Discovery And Optimization Of Superactive Tsh Analogs

Protein engineering using recombinant DNA methods started in 1982, after the first results of oligonucleotide-directed mutagenesis had been published. Despite numerous site-directed mutagenesis studies, successful examples of engineering proteins with improved receptor binding affinity are quite rare. The first superactive analogs of hTSH with significant increases in receptor binding affinity, in vitro and in vivo bioactivity were constructed in 1994 (Table 1).

It was recognized previously that human TSH binding to TSH receptor is relatively a low-affinity interaction resulting in an extended 4-5 log unit competition curve for

Table 1. Chronology of superactive analog development and related projects

Year (Institution) Achievements/Studies

1994 (NIH) First TSH superactive analogs with single amino acid substitutions

1995 (NIH) Superactive analogs with combined substitutions in TSH and hCG

1996 (NIH) Initial superactive analogs ofLH and FSH

1996 (NIH) Biological activity of modified free-alpha subunit

1996-1998 (UMBI) Rescuing "loss of function" mutations

1996—2001 (UMBI) Studies on the mechanism of TSH receptor activation

TSH binding, with IC50 in high nanomolar range. However, Scatchard transformation of the equilibrium binding data produced a two-component curve which translates into a high and low affinity binding site, cellular bioassays always required low ionic strength buffer to achieve adequate sensitivity and reliability (Willey 1999). Although reasons for relatively low affinity of human TSH-TSH receptor interaction were not completely understood (Rommerts et al. 1992), in order to circumvent low affinity of human TSH all bindings studies were performed using bovine TSH. Interestingly, bovine and rat TSH were previously found 10-100 fold more potent than human TSH and such differences were observed at human and rodent TSH receptors, both in vitro and in vivo (Rapoport and Seto, 1985; East-Palmer et al. 1995). Thus, TSH from rodents was considered more bioactive than human TSH, but the exact mechanism of such differences were unknown. Some investigators attributed differences to variable purity, carbohydrate structures or other posttranslational modifications of pituitary TSH preparations.

Our studies on the role of carbohydrate residues in TSH bioactivity performed at NIH from 1990 to 1993 indicated that species- or production-dependent variability of TSH carbohydrate residues could not provide adequate explanation for observed differences in bioactivity between human TSH and TSH from various non-primate species (Szkudlinski et al. 1993). While exploring the role of carbohydrate residues by using subunit hybrids we observed that [(bovine alpha) - (human TSH beta)] het-erodimer is at least 10-fold more bioactive in vitro that human TSH or [(human alpha— bovine TSH beta)] heterodimer (Szkudlinski, Thotakura - unpublished data). These and other studies led Szkudlinski et al. in 1994 to substitute various amino acids in human alpha subunit according to the amino acid sequence of the bovine alpha subunit. After the first three "gain of activity" mutations were identified (Q13K, P16K, Q20K) more detailed sequence alignments, homology modeling and sequencing of subunit genes in various species, including primates, were performed. This resulted in an identification of additional targets within the sequence of both subunits including residue 14 in the alpha and 69 in the TSH beta subunit. Further development included combination of single mutations, alternative substitutions with arginines or histidines as well as studies on cooperative effects of different substitutions. hTSH with quadruple mutations in the a-subunit (Q13K+E14K+P16K+Q20K) and an additional replacement in the (L69R) showed 95-fold higher potency and more than

Figure 2. Superactive Analogs of TSH. Reprinted from Leitolf et al. (Leitolf et al. 2000). Gradual increase in the in vitro bioactivity (A) and receptor binding (B) for mutants with one. two and three engineered peripheral loops. Combination of the aL3 loop analog "with our previously optimized aL1-PL3 loop combination (Grossmann, et al. 1998) results in a further gain of hormone potency.

Figure 2. Superactive Analogs of TSH. Reprinted from Leitolf et al. (Leitolf et al. 2000). Gradual increase in the in vitro bioactivity (A) and receptor binding (B) for mutants with one. two and three engineered peripheral loops. Combination of the aL3 loop analog "with our previously optimized aL1-PL3 loop combination (Grossmann, et al. 1998) results in a further gain of hormone potency.

1.5-fold increase in efficacy compared to the in vitro bioactivity of the wild-type hormone (Szkudlinski et al. 1996). Moreover, the combination of these 4 mutations in the a-subunit with 3 mutations in the (3-subunit (I58R+E63R+L69R) resulted in an analog with greater than 1000-fold increase in receptor binding and in vitro bioactivity and 100-fold increase in the in vivo activity (Grossmann et al. 1998). Subsequently seven new site-specific "gain-of-activity" mutations were identified. Four in the ocL3 loop (S64K, N64K, G73K, A81K) and three in the (3L1 loop (Leitolf et al. 2000) (Figure 2). TSH analogs with optimized combinations of these substitutions are significantly more potent and efficacious than any known species of TSH and hold great promise as a second generation therapeutic forms of recombinant TSH. The relative increase in potency and efficacy (Vmax) of superactive analogs in comparison to the unmodified hormone are assay system dependent as illustrated in Figure 3. Because of the specious argument that increasing dose of the unmodified hormone should result in comparable to analog maximal effect, it is important to emphasize that such possibility is limited to extremely sensitive (rarely physiological) systems with high receptor number and/or highly efficient coupling.

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