Na busca por agonistas, antagonistas e inibidores de natureza peptídica do fator de crescimento de fibroblastos ácido humano (hFGF-1), iniciamos o presente trabalho fazendo uma análise conformacional teórica do peptídeo Ac- WFVGLKKNGSSKRGPRT-NH2 (107-123 [hFGF-1]). Em trabalho anterior, este composto havia se mostrado um agonista da atividade mitogênica da proteína capaz de inibir a ligação de 125I-hFGF-1 aos seus receptores celulares e de se ligar à resina heparina-Sepharose (Oyama et al. 1996). Os cálculos das propriedades dinâmicas deste peptídeo (I; Tabela 1) demonstraram que ele não adotava nenhuma conformação preferencial, o que poderia justificar a baixa atividade apresentada pelo mesmo (104 vêzes menor do que a da proteína nativa). Este peptídeo foi ressintetizado, purificado, caracterizado química e biologicamente, confirmando os resultados anteriores. Seu comportamento randômico foi comprovado experimentalmente através de uma análise estrutural parcial por 1H-RMN. O resultado desta análise demonstrou que este peptídeo exibe uma configuração random coil, em solução aquosa (Kiyota et al., 1996, 1999). Diante desta constatação e com base em dados descritos na literatura (Harper & Lobb 1988; Burgess et al., 1991; Pantoliano et al., 1994, Springer et al., 1994; Thompson et al., 1994; Imamura et al., 1995; Blaber et al., 1996; Ornitz et al., 1996; Zhu et al.,1991, 1995, 1997; Schwizer, 1995; Sieber & Moe, 1996; Rizo et al., 1996); desenhamos dezessete peptídeos relacionados ao Sítio 2 (97-132) do hFGF-1 mostrados na Tabela 1 (ver arquivo PDF). Todos os peptídeos foram sintetizados manualmente por síntese em fase sólida, sempre que possível purificados à homogeneidade por RP-HPLC e caracterizados quimicamente por RP-HPLC, análise de aminoácidos e espectrometria de massas. Os peptídeos cíclicos foram obtidos por: a) oxidação das sulfidrilas das cisteínas e formação de ponte dissulfeto intramolecular; b) reação entre os grupos amina e carboxila de cadeias laterais de dois diferentes resíduos de aminoácidos com formação de uma ligação lactama. Os testes de atividade mitogênica foram realizados sempre que os peptídeos eram obtidos com pureza ≥90% determinada por RP-HPLC analítica em dois sistemas diferentes de solventes. Os resultados obtidos em culturas de fibroblastos de camundongos Balb/c 3T3 mostraram que: 1) II, III, VI-IX e XIII eram inativos; 2) o cíclico IV era mitogênico (ED50 ~50 µM) ao contrário do seu análogo linear correspondente (III); 3) V, um análogo de IV que apresenta deleção de um resíduo (Asn), exibia uma atividade mitogênica menor do que a de IV; 4) X exibia uma atividade mitogênica menor do que a do I; 5) os cíclicos XII e XV exibiam atividades comparáveis a do I, enquanto que os seus análogos lineares correspondentes (XI e XIV) eram inativos; 6) XVI e XVII exibiam atividades mitogênicas também comparáveis à de I. Paralelamente a estes estudos, foi desenvolvido um modelo teórico dos domínios extracelulares DII e DIII do FGFR-1β. A partir do posicionamento gráfico das moléculas de hFGF-1 e de um hexassacarídeo de heparina junto a este modelo do receptor, foram desenhados os peptídeos Ac-170NTTDKENEVLH180-NH2 (XVIII) e Ac-194SLAGNSIGLSH204-NH2 (XIX) (Oyama et al., 1997). Estes dois peptídeos cujas seqüências primárias são, respectivamente, relacionadas àquelas dos loops DE e FG do domínio DIII, foram também sintetizados e testados neste trabalho como possíveis ligantes do sítio 2 do hFGF-1.Os testes biológicos demonstraram que o peptídeo XIX, na faixa de concentração testada, não exibia nenhuma atividade inibitória sobre as atividades mitogênicas dos FGFs -1 e -2. Por outro lado, notou-se um claro efeito inibitório de XVIII sobre a atividade mitogênica de ambas as proteínas, sendo este efeito mais significativo para o FGF-2 (Kiyota et al., 1998). Alguns dos peptídeos estudados foram submetidos a análises espectroscópicas com o objetivo de determinar suas conformações em solução. Este conhecimento forneceria subsídios para o desenho de novos peptídeos mitogênicos e, mais ainda, para determinação dos requisitos estruturais destes peptídeos e, como reflexo, do hFGF1 para expressão de suas atividades mitogênicas. Assim, foi feita uma análise parcial do peptídeo mitogênico IV e de seu análogo linear III inativo em solução aquosa empregando fluorescência e 1HRMN ( (Kiyota et al., 1998). Estes peptídeos foram analisados também por técnicas de CD e 1H-NMR (Kiyota et al., 1999). Os resultados obtidos mostraram que III e IV parece se organizarem de forma semelhante em suas porções N-terminais, em estruturas correspondentes a β-turns. Por outro lado, as conformações das porções C-terminais destes peptídeos diferiram; somente em IV, observouse a presença de uma família de confôrmeros com estruturas helicoidais nessa porção do esqueleto e que eram superponíveis. O mesmo não foi observado na porção C-terminal de III. A análise conformacional do peptídeo XVIII em solução foi também realizada empregando-se CD e 1H-RMN. Os resultados obtidos indicaram que este peptídeo tem forte tendência em assumir uma estrutura helicoidal em solução aquosa contendo 50% CD3OH. O conjunto dos resultados obtidos neste trabalho nos permitem concluir que: 1) a presença de W107 e F108 é, de fato, essencial para a expressão da atividade mitogênica de peptídeos derivados do sítio 2 do hFGF-1; 2) a presença de N106 adjacente ao core hidrofóbico constituído pelos resíduos W107F108 é importante; 3) a ciclização do peptídeo IV foi decisiva para a expressão de sua atividade, indicando que, não apenas a presença, mas o posicionamento adequado das cadeias laterais de N106, W107 e F108 são determinantes; 4) apenas uma das lisinas (K112 ou K113) é essencial para a atividade mitogênica (X, XVI e XVII); 5) o importante parece ser a manutenção do posicionamento das cadeias laterais em relação aos dos outros resíduos contidos na seqüência, uma vez que, a substituição do segmento 112KKNGS116 por uma prolina e/ou deleção de 120GPRT123 (XI e XIV) abolem a atividade mitogênica do peptídeo; 6) a ciclização que mantem a distância e as orientações relativas entre WF e KR (considerados essenciais para a atividade) em seqüência (XII e XV), leva à recuperação da atividade; 7) a atividade inibitória específica para o FGF-2 exibida pelo peptídeo XVIII parece ser um indicativo de que a alça DE do domínio DIII do receptor pode estar envolvido na ligação a esta proteína. Estas conclusões são relevantes e essenciais para: 1) o entendimento dos requisitos estruturais para a atividade mitogênica dos peptídeos estudados e, como reflexo, do hFGF-1, 2) o desenho de novos agonistas, antagonistas ou inibidores do sistema FGF. / In our search for small potent agonists or inhibitors related to hFGF-1(97-132), we first investigated the preferred conformation in solution of Ac -WFVGLKKNGSSKRGPRT-NH2 (I), by 1H-NMR. This compound has been described as a weak agonist of the mitogenic activity of this growth factor able to inhibit the binding of 125I-hFGF-1 to their cellular receptors, and to heparin-Sepharose columns (Oyama et al., 1996). We found that this peptide is in a random coil configuration, which could explain its low activity (104 times less potent than the native protein). On the basis on these results and on several data available in the literature (Harper & Lobb 1988; Burgess et al., 1991; Pantoliano et al., 1994, Springer et al., 1994; Thompson et al., 1994; Imamura et al., 1995; Ornitz et al., 1996; Blaber et al., 1996; Zhu et al., 1991, 1995; 1997; Schwizer, 1995; Sieber & Moe, 1996; Rizo et al., 1996), we designed seventeen peptides related to the Site 2 (97-132)[hFGF-1] listed on the Table 1: some were linear and some were cyclic. They were synthesized manually using the solid phase method, purified by RP-HPLC, and chemically characterized by RP-HPLC, amino acid analysis and mass spectrometry. Conformational constraints of certain peptides were achieved by cyclization. Intramolecular dissulfide bonds were formed by the oxidation of the thiol groups of two cysteins residues with air oxigen and/or K2Fe(CN)6. Lactama bonds were formed between the functional side chain group of acidic and basic residue. The synthetic peptides were tested in of their ability to inducing mitogenic response on Balb/c 3T3, A-31 clone fibroblasts cultures. The results obtained were the following: 1) peptides II, III, VI-IX were essentially inactive on Balb/c 3T3 fibroblasts in the range of concentrations used (up to 200 µM), 2) in the same range of concentration, peptide IV showed an ED50 60µM (similar to that found for peptide I) while its correspondent linear analog (III) was inactive; 3) V, analog of IV, that has Asn deleted, exibihited mitogenic activity lower than IV; 4) X showed a mitogenic activity on Balb/c fibroblasts lower than I, 5) cyclic peptides XII and XV showed mitogenic activities similar to that of I, while their correspondent linear (XI and XIV) and cyclic (XIII) analogs were inactive; 6) XVI and XVII showed mitogenic activities similar to that found for I. In parallel, two peptides [Ac-170NTTDKENEVLH180-NH2 (XVIII) and Ac-194SLAGNSIGLSH204-NH2 (XIX)], derived from DIII FGFR-1β and designed as putative ligands of Site2 hFGF-1, were synthesized and tested. In the range of concentration used (up to 200 µM), XIX was inactive and exhibited no inhibitory effect on FGF-1 and FGF-2 mitogenic activities. Nevertheless, XVIII inhibited the mitogenic activity of both proteins, being this effect clearly more significant for the FGF-2 (Kiyota et al., 1998). Some of synthetic peptides have been spectroscopically analyzed in order to disclose the structural features that characterize the active (Kiyota et al., 1999). A detailed analysis was undertaken with peptides III and IV using circular dichroism (CD) and 1H-NMR. Although the similarities in their primary sequences, III has shown inactive when tested on Balb/c 3T3 fibroblasts culture while IV was mitogenic with ED50 values around 50 µM. I was also not capable of inhibiting the binding of hFGF-1 to its cellular receptors, I was inactive while II inhibits it with ID50 values of about 30 µM. Circular dichroism study showed that while at increasing SDS concentrations the spectra of III suggested the presence of an equilibrium among partially structured states, those of IV indicated that this peptide exists in unordered extended conformation, folds into a β-conformation and, finally, assumes a helix rich structure. 1H-NMR analysis revealed the existence of a well defined γ-turn encompassing residues 4-6 that nicely fits with that present in the same portion of the crystallized hFGF-1. Superposition of the final structures of III and IV over the entire sequence revealed that only the C-terminal portion of III has the tendency to fold into a regular structure. Together these data indicate that the turn existence in IV allowed it to acquire the structural determinants for the expression of mitogenicity probably through a more appropriate arrangement of residues 8-10. More importantly, they demonstrate that we have found a short agonist of hFGF-1 able to structurally mimic its corresponding stretch. Conformational analysis of XVIII in solution was undertaken also by using CD e 1HRMN. The results obtained indicated that it has a strong tendency to assume helicoidal configuration in aqueous solution containing 50% CD3OH. Altogether these data led us to conclude that: 1) the presence of Asn106 adjacent to hydrophobic core constituted by W107F108 is essential for the mitogenic activity of IV; 2) conformation constraint by cyclization was efficient for the correct N106, W107 and F108 side chains orientation in peptide IV for an effective cellular receptors binding; 3) peptides related to hFGF-1 (114-123) seem to be promising mitogenic agonists; 4) the only one lysine between L126 and N129 is enough for the mitogenic activity expression (X, XVI and XVII); 5) deletions of residues, replacement of deleted fragment by Pro followed by restriction of peptide conformation might keep the frame of the residues considered essential for the mitogenic activity along the peptides backbones; 6)The inhibitory effect on the FGF-2 mitogenic activity observed in peptide XVIII was indicative that the loop DE of DIII FGFRs seems to be involved in the binding of this protein.These conclusions are very relevant in terms of the knowledge of the structural requirements for the mitogenic activity of studied peptides and, as a reflex, of the hFGF-1. Furthermore, they constitute additional guidelines for designing new constrained peptides derived from this segment of FGF-1, which may result in more potent agonists, antagonists or inhibitors of such important target.

Introdução: A falta de previsibilidade no tratamento periodontal regenerativo de defeito de furca grau III, têm estimulado o estudo de alternativas para melhorar os resultados, através do emprego de diferentes técnicas e biomateriais. Um novo enxerto ósseo enriquecido com peptídeos - Matriz óssea bovina inorgânica/P15 (PepGen P15) - foi desenvolvido recentemente e, segundo a literatura, mostrou resultados significantes em relação à neoformação tecidual nos defeitos infra-ósseos testados. Assim, o presente estudo teve como objetivo verificar o potencial regenerativo da matriz óssea inorgânica/P15 particulada, no tratamento de defeitos de furca grau III em cães associado ou não ao uso de membrana de PTFE-e Material e Métodos: Foram utilizados seis cães, nos quais defeitos de furca grau III nos pré-molares inferiores foram confeccionados, sendo que no grupo teste foi utilizado a membrana de PTFE-e e a matriz óssea inorgânica/P15, no grupo controle foi utilizada somente a membrana e no grupo controle negativo, nos 2° prémolares, não foi colocado biomaterial. Os defeitos foram confeccionados e preenchidos com material de impressão (Impregum F) e após 21 dias foram debridados, as raízes aplainadas com cureta Gracey (Hu-friedy) e os dentes submetidos à profilaxia semanal com ultra-som e limpeza diária com digluconato de clorexidina a 0,12% durante 15 dias. Na segunda fase cirúrgica, foram realizadas marcações do nível ósseo nas raízes mesial e distal dos dentes P2, P3 e P4, colocação das membranas e do enxerto ósseo nos respectivos defeitos. Os dentes P3 e P4 foram aleatoriamente escolhidos para ser o grupo teste ou controle. Quatro semanas após a colocação das membranas, estas foram retiradas e, doze semanas após a remoção, os animais foram sacrificados. Os dentes e seus tecidos periodontais de proteção e suporte foram removidos, fixados em formalina tamponada a 10%, descalcificados em ácido tricloroacético a 10%, desidratados e seccionados no plano mésio-distal em cortes semi-seriados com 7m de espessura cada. Os cortes foram corados com hematoxilina e eosina (HE) ou tricrômico de Mallory (TM) sendo selecionados para a análise histomorfométrica 5 cortes representativos da porção central da bifurcação. Foram realizadas medidas da área total da bifurcação (AT), área de novos tecidos formados (ANT), área de epitélio (AE), área de tecido conjuntivo (ATC), área de novo osso (ANO) e medidas lineares da extensão representativa da altura do defeito (ED), extensão do novo tecido extensão (ENT), extensão do novo osso (EO), extensão da bifurcação (EB) e extensão do novo cemento (EC). Resultados: A análise histológica demonstrou características morfológicas similares entre os grupos avaliados. Adicionalmente, o grupo teste apresentou grânulos do enxerto ósseo, envoltos por uma matriz óssea imatura, aprisionado entre os tecidos neoformados. Os resultados das médias das medidas lineares (mm) e das medidas de área (mm2) foram respectivamente os seguintes para o grupo teste: 14,11 ± 1,74 (EF) e 17,62 ± 2,39 (ATF); 8,61 ± 3,24 (EC) e 14,66 ± 3,73 (ANT); 4,71 ± 0,54 (ED) e 0,90 ± 0,80 (AE); 3,78 ± 0,85 (ENT) e 5,36 ± 2,41 (ATC); 1,77 ± 1,54 (EO) e 6,52 ± 5,69 (ANO); 4,82 ± 2,98 (DO). As seguintes médias lineares e de área para o grupo controle respectivamente: 13,19 ± 2,03 (EF) e 15,11 ± 3,29 (ATF); 8,52 ± 3,54 (EC) e 11,88 ± 2,09 (ANT); 4,59 ± 0,65 (ED) e 0,95 ± 0,71 (AE); 3,54 ± 0,61 (ENT) e 5,19 ± 2,17 (ATC); 1,64 ± 1,06 (EO) e 4,17 ± 3,40 (ANO); 2,58 ± 1,71 (DO). E as seguintes médias lineares e de área para o grupo controle negativo respectivamente: 13,54 ± 1,41 (EF) e 14,99 ± 3,13 (ATF); 6,56 ± 2,11 (EC) e 11,13 ± 3,25 (ANT); 4,62 ± 0,47 (ED) e 0,95 ± 0,78 (AE); 3,59 ± 0,28 (ENT) e 5,89 ± 0,87 (ATC); 0,98 ± 0,48 (EO) e 2,88 ± 2,49 (ANO); 2,35 ± 2,00 (DO). A análise estatística dos dados, realizada através da aplicação do teste de Friedman Test (< 0,05), demonstrou haver diferenças estatisticamente significantes entre o grupo teste e controle negativo no parâmetro referente à ANO. Conclusão: Pode-se concluir que a matriz óssea inorgânica/P15 apresentou resultados semelhantes aos outros grupos em relação à regeneração periodontal do defeito. No entanto, quando a matriz óssea permaneceu no defeito, apresentou resultados satisfatórios através da formação óssea que circunscreveu as partículas. / Background: The aim of this study was to verify the regenerative potential of particulate ABM/Synthetic Peptide in class III furcation defects associated or not with ePTFE membranes. Material and Methods: Six dogs were used and class III furcation defects were produced in the lower pre-molars and filled with impression material. After 21 days, the membranes and the bone grafts were inserted and P3 and P4 were randomized to form the test and control groups. P2 was the negative control. The animals were sacrificed 3 months post-treatment. Results: Comparisons between groups by the Friedman Test (<0.05) showed statistically significant differences between the test and negative control groups, based on NBA parameter. In the negative control group, a new bone area (NBA) of 20% was observed. In the test group, a new bone area of 37% was observed. There was no statistically significant difference between the groups to the others parameters. Conclusions: The statistical analysis using Friedman Test (<0.05) showed statistically significant differences between the test and negative control groups, based on NBA parameter. Furthermore, when the Inorganic Bone Matrix/P15 remained inside the defects, it could be clearly observed that new bone formation not only circumscribed the particles but formed above the level of the particles.

Na busca por agonistas, antagonistas e inibidores de natureza peptídica do fator de crescimento de fibroblastos ácido humano (hFGF-1), iniciamos o presente trabalho fazendo uma análise conformacional teórica do peptídeo Ac- WFVGLKKNGSSKRGPRT-NH2 (107-123 [hFGF-1]). Em trabalho anterior, este composto havia se mostrado um agonista da atividade mitogênica da proteína capaz de inibir a ligação de 125I-hFGF-1 aos seus receptores celulares e de se ligar à resina heparina-Sepharose (Oyama et al. 1996). Os cálculos das propriedades dinâmicas deste peptídeo (I; Tabela 1) demonstraram que ele não adotava nenhuma conformação preferencial, o que poderia justificar a baixa atividade apresentada pelo mesmo (104 vêzes menor do que a da proteína nativa). Este peptídeo foi ressintetizado, purificado, caracterizado química e biologicamente, confirmando os resultados anteriores. Seu comportamento randômico foi comprovado experimentalmente através de uma análise estrutural parcial por 1H-RMN. O resultado desta análise demonstrou que este peptídeo exibe uma configuração random coil, em solução aquosa (Kiyota et al., 1996, 1999). Diante desta constatação e com base em dados descritos na literatura (Harper & Lobb 1988; Burgess et al., 1991; Pantoliano et al., 1994, Springer et al., 1994; Thompson et al., 1994; Imamura et al., 1995; Blaber et al., 1996; Ornitz et al., 1996; Zhu et al.,1991, 1995, 1997; Schwizer, 1995; Sieber & Moe, 1996; Rizo et al., 1996); desenhamos dezessete peptídeos relacionados ao Sítio 2 (97-132) do hFGF-1 mostrados na Tabela 1 (ver arquivo PDF). Todos os peptídeos foram sintetizados manualmente por síntese em fase sólida, sempre que possível purificados à homogeneidade por RP-HPLC e caracterizados quimicamente por RP-HPLC, análise de aminoácidos e espectrometria de massas. Os peptídeos cíclicos foram obtidos por: a) oxidação das sulfidrilas das cisteínas e formação de ponte dissulfeto intramolecular; b) reação entre os grupos amina e carboxila de cadeias laterais de dois diferentes resíduos de aminoácidos com formação de uma ligação lactama. Os testes de atividade mitogênica foram realizados sempre que os peptídeos eram obtidos com pureza &#8805;90% determinada por RP-HPLC analítica em dois sistemas diferentes de solventes. Os resultados obtidos em culturas de fibroblastos de camundongos Balb/c 3T3 mostraram que: 1) II, III, VI-IX e XIII eram inativos; 2) o cíclico IV era mitogênico (ED50 ~50 &#181;M) ao contrário do seu análogo linear correspondente (III); 3) V, um análogo de IV que apresenta deleção de um resíduo (Asn), exibia uma atividade mitogênica menor do que a de IV; 4) X exibia uma atividade mitogênica menor do que a do I; 5) os cíclicos XII e XV exibiam atividades comparáveis a do I, enquanto que os seus análogos lineares correspondentes (XI e XIV) eram inativos; 6) XVI e XVII exibiam atividades mitogênicas também comparáveis à de I. Paralelamente a estes estudos, foi desenvolvido um modelo teórico dos domínios extracelulares DII e DIII do FGFR-1&#946;. A partir do posicionamento gráfico das moléculas de hFGF-1 e de um hexassacarídeo de heparina junto a este modelo do receptor, foram desenhados os peptídeos Ac-170NTTDKENEVLH180-NH2 (XVIII) e Ac-194SLAGNSIGLSH204-NH2 (XIX) (Oyama et al., 1997). Estes dois peptídeos cujas seqüências primárias são, respectivamente, relacionadas àquelas dos loops DE e FG do domínio DIII, foram também sintetizados e testados neste trabalho como possíveis ligantes do sítio 2 do hFGF-1.Os testes biológicos demonstraram que o peptídeo XIX, na faixa de concentração testada, não exibia nenhuma atividade inibitória sobre as atividades mitogênicas dos FGFs -1 e -2. Por outro lado, notou-se um claro efeito inibitório de XVIII sobre a atividade mitogênica de ambas as proteínas, sendo este efeito mais significativo para o FGF-2 (Kiyota et al., 1998). Alguns dos peptídeos estudados foram submetidos a análises espectroscópicas com o objetivo de determinar suas conformações em solução. Este conhecimento forneceria subsídios para o desenho de novos peptídeos mitogênicos e, mais ainda, para determinação dos requisitos estruturais destes peptídeos e, como reflexo, do hFGF1 para expressão de suas atividades mitogênicas. Assim, foi feita uma análise parcial do peptídeo mitogênico IV e de seu análogo linear III inativo em solução aquosa empregando fluorescência e 1HRMN ( (Kiyota et al., 1998). Estes peptídeos foram analisados também por técnicas de CD e 1H-NMR (Kiyota et al., 1999). Os resultados obtidos mostraram que III e IV parece se organizarem de forma semelhante em suas porções N-terminais, em estruturas correspondentes a &#946;-turns. Por outro lado, as conformações das porções C-terminais destes peptídeos diferiram; somente em IV, observouse a presença de uma família de confôrmeros com estruturas helicoidais nessa porção do esqueleto e que eram superponíveis. O mesmo não foi observado na porção C-terminal de III. A análise conformacional do peptídeo XVIII em solução foi também realizada empregando-se CD e 1H-RMN. Os resultados obtidos indicaram que este peptídeo tem forte tendência em assumir uma estrutura helicoidal em solução aquosa contendo 50% CD3OH. O conjunto dos resultados obtidos neste trabalho nos permitem concluir que: 1) a presença de W107 e F108 é, de fato, essencial para a expressão da atividade mitogênica de peptídeos derivados do sítio 2 do hFGF-1; 2) a presença de N106 adjacente ao core hidrofóbico constituído pelos resíduos W107F108 é importante; 3) a ciclização do peptídeo IV foi decisiva para a expressão de sua atividade, indicando que, não apenas a presença, mas o posicionamento adequado das cadeias laterais de N106, W107 e F108 são determinantes; 4) apenas uma das lisinas (K112 ou K113) é essencial para a atividade mitogênica (X, XVI e XVII); 5) o importante parece ser a manutenção do posicionamento das cadeias laterais em relação aos dos outros resíduos contidos na seqüência, uma vez que, a substituição do segmento 112KKNGS116 por uma prolina e/ou deleção de 120GPRT123 (XI e XIV) abolem a atividade mitogênica do peptídeo; 6) a ciclização que mantem a distância e as orientações relativas entre WF e KR (considerados essenciais para a atividade) em seqüência (XII e XV), leva à recuperação da atividade; 7) a atividade inibitória específica para o FGF-2 exibida pelo peptídeo XVIII parece ser um indicativo de que a alça DE do domínio DIII do receptor pode estar envolvido na ligação a esta proteína. Estas conclusões são relevantes e essenciais para: 1) o entendimento dos requisitos estruturais para a atividade mitogênica dos peptídeos estudados e, como reflexo, do hFGF-1, 2) o desenho de novos agonistas, antagonistas ou inibidores do sistema FGF. / In our search for small potent agonists or inhibitors related to hFGF-1(97-132), we first investigated the preferred conformation in solution of Ac -WFVGLKKNGSSKRGPRT-NH2 (I), by 1H-NMR. This compound has been described as a weak agonist of the mitogenic activity of this growth factor able to inhibit the binding of 125I-hFGF-1 to their cellular receptors, and to heparin-Sepharose columns (Oyama et al., 1996). We found that this peptide is in a random coil configuration, which could explain its low activity (104 times less potent than the native protein). On the basis on these results and on several data available in the literature (Harper & Lobb 1988; Burgess et al., 1991; Pantoliano et al., 1994, Springer et al., 1994; Thompson et al., 1994; Imamura et al., 1995; Ornitz et al., 1996; Blaber et al., 1996; Zhu et al., 1991, 1995; 1997; Schwizer, 1995; Sieber & Moe, 1996; Rizo et al., 1996), we designed seventeen peptides related to the Site 2 (97-132)[hFGF-1] listed on the Table 1: some were linear and some were cyclic. They were synthesized manually using the solid phase method, purified by RP-HPLC, and chemically characterized by RP-HPLC, amino acid analysis and mass spectrometry. Conformational constraints of certain peptides were achieved by cyclization. Intramolecular dissulfide bonds were formed by the oxidation of the thiol groups of two cysteins residues with air oxigen and/or K2Fe(CN)6. Lactama bonds were formed between the functional side chain group of acidic and basic residue. The synthetic peptides were tested in of their ability to inducing mitogenic response on Balb/c 3T3, A-31 clone fibroblasts cultures. The results obtained were the following: 1) peptides II, III, VI-IX were essentially inactive on Balb/c 3T3 fibroblasts in the range of concentrations used (up to 200 &#181;M), 2) in the same range of concentration, peptide IV showed an ED50 60&#181;M (similar to that found for peptide I) while its correspondent linear analog (III) was inactive; 3) V, analog of IV, that has Asn deleted, exibihited mitogenic activity lower than IV; 4) X showed a mitogenic activity on Balb/c fibroblasts lower than I, 5) cyclic peptides XII and XV showed mitogenic activities similar to that of I, while their correspondent linear (XI and XIV) and cyclic (XIII) analogs were inactive; 6) XVI and XVII showed mitogenic activities similar to that found for I. In parallel, two peptides [Ac-170NTTDKENEVLH180-NH2 (XVIII) and Ac-194SLAGNSIGLSH204-NH2 (XIX)], derived from DIII FGFR-1&#946; and designed as putative ligands of Site2 hFGF-1, were synthesized and tested. In the range of concentration used (up to 200 &#181;M), XIX was inactive and exhibited no inhibitory effect on FGF-1 and FGF-2 mitogenic activities. Nevertheless, XVIII inhibited the mitogenic activity of both proteins, being this effect clearly more significant for the FGF-2 (Kiyota et al., 1998). Some of synthetic peptides have been spectroscopically analyzed in order to disclose the structural features that characterize the active (Kiyota et al., 1999). A detailed analysis was undertaken with peptides III and IV using circular dichroism (CD) and 1H-NMR. Although the similarities in their primary sequences, III has shown inactive when tested on Balb/c 3T3 fibroblasts culture while IV was mitogenic with ED50 values around 50 &#181;M. I was also not capable of inhibiting the binding of hFGF-1 to its cellular receptors, I was inactive while II inhibits it with ID50 values of about 30 &#181;M. Circular dichroism study showed that while at increasing SDS concentrations the spectra of III suggested the presence of an equilibrium among partially structured states, those of IV indicated that this peptide exists in unordered extended conformation, folds into a &#946;-conformation and, finally, assumes a helix rich structure. 1H-NMR analysis revealed the existence of a well defined &#947;-turn encompassing residues 4-6 that nicely fits with that present in the same portion of the crystallized hFGF-1. Superposition of the final structures of III and IV over the entire sequence revealed that only the C-terminal portion of III has the tendency to fold into a regular structure. Together these data indicate that the turn existence in IV allowed it to acquire the structural determinants for the expression of mitogenicity probably through a more appropriate arrangement of residues 8-10. More importantly, they demonstrate that we have found a short agonist of hFGF-1 able to structurally mimic its corresponding stretch. Conformational analysis of XVIII in solution was undertaken also by using CD e 1HRMN. The results obtained indicated that it has a strong tendency to assume helicoidal configuration in aqueous solution containing 50% CD3OH. Altogether these data led us to conclude that: 1) the presence of Asn106 adjacent to hydrophobic core constituted by W107F108 is essential for the mitogenic activity of IV; 2) conformation constraint by cyclization was efficient for the correct N106, W107 and F108 side chains orientation in peptide IV for an effective cellular receptors binding; 3) peptides related to hFGF-1 (114-123) seem to be promising mitogenic agonists; 4) the only one lysine between L126 and N129 is enough for the mitogenic activity expression (X, XVI and XVII); 5) deletions of residues, replacement of deleted fragment by Pro followed by restriction of peptide conformation might keep the frame of the residues considered essential for the mitogenic activity along the peptides backbones; 6)The inhibitory effect on the FGF-2 mitogenic activity observed in peptide XVIII was indicative that the loop DE of DIII FGFRs seems to be involved in the binding of this protein.These conclusions are very relevant in terms of the knowledge of the structural requirements for the mitogenic activity of studied peptides and, as a reflex, of the hFGF-1. Furthermore, they constitute additional guidelines for designing new constrained peptides derived from this segment of FGF-1, which may result in more potent agonists, antagonists or inhibitors of such important target.

Introdução: A falta de previsibilidade no tratamento periodontal regenerativo de defeito de furca grau III, têm estimulado o estudo de alternativas para melhorar os resultados, através do emprego de diferentes técnicas e biomateriais. Um novo enxerto ósseo enriquecido com peptídeos - Matriz óssea bovina inorgânica/P15 (PepGen P15) - foi desenvolvido recentemente e, segundo a literatura, mostrou resultados significantes em relação à neoformação tecidual nos defeitos infra-ósseos testados. Assim, o presente estudo teve como objetivo verificar o potencial regenerativo da matriz óssea inorgânica/P15 particulada, no tratamento de defeitos de furca grau III em cães associado ou não ao uso de membrana de PTFE-e Material e Métodos: Foram utilizados seis cães, nos quais defeitos de furca grau III nos pré-molares inferiores foram confeccionados, sendo que no grupo teste foi utilizado a membrana de PTFE-e e a matriz óssea inorgânica/P15, no grupo controle foi utilizada somente a membrana e no grupo controle negativo, nos 2° prémolares, não foi colocado biomaterial. Os defeitos foram confeccionados e preenchidos com material de impressão (Impregum F) e após 21 dias foram debridados, as raízes aplainadas com cureta Gracey (Hu-friedy) e os dentes submetidos à profilaxia semanal com ultra-som e limpeza diária com digluconato de clorexidina a 0,12% durante 15 dias. Na segunda fase cirúrgica, foram realizadas marcações do nível ósseo nas raízes mesial e distal dos dentes P2, P3 e P4, colocação das membranas e do enxerto ósseo nos respectivos defeitos. Os dentes P3 e P4 foram aleatoriamente escolhidos para ser o grupo teste ou controle. Quatro semanas após a colocação das membranas, estas foram retiradas e, doze semanas após a remoção, os animais foram sacrificados. Os dentes e seus tecidos periodontais de proteção e suporte foram removidos, fixados em formalina tamponada a 10%, descalcificados em ácido tricloroacético a 10%, desidratados e seccionados no plano mésio-distal em cortes semi-seriados com 7m de espessura cada. Os cortes foram corados com hematoxilina e eosina (HE) ou tricrômico de Mallory (TM) sendo selecionados para a análise histomorfométrica 5 cortes representativos da porção central da bifurcação. Foram realizadas medidas da área total da bifurcação (AT), área de novos tecidos formados (ANT), área de epitélio (AE), área de tecido conjuntivo (ATC), área de novo osso (ANO) e medidas lineares da extensão representativa da altura do defeito (ED), extensão do novo tecido extensão (ENT), extensão do novo osso (EO), extensão da bifurcação (EB) e extensão do novo cemento (EC). Resultados: A análise histológica demonstrou características morfológicas similares entre os grupos avaliados. Adicionalmente, o grupo teste apresentou grânulos do enxerto ósseo, envoltos por uma matriz óssea imatura, aprisionado entre os tecidos neoformados. Os resultados das médias das medidas lineares (mm) e das medidas de área (mm2) foram respectivamente os seguintes para o grupo teste: 14,11 ± 1,74 (EF) e 17,62 ± 2,39 (ATF); 8,61 ± 3,24 (EC) e 14,66 ± 3,73 (ANT); 4,71 ± 0,54 (ED) e 0,90 ± 0,80 (AE); 3,78 ± 0,85 (ENT) e 5,36 ± 2,41 (ATC); 1,77 ± 1,54 (EO) e 6,52 ± 5,69 (ANO); 4,82 ± 2,98 (DO). As seguintes médias lineares e de área para o grupo controle respectivamente: 13,19 ± 2,03 (EF) e 15,11 ± 3,29 (ATF); 8,52 ± 3,54 (EC) e 11,88 ± 2,09 (ANT); 4,59 ± 0,65 (ED) e 0,95 ± 0,71 (AE); 3,54 ± 0,61 (ENT) e 5,19 ± 2,17 (ATC); 1,64 ± 1,06 (EO) e 4,17 ± 3,40 (ANO); 2,58 ± 1,71 (DO). E as seguintes médias lineares e de área para o grupo controle negativo respectivamente: 13,54 ± 1,41 (EF) e 14,99 ± 3,13 (ATF); 6,56 ± 2,11 (EC) e 11,13 ± 3,25 (ANT); 4,62 ± 0,47 (ED) e 0,95 ± 0,78 (AE); 3,59 ± 0,28 (ENT) e 5,89 ± 0,87 (ATC); 0,98 ± 0,48 (EO) e 2,88 ± 2,49 (ANO); 2,35 ± 2,00 (DO). A análise estatística dos dados, realizada através da aplicação do teste de Friedman Test (< 0,05), demonstrou haver diferenças estatisticamente significantes entre o grupo teste e controle negativo no parâmetro referente à ANO. Conclusão: Pode-se concluir que a matriz óssea inorgânica/P15 apresentou resultados semelhantes aos outros grupos em relação à regeneração periodontal do defeito. No entanto, quando a matriz óssea permaneceu no defeito, apresentou resultados satisfatórios através da formação óssea que circunscreveu as partículas. / Background: The aim of this study was to verify the regenerative potential of particulate ABM/Synthetic Peptide in class III furcation defects associated or not with ePTFE membranes. Material and Methods: Six dogs were used and class III furcation defects were produced in the lower pre-molars and filled with impression material. After 21 days, the membranes and the bone grafts were inserted and P3 and P4 were randomized to form the test and control groups. P2 was the negative control. The animals were sacrificed 3 months post-treatment. Results: Comparisons between groups by the Friedman Test (<0.05) showed statistically significant differences between the test and negative control groups, based on NBA parameter. In the negative control group, a new bone area (NBA) of 20% was observed. In the test group, a new bone area of 37% was observed. There was no statistically significant difference between the groups to the others parameters. Conclusions: The statistical analysis using Friedman Test (<0.05) showed statistically significant differences between the test and negative control groups, based on NBA parameter. Furthermore, when the Inorganic Bone Matrix/P15 remained inside the defects, it could be clearly observed that new bone formation not only circumscribed the particles but formed above the level of the particles.

La détection de lymphocytes T (LT) CD8+ infiltrant les tumeurs (TIL), est généralement associée à un bon pronostic chez des patients atteints du cancer. A l’inverse, l'infiltration des tumeurs par des LT CD4+ régulateurs (Treg), est quant à elle, souvent corrélée à un mauvais pronostic. Plusieurs vaccins « thérapeutiques » capables d’induire des réponses T CD8+ spécifiques d’antigènes tumoraux ont été développés. Cependant, à ce jour, les résultats cliniques obtenus par ces vaccins restent décevants. Dans un premier travail, nous avons développé puis testé un vaccin thérapeutique composé de trois longs peptides synthétiques (LSP) dérivés de la protéine survivine (SVX). La survivine est une protéine surexprimée dans la majorité des cancers humains, mais absente dans les tissus adultes sains, ce qui fait d’elle une cible thérapeutique privilégiée pour les vaccins anti-tumoraux. Nous avons démontré l'efficacité thérapeutique élevée du vaccin SVX contre diverses tumeurs murines. Ceci a été associé à l'induction de réponses T spécifiques de la survivine, un prolongement de la survie et la génération de réponses mémoires antitumorales. De plus, SVX a induit à la fois des LT CD8+ cytotoxiques spécifiques et LT CD4+ auxiliaires de type 1 multifonctionnelles, dans la rate et au sein des tumeurs. Il a aussi induit une diminution des Treg, favorisant ainsi une réponse immunitaire très efficace. Enfin, une étude préliminaire menée chez des patients atteints de différents types de cancers, nous a révélé la présence de taux élevés de précurseurs spontanés de LT spécifiques du vaccin SVX. Ces résultats suggèrent que SVX pourrait potentiellement stimuler l'activation de ces précurseurs spécifiques. Ces résultats constituent une preuve de concept pour amener ce vaccin comme un produit de première génération en essai clinique chez l’homme. Dans le but d’étudier plus en détails la cinétique des réponses immunitaires T spécifiques, associées aux vaccins LSP, nous avons étudié dans un second travail, l’efficacité d’un vaccin LSP dérivé de la protéine Ovalbumine (OVA). Nous avons montré dans plusieurs modèles de tumeurs murines, que la combinaison du vaccin LSP-OVA à un adjuvant approprié, induisait une forte régression de la croissance tumorale, l’expansion des cellules spécifiques T CD4+ et T CD8+ dans les organes lymphoïdes, ainsi que leur migration vers les tumeurs. De plus, le vaccin a induit des cellules T spécifiques fonctionnelles, comme le témoignent les niveaux élevés de cytokines cytotoxiques mesurées. De manière intéressante, le vaccin n’a pas induit de Treg spécifiques d’OVA ou de Treg polyclonaux et ce, malgré la présence de la tumeur. Enfin, lorsque LSP-OVA ne parvenait pas à induire une régression complète de la tumeur, celle-ci était infiltrée par des TIL CD4+ conventionnels et CD8+ exprimant fortement les récepteurs inhibiteurs (PD-1, TIM-3 et TIGIT). Nos résultats suggèrent que pour optimiser ce vaccin LSP, une association à des anticorps bloquant un ou plusieurs de ces récepteurs inhibiteurs, devrait être envisagée. / The presence of tumor-infiltrating CD8+ T lymphocytes (TIL) is generally associated with a good prognosis in cancer patients. Conversely, the infiltration of tumors by CD4+ regulators T cells (Treg), is often associated with poor prognosis. Several "therapeutic" vaccines able to induce tumor-specific CD8+ T cell responses have been developed. However, to date, the clinical results of these vaccines remain insufficient. In a first work, we developed and analyzed the immunogenicity and therapeutic efficacy of a new survivin vaccine (SVX) composed of three long synthetic peptides (LSP) containing several CD4 and CD8 T-cell epitopes. Survivin is over-expressed by most human cancers, but absent in healthy adult tissues, making it an interesting therapeutic target for cancer vaccines. We demonstrated the high therapeutic efficacy of SVX vaccine against various established murine tumor models, associated with its capacity to generate both specific cytotoxic CD8+ and multifunctional Th1 CD4+ T-cell responses but also effective memory T-cell responses for long-term protection against relapses. Treatment with SVX vaccine was also found to strongly increase the tumor infiltration of both CD4+ and CD8+ T cells over Treg cells therefore tipping the balance toward a highly efficient immune response. Finally, a preliminary study in patients with different types of cancer revealed the presence of high levels of SVX-specific spontaneous T-cell precursors. This suggests that SVX can potentially stimulate the activation of these specific precursors. Altogether, our results strongly suggest that SVX is a promising cancer vaccine and warrants its further clinical development. In order to study the kinetics of tumor-specific immune responses associated with LSP vaccines, we studied the efficacy of a LSP vaccine derived from the Ovalbumin (OVA) protein. We showed in two tumor models that the combination of LSP-OVA with a suitable adjuvant induced a strong tumor regression, an important expansion of both OVA-specific CD4+ and CD8+ T cells in the lymphoid organs, as well as their migration to the tumor. In addition, the vaccine induced functional specific T cells, as shown by the high levels of cytotoxic cytokines. Interestingly, the vaccine did not induce either OVA-specific or polyclonal Treg, despite the presence of the tumor. Finally, when LSP-OVA failed to induce a complete depletion of the tumor, we observed an important expression of inhibitory receptors (PD-1, TIM-3 and TIGIT) on conventional CD4+ and CD8+ TIL. Our results suggest that to optimize this LSP vaccine, a combination with one or more immune checkpoint blockade agents should be considered.

Investigations detailed in this thesis constitute a part of the continuing programme of research undertaken in our laboratory on the riboflavin carrier protein (RCP) with partic­ular reference to identification and synthesis of neutralizing antigenic determinants, design of relevant epitope mimetics with improved immunogenic characteristics and relationship between their secondary structures and immunological properties. The riboflavin carrier protein is elaborated as a reproductive stratagem to ensure ade­quate vitamin deposition in the developing oocyte in the chickens. The protein is scrupu­lously conserved through evolution in terms of physico chemical and immunological char­acteristics from fish through birds to mammals, including primates. In rodents and sub­human primates immunization with the heteroantigen viz., chicken egg white RCP leads to functional neutralization of the endogenous maternal protein resulting in curtailment of early pregnancy. Thus, the crucial role of RCP in maintenance of pregnancy is established and the protein identified as a potential candidate vaccine for immimocontraception. Fur­ther studies with the reduced and carboxymethylated (RCM) RCP as the immunogen re­veal that antibodies induced by RCM-RCP are equally effective in bioneutralization of the endogenous protein. So it can be surmised that the native folded structure of RCP is not obligatory for eliciting bioneutralizing antibodies. In an attempt to identify functionally relevant regions of the protein, a panel of monoclonal antibodies (MAbs) have been raised and characterized. One of the MAbs viz., 6J32Ci2 could bring about early fetal resorp-tion when injected to mice with confirmed pregnancies. These results prompted a detail molecular immunological approach to understand underlying mechanisms. The principal aims of the present investigations include: (1) identification of neutralizing epitopes; (2) synthesis of peptidyl sequences incorporating these determinants; (3) an understanding of the structure, antigenic and immunogenic characteristics of these peptides; (4) correlation of conformational and antigenic characteristics; (5) rational design and synthesis of peptide analogs with greater propensity to assume predicted secondary structures; (6) analysis of conformation dependency of peptide antigens and the importance of such conformation in generating an optimal B-cell response; (7) the efficacy of the antibodies elicited by these Peptide antigens in neutralizing endogenous protein with the ultimate aim of designing synthetic vaccines. Chapter 1 of this thesis deals with a general introduction summarizing the current status of knowledge regarding the chemistry and biology of RCP as well as synthetic pep­tides as potential immunogens. Chapter 2 outlines details of the experimental procedures adopted. Chapter 3 describes the results of investigations on the C-terminal fragment (residues 200-219) of cRCR The main consideration in selecting this sequence for the design of a potential peptide-based vaccine relied on the epitopic specificity of the neu­tralizing MAb 6S2C12. Epitope mapping using the Pepscan method revealed that the monoclonal antibody recognizes a core sequence corresponding to residues 203-210 of the cRCP. A 21-residue synthetic peptide (C-21) comprising this epitope was synthesized and antibodies elicited to the peptide conjugated to two different carriers, namely diphtheria toxoid and purified protein derivative (PPD) for T-cell help. In both active and passive immunoneutralization experiments, the peptide specific neutralizing antibodies interfered with the biological function of the protein and hence either protected from pregnancy or caused early fetal resorption in rodents as well as in sub-human primates. The conforma-tional properties of the peptide in aqueous buffers were analyzed from circular dichroism which revealed the absence of any ordered structure in the native C-21 peptide. Theoreti­cal predictions of secondary structure suggested a propensity for an t*-helical structure for this fragment in the native protein. Therefore, influence of the helix-promoting solvent, vizM 2,2,2,trifluroethanol (TFE) on the C-21 peptide was investigated. Addition of TFE resulted in spectral changes with negative bands at 208 and 222 nm and a positive band at 190 rim which are typical of an a-helix. To gain more information on the conformational characteristics of this peptide, it was considered worthwhile to stabilize the native peptide in an a-helical conformation based on simple rational design principles. Towards this end, four analogs of the parent peptide were synthesized and helix stabilization was sought to be achieved by introducing either salt bridges or back-bone conformational constraints such as by incorporating a-amino isobutyric acid at appropriate positions. In all the analogs, the core sequence, recognised by the neutralizing MAb 6B2C12 was maintained intact to ensure induction of antibodies capable of recognizing the native protein. CD spectral analysis of the analog peptides indicated that all the engineered peptides had varying degrees of enhanced helicities as compared to the parent peptide. The immunogenicity of each analog was studied by to the relevant peptide-diphtheria toxoid conjugates and analyzing their reactivities with the native protein by direct and competitive ELISA. The results revealed that these engi­neered conformational analogs axe highly immunogenic eliciting high titers of anti-protein antibodies. The relative affinities of these antibodies to bind cRCP were investigated. The antibodies to peptide analogs had higher affinities for the native protein and a positive correlation was found between the helical content of the peptide antigen in question and the relative affinity of corresponding antibody. The antibodies directed to all the peptide analogs could block the function of RCP resulting in early embryonic resorption when ad­ministered to pregnant mice. An interesting pattern of immunological cross-reactivity has been observed with the native and designed peptides. Antibodies raised to constrained helical analogs could bind the C-21 peptide which is structurally flexible. In contrast, the antibodies raised to the flexible native peptide antigen were inefficient in recognizing the structured peptides. The ability of all the peptide antibody to bind the native protein has been interpreted in terms of a conformationally flexible C-terminus region in cRCP. Chapter 4 details investigations on a 21-residue peptide (N- 21) from the N-terminiis (4-24) of the protein. Selection of this peptidyl sequence relied on theoretical prediction of potential sequential determinants on RCP other than at C-terminus as well as on the outcome of immunoneutralisation experiments using antibodies to egg yolk RCP which lacks the relevant C-terminal determinants. The structure of this peptide in solution was analyzed by two dimensional NMR and CD. NMR experiments revealed the presence of two structured regions in the peptide. Diagnostic nuclear Overhauser effects characteristics of reverse turns or short frayed helical segments over residues 3-9 and 18-21 of the peptide were obtained. CD spectra showed the presence of a strong, negative band at 204 nm over a wide range of solvent conditions, a feature which has been interpreted in terms of a "polyproline Il-like" segment encompassing residues 11-16 which corresponds to an interesting (X-Pro)^ repeat in the N-21 sequence. Specific antibodies were generated to this peptide as a conjugate with diphtheria tox­oid. Administration of the antipeptide antibodies could neutralize the protein in vivo as demonstrated by early embryonic loss in pregnant mice. In limited experiments the anti­peptide antibodies showed propensity to protect bonnet monkeys from pregnancy over a few consecutive ovulatory cycles when titres are maintained elevated by periodic boosting. To address the relationship between peptide structures and antigenicity, epitope mapping of this antipeptide antibodies as well- as the polyclonal antibodies to native RCP was undertaken using the Pepscan method. The results reveal that antigenic regions correspond well to conformationally well-defined elements of structure with the polyproline II-like seg­ment being a common antigenic determinant on both the peptide and the native protein. These observations are suggestive of the involvement of both the N and C-terminal regions of RCP in terms of its binding to putative plasma membrane receptors.

Synthetic construction of protein molecules has been widely pursued over the last two decades. A primary goal behind de novo protein design has been to build minimal systems by capturing the essential features of protein structures. Such minimal models can be used to understand underlying principles governing folding, structure, and function of proteins molecules. Several approaches envisioning successful construction of synthetic proteins have been described over the years, some of them being admirably successful (DeGrado et al, 1999; Richardson et al> 1992; Baltzer, 1998). Specific patterning of polar and apolar residues in synthetic sequences has been widely used to achieve designed polypeptide structures like helix bundles (DeGrado et ah, 1999) and (3-sheets (Smith and Regan, 1997; Lacroix et a/., 1998), with reliance on hydrophobic driving forces for folding. Our laboratory has been pursuing a distinctly alternative approach, that employs stereochemically constrained amino acids to generate specific secondary structures which can then be assembled into composite structures by appropriately chosen linking segments. This approach, which involves linking prefabricated modules of secondary structures can be termed as a "Meccano set" approach to protein design (Balaram, 1992). The studies embodied in the present thesis describe attempts at construction of synthetic polypeptide motifs using the stereochemically directing influence of conformationally constrained amino acid residues, such as DPro or Aib (α-aminoisobutyric acid). This thesis is subdivided into 8 chapters, with Chapter 1 providing a perspective of the field of protein design. Subsequent chapters (2-8) describe studies directed towards the specific goal of construction of polypeptide motifs. Chapter 2 describes synthesis and conformational characterization of two octapeptides Boc-Leu-Val-Val-DPro-LAla-Leu-Val-Val-OMe (1) and Boc-Leu-Val-Val-DPro-DAla-Leu-Val-Val-OMe (2), designed to investigate the effect of specific β-turn stereochemistry on β-hairpin structures. 500 MHz NMR studies establish that both peptides 1 and 2 adopt predominantly β-hairpin conformations in chloroform and methanol solutions, with interstrand registry established by observation of long-range nuclear Overhauser effects (NOEs). Specific NOEs provide evidence for a type II' β-turn conformation for the DPro-LAla segment in 1, while the NMR data suggest that a type I' DPro-DAla β-turn conformation predominates in the peptide 2. The crystal structure of 1 reveals two independent molecules in the crystallographic asymmetric unit, both of which adopt β-hairpin conformations nucleated by a type II’ β-turn across DPro-LAla and stabilized by 3 cross strand hydrogen bonds. These designed β-hairpins with defined tight turns produce characteristic vibrational circular dichroism (VCD) patterns, demonstrating the utility of VCD as a probe for conformational analysis of β-hairpins. In Chapter 3, we present conformational analysis on designed β-hairpin sequences incorporating a 'Phe-Phe' residue pair at a non-hydrogen bonding position. Two octapeptides Boc-Leu-Phe-Val-DPro-Gly-Leu-Phe-Val-OMe and Boc-Leu-Phe-Val-DPro-Ala-Leu-Phe-Val-OMe were synthesized and conformationally characterized by 500 MHz NMR spectroscopy. Specific NOEs observed in solution provide conclusive evidence favoring specific orientation effects pertaining to the 'Phe-Phe' pair. The peptides exhibited anomalous electronic CD, which has been explained in terms of aromatic contributions by the side chain chromophores. Interestingly, the VCD patterns obtained for these peptides were almost identical to those obtained for other β-hairpins, described in Chapter 2. Chapter 4 describes the synthesis and conformational analysis of designed decapeptide sequences with centrally located DPro-Xxx β-trun segments. Two sequences Boc-Met-Leu»Phe-Val'DPro-Ala-Leu-Val-Val-Phe-OMe (1) and Boc-Met-Leu-Val-Val-^ro-Gly-Leu-Val-Val-Phe-OMe (2) were designed to study the effect of chain length elongation, of β-strands, on designed β-hairpin structures. 500 MHz NMR studies establish β-hairpin folds in both these sequences, with strand segments aligned even at the termini of the structures. Multi-stranded, antiparallel β-sheet structures can be generated by successive placement of β-hairpin sequences in a single polypeptide chain. The successful construction of three stranded β-sheet structures is described in Chapter 5 of this dissertation. A 14-residue peptide Boc-Leu-Phe-Val-DPro-Gly-Leu-Val-Leu-Ala-DPro-Gly-Phe-Val-Leu-OMe (LFV14) was designed such that it is composed of three strand segments linked by two DPro-Gly turn segments. The peptide showed excellent solubility in apolar media, permitting detailed conformational analysis by 500 MHz NMR spectroscopy in organic solvents. Observation of long-range, interstrand NOEs, diagnostic of multiple hairpin structures, provides conclusive evidence for a predominantly populated three stranded β-sheet structure in solution. Extension of this strategy has been described in which an 18-residue peptide, Arg-Gly-Thr-Ile-Lys-DPro-Gly-Val-Thr-Phe-Ala-DPro-Ala-Thr-Lys-Tyr-Gly-Arg, was designed with enhanced solutility in water to probe (β-sheet structure formation in aqueous and mixed aqueous-methanol systems. NMR data provided conclusive evidence in favor of the desired structure being significantly populated in methanol and methanol-water mixtures (50 %, v/v). In water, spectroscopic evidence suggests that the long-range order expected of a three-stranded structure is lost, possibly due to water invading the interstrand hydrogen bonds. Successful construction of a four-stranded antiparallel β-sheet structure has been demonstrated in Chapter 6. A 26-residue peptide Arg-Gly-Thr-Ile-Lys»DPro-Gly-Ile-Thr- Phe-Ala-DPro-Ala-Thr-Val-Leu-Phe-Ala-Val-DPro-Gly-Lys-Thr-Leu-Tyr-Arg was designed to have four strand segments linked by three DPro-Xxx turn segments. The peptide exhibited excellent NMR properties permitting structure determination by analysis of NOE data, which revealed that a four stranded β-sheet structure is indeed populated in methanol. Structural studies on this peptide in mixed methanol-water established that the four stranded β-sheet is appreciably populated at a composition of 50 % (v/v) methanol-water mixture, with the β-sheet structure still detectable even at a composition of 70 % water-30 % methanol. In a completely aqueous environment, the β-sheet structures is significantly disrupted, presumably due to solvent invasion. The nucleating β-turns, however, appear to have retained their structural integrity even in this competitive environment. Chapter 7 describes the insertion of L-Lactic acid (Lac), a hydroxy acid, into polypeptide helices stabilized by a-aminoisobutyricacid (Aib). This study was undertaken to investigate the effect of hydrogen bond deletion on peptide helices. Crystal structure determination of three oligopeptides containing Lac residues has been performed. Peptide 1, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe, and peptide 2, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Leu-OMe adopt completely helical conformations in the crystalline state, with the Lac(6) residue comfortably accommodated in the center of a helix. NMR studies of peptide 1 and its all amide analog 4, Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe, provide firm evidence for a continuous helical segment in both the cases. In a 14-residue peptide 3, Boc-Val-Ala-Leu-Aib- Val- Ala-Leu- Val- Ala-Leu- Aib-Val-Lac-Leu-OMe, residues Val( 1 )-Leu( 10) adopt a helical conformation, which is terminated by formation of a Schellman motif, with Aib(ll) as the site of chiral reversal. The loss of the hydrogen bond at the C-terminus appears to facilitate the chiral reversal at Aib(l 1). In the final section of this thesis, Chapter 8, successful construction of a synthetic motif containing two distinct elements of secondary structure, a (β-hairpin and a helix, has been described. The design of a 17-residue peptide Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Gly-Gly-Leu-Phe-Val-DPro-Gly-Leu-Phe-Val-OMe, BH17, is based on a modular approach, in which previously characterized β-hairpin (Leu-Phe-Val-DPro-Gly-Leu-Phe-Val) and helix (Val-Ala-Leu-Aib-Val-Ala-Leu) modules are linked by a Gly-Gly linker. The positioning of the achiral Gly residue at position 8 facilitates termination of the potential helical segment (residues 1-7) by formation of a Schellman motif. Gly(9) is anticipated to be the sole conformationally flexible residue. NMR studies on BH17 indicated the presence of both the helix (residues 1-7) and the β-hairpin (residues 10-17) structures in the sequence, with four major conformational possibilities at the linking segment. Crystal structure determination of BH17 revealed that the two elements of structure are approximately arranged in an orthogonal fashion. The crystal structure validates the original premise that a modular assembly strategy may be viable for the construction of larger synthetic structures. Chapter 9 summarises the major results of this thesis. (For formulae, please refer "pdf" format)