TNF-a is by conformational restriction (34). At least four anti-TNF antibodies, Di62 (119), 007, 189, and 004 (120), have been reported and have been sequenced. Similarities between the primary structure of all relevant antibodies and receptor molecules have been compared. Peptides derived from the CDR3 region of the light chain of Di62 have been shown to possess antagonistic activity (119).

The anti-TNF-a monoclonal antibody Di62 analysis led to CDR-based TNF-a antagonists (119). The peptides inhibited TNF-a binding to both TNFR-I and TNFR-II receptors. Inhibition of apoptosis in L929 cells at micromolar concentrations (IC50 = 6-^M) were noted. Interestingly, these unconstrained peptides show much higher activity than constrained ones (119), suggesting that peptide oligomers may be the active form.

We have modeled the three-dimensional structure of all the four antibodies (101) using AbM (Oxford Molecule, Inc., Hunt Valley, Maryland). The structures of all the three CDR loops were analyzed. Comparison of the sequences of Di62 revealed about 36% homology to the TNF receptor in the region of residues 75-85 whereas the CDR3H of 004 antibodies share 50% homology to a region in TNF-a (128-139) which is located at the interface of the trimer in the ligand complex. Since Di63 and 004 antibodies have some similarities to receptor and TNF-a, respectively, the CDR3L of Di63 and CDR3H of 004 loops were considered as a template. Similarities in the loop structures were analyzed normally on a graphics workstation. The following peptides were designed from the CDR of the two monoclonal antibodies.





Original sequence

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Arthritis Joint Pain

Arthritis Joint Pain

Arthritis is a general term which is commonly associated with a number of painful conditions affecting the joints and bones. The term arthritis literally translates to joint inflammation.

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