Protein Engineering And Sitedirected Mutagenesis

Rapid developments in the technique of site-directed mutagenesis have created the ability to change essentially any amino acid, or even substitute or delete whole domains, in any protein, with the goal of designing and constructing new proteins with novel binding, clearance, or catalytic activities (31, 32). The concomitant changes in protein folding and tertiary structure, protein physiology, binding affinities (for a receptor or hormone), binding specificities (either for substrate or receptor), or catalytic activity (for enzyme active site mutants) are all effects that are measurable against the "wild-type" parent, assuming that expression of the gene and subsequent proper folding have successfully occurred. Several surprising observations have been made during the short period that this technology has been available: amino acid substitutions lead, in general, to highly localized changes in protein structure with few global changes in overall folding; substitutions of residues not involved in internal hydrophobic contacts are extremely well accommodated, leading to few unsynthesizable mutants; and proteins seem extremely tolerant of domain substitution, even among unrelated proteins, allowingoften even crude first attempts at producing chimeric proteins to be successful. The implications of this technology for the discovery of new pharmaceuticals lie in two areas: second-generation protein therapeutics and site- or domain-specific mutant proteins for structure-function investigations.

Throughout this chapter, amino acids are denoted by their standard one-letter codes; and site-specific mutations are represented by the code for the wild-type amino acid, the residue number, and the code for the replacement amino acid (31).

3.1 Second-Generation Protein Therapeutics

The cloning, expression, and manufacture cf proteins as therapeutics involve the same problems encountered in the development and successful clinical approval of any drug. Potency, efficacy, bioavailability, metabolism, and pharmaceutical formulation challenges presented by the natural protein suggest that second-generation products might be engineered to alleviate the particular problem at hand, producing desired therapeutic improvements. The parent proteins to which this technology has been applied extend across the range of recombinant products already approved and those in advanced stage of clinical evaluation (33,34a). As an example,for tissue-type plasminogen activator (t-PA), one of the most studied recombinant products (34b-36), four properties functioning in concert [i.e., substrate specificity, fibrin affinity, stimulation of t-PA activity by fibrin and fibrinogen, and sensitivity of the enzyme to inhibition by plasminogen activator inhibitors (PAIs)] are responsible for the localization and potentiation cf the lytic reaction at a clot surface and are readily analyzed using molecular variants (37).A consensus structure combining the major domains of t-PA has been predicted based on the significant sequence homology with other serum proteins and serine proteases. The complexity of this structure is reflected in its functional multiplicity: efficient production of plasmin by cleavage of the R560-V561 bond of plasminogen, very low binding to plasminogen in the absence of fibrin, moderately high affinity for fibrin, increase in the efficiency of plasminogen activation by 500-fold in the presence of fibrin, rapid inactivation by PAI-1, and rapid hepatic elimination by receptor-mediated endocytosis (38). BM 06.022, a recombinantly engineered t-PA deletion mutant [t-PA del (V4-E175)], made up of the Kringle 2 and protease domains, has been reported to have the same plasminogenolytic activity but a lower fibrin affinity compared with wild type t-PA (39). Another variant of t-PA (T103N, KHRR 296-299 AAAA) was demonstrated to have the combined desirable properties of decreased plasma clearance, increased fibrin specificity, resistance to PAI-1, and in vivo increased potency and decreased systemic activation of plasminogen when administered by bolus dose (40).

Although the systematic changes exemplified by t-PA site-directed mutagenesis studies are the rDNA equivalents of medicinal chemistry [multiple analog synthesis for structure-activity relationship (SAR) development], more recent applications of this technology bear a less straight-forward resemblance to medicinal chemistry-driven drug discovery paradigms. However, these same recombinant techniaues can be used to combine domains from different proteins to produce chimeric constructs that incorporate multiple desired properties into a single final product or reagent. For instance, in an effort to overcome the short plasma half-life associated with soluble CD4, chimeric molecules termed immu-noadhesins (Fig. 4.1) have been recombi-nantly constructed from the gpl20-specific domains of CD4 and the effector domains of various immunoglobulin classes (41, 42). In addition to dramatically improved pharmacokinetics, these chimeric constructs incorporate functions such as Fc receptor binding, protein A binding, complement fixation, and placental transfer, all of which are imparted by the Fc portion of immunoglobulins. Dimeric constructs from human (CD4-27I and CD4-4-yl) and mouse (CD4-My2a) IgG and a pentameric chimera (CD4-M|ll) from mouse IgM exhibit evidence of retained gpl20 binding and anti-HIV infectivity activity. Both CD4-271 and CD4-4-yl show significantly increased plasma half-lives of 6.7 and 48 h, respectively, compared with 0.25 h for rCD4. Furthermore, the immunoadhesin CD4-2yl (CD4-IgG) mediates antibody-dependent, cellmediated cytotoxicity (ADCC) toward HIV-in-fected cells and is efficiently transferred across the placenta of primates (43).

It is becoming clear that genetic variations play critical roles in patients' response to certain medications. Differential expression of drug targets and or metabolic enzymes has been shown to lead to differences in efficacy and toxicity profiles of drugs in section of population that harbors this genetic variation (44). Molecular biology and its associated techniques feature prominently in bringing to birth an interdisciplinary field, pharmacogenetics, which promises to unravel how genetic make up and variation thereof affect human response to medication (45). It is widely held that advances in pharmacogenetics will revolutionize drug dispensation and drug discovery processes. When fully realized, the gains of pharmacogenetics will positively impact drug discovery processes in numerous ways including the following: (1)i dentification of new and novel therapeutic targets; (2)an increased understanding of the molecular "uniqueness" of diseases; (3) genetic tagging of diseases with the consequence of developing designer medications that best combat an ailment; and (4) efficient design of clinical trials, with a better chance of success, aided by a genetic pre-screening of candidates. Medications that were judged ineffective by traditional validation methods in a random patient population may be found beneficial to a population of patients having an overexpressed "susceptibility gene(s)."

The role cf recombinant DNA technology in medicinal chemistry and drug discovery

CD4 immunoadhesin

Soluble rCD4

If ss lgG1 heavy chain lgG1 heavy chain

Figure 4.1. Structure of CD4 immunoadhesin, soluble rCD4, and the parent human CD4 and IgGl heavy-chain molecules. CD4- and IgGl-derived sequences are indicated by shaded and unshaded regions, respectively. The immunoglobulin-like domains are numbered 1 to 4, TM is the transmembrane domain, and CYT is the cytoplasmic domain. Soluble CD4 is truncated after P368 of the mature CD4 polypeptide. The variable (VH) and constant (CH1, Hinge, CH2, and CH3) regions of IgGl heavy chains are shown. Disulfide bonds are indicated by S-S. CD4 immunoadhesin consists of residues 1-180 of the mature CD4 protein fused to IgGl sequences, beginning at D216, which is the first residue in the IgGl hinge after the tysteine residue involved in heavy-light chain bonding. The CD4 immunoadhesins shown, which lacks a CH1 domain, was derived from a CH1-containing CD immunoadhesin by oligonucleotide-directed deletional mutagenesis, expressed in Chinese hamster ovary cells and purified to >99% purity using protein A-sepharose chromatography (42).

Figure 4.1. Structure of CD4 immunoadhesin, soluble rCD4, and the parent human CD4 and IgGl heavy-chain molecules. CD4- and IgGl-derived sequences are indicated by shaded and unshaded regions, respectively. The immunoglobulin-like domains are numbered 1 to 4, TM is the transmembrane domain, and CYT is the cytoplasmic domain. Soluble CD4 is truncated after P368 of the mature CD4 polypeptide. The variable (VH) and constant (CH1, Hinge, CH2, and CH3) regions of IgGl heavy chains are shown. Disulfide bonds are indicated by S-S. CD4 immunoadhesin consists of residues 1-180 of the mature CD4 protein fused to IgGl sequences, beginning at D216, which is the first residue in the IgGl hinge after the tysteine residue involved in heavy-light chain bonding. The CD4 immunoadhesins shown, which lacks a CH1 domain, was derived from a CH1-containing CD immunoadhesin by oligonucleotide-directed deletional mutagenesis, expressed in Chinese hamster ovary cells and purified to >99% purity using protein A-sepharose chromatography (42).

A recent success story in new target identification and validation is seen in the introduction of two new nonsteroidal antiinflammatory drugs (NSAIDs),Vioxx and Celebrex, by Merck Frosst and Monsanto-Pfizer, respectively. Until recently, the onset of inflammation and pain has been linked to one cyclooxy-genase enzyme, COX. Clinically useful NSAIDs such as aspirin, diclofenac, and ibu-profen exhibit their anti-inflammatory and antipyretic activity by inhibiting COX. A prolonged use of most NSAIDs results in gastrointestinal (GI) toxicity (46), which may be debilitating enough to require hospitalization in many patients. Recent progresses in the understanding of the biology of COX, championed by elegant biochemical and recombinant DNA studies, revealed that it exists in two iso-forms: constitutive COX-1 and inducible COX-2 (Fig. 4.2). COX-1 is always expressed and mediates the synthesis of prostaglandins that regulate normal cell functions, whereas COX-2 is active only at the onset of inflamma tion (47, 48). However, early NSAIDs inhibit both COX isoforms, thereby interfering with the production of the protective prostaglandin . products of COX-1 in addition to their pain. relieve activity. It was hypothesized that selective inhibitors of inflammation associated COX-2 may produce a better drug profile and possibly avoid many side effects caused by non-selective NSAIDs, especially GI tract toxicity (49). The discovery of the COX-2 gene stimulated intensive studies aimed at verifying this hypothesis (50, 51). The clinical and. commercial success of Vioxx and Celebrex (1 and 2), two highly potent COX-2 specific inhibitors, validated COX-2 as a new target for anti-inflammation and antipyretic therapy. Furthermore, the safety profiles of these drug showed that they do not have many toxic side effects of traditional non-selective NSAIDs (52-54).

Alternatively, recombinant DNA technology is making it possible to decipher the roles of disease subtypes and genetic variability in a) Prostaglandin synthesis from arachidonic acid fyr 285

fyr 285

(b) Ncn specific NSAIDs bind to COX 1 and COX 2 to block entry (f arachidonic acid

COX 1 COX 2

(c) cox 1 and cox 2 are different cox i cox 2

(c) cox 1 and cox 2 are different cox i cox 2

irreversible inhibition

(d) cox 2 selective agents bind in cox 2 pocket COX 1 COX 2

(b) Ncn specific NSAIDs bind to COX 1 and COX 2 to block entry (f arachidonic acid

COX 1 COX 2

(d) cox 2 selective agents bind in cox 2 pocket COX 1 COX 2

Figure 4.2. Prostaglandin synthesis and inhibition in COX-1 and COX-2. (a) Initial stages of prostaglandin synthesis, (b) Binding stages of standard NSAIDs to arginine 120 to inhibit prostaglandin synthesis by direct blockade of cyclo-oxygenase channel, (c) Differences between COX-1 and COX-2. id) Specific blockade of COX-2.

Figure 4.2. Prostaglandin synthesis and inhibition in COX-1 and COX-2. (a) Initial stages of prostaglandin synthesis, (b) Binding stages of standard NSAIDs to arginine 120 to inhibit prostaglandin synthesis by direct blockade of cyclo-oxygenase channel, (c) Differences between COX-1 and COX-2. id) Specific blockade of COX-2.

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