The WHO-designated neglected-disease pathogen Chlamydia trachomatis (CT) is a gram-negative bacterium responsible for the most frequently diagnosed sexually transmitted infection worldwide. CT infections can lead to infertility, blindness and reactive arthritis, among others. CT acts as an infectious agent by its ability to evade the immune response of its host, which includes the impairment of the NF-κB mediated inflammatory response and the Mcl1 pro-apoptotic pathway through its deubiquitylating, deneddylating and transacetylating enzyme ChlaDUB1 (Cdu1). Expression of Cdu1 is also connected to host cell Golgi apparatus fragmentation, a key process in CT infections. Cdu1 may this be an attractive drug target for the treatment of CT infections. However, a lead molecule for the development of novel potent inhibitors has been unknown so far. Sequence alignments and phylogenetic searches allocate Cdu1 in the CE clan of cysteine proteases. The adenovirus protease (adenain) also belongs to this clan and shares a high degree of structural similarity with Cdu1. Taking advantage of topological similarities between the active sites of Cdu1 and adenain, a target-hopping approach on a focused set of adenain inhibitors, developed at Novartis, has been pursued. The thereby identified cyano-pyrimidines represent the first active-site directed covalent reversible inhibitors for Cdu1. High-resolution crystal structures of Cdu1 in complex with the covalently bound cyano-pyrimidines as well as with its substrate ubiquitin have been elucidated. The structural data of this thesis, combined with enzymatic assays and covalent docking studies, provide valuable insights into Cdu1s activity, substrate recognition, active site pocket flexibility and potential hotspots for ligand interaction. Structure-informed drug design permitted the optimization of this cyano-pyrimidine based scaffold towards HJR108, the first molecule of its kind specifically designed to disrupt the function of Cdu1. The structures of potentially more potent and selective Cdu1 inhibitors are herein proposed. This thesis provides important insights towards our understanding of the structural basis of ubiquitin recognition by Cdu1, and the basis to design highly specific Cdu1 covalent inhibitors. / Der Krankheitserreger Chlamydia trachomatis (CT) - ein gramnegatives Bakterium - ist verantwortlich für die häufigste sexuell übertragene Infektionskrankheit weltweit, die CT basierte Chlamydiose. Sie wird von der Weltgesundheitsorganisation zu den vernachlässigten Krankheiten gezählt. CT Infektionen können unter anderem zu Unfruchtbarkeit, Erblindung und reaktiver Arthritis führen. CT agiert als Krankheitserreger mittels seiner Fähigkeit, die Immunantwort des Wirts zu umgehen. Dies umfasst unter anderem die Schwächung und Störung der NF-κB vermittelten Entzündungsantwort und des Mcl1 pro-Apoptoseweges über ihr deubiquitinierendes, deneddylierendes und trans-acetylierendes Enzym ChlaDub1 (Cdu1). Die Expression von Cdu1 ist aber auch mit der Fragmentierung des Golgi-Apparates des Wirtes verknüpft, ein Schlüsselprozess bei Infektionen mit CT. Cdu1 ist daher vermutlich ein attraktives Zielprotein für die Entwicklung von Wirkstoffen, um CT Infektionen zu behandeln. Eine Leitstrukturverbindung zur Entwicklung neuer wirksamer Inhibitoren war bislang jedoch noch nicht bekannt. Sequenzvergleiche und phylogenetische Untersuchungen verorten Cdu1 im CE Clan der Cysteinproteasen. Die Adenovirus-Protease (Adenain) gehört ebenfalls diesem Clan an und besitzt strukturelle Ähnlichkeit mit Cdu1. Unter Ausnutzung der topologischen Ähnlichkeiten der aktiven Zentren von Cdu1 und Adenain wurde ein Target-Hopping Ansatz mit einem klar definierten und fokussierten Satz von bei Novartis entwickelten Adenain-Inhibitoren verfolgt. Die hierbei identifizierten Cyano-Pyrimidine stellen die ersten kovalenten Inhibitoren von Cdu1 dar, die an das aktive Zentrum von Cdu1 binden und es direkt adressieren. Hochauflösend wurden Kristallstrukturen sowohl von Komplexen von Cdu1 mit kovalent gebundenen Cyano-Pyrimidinen als auch mit Cdu1’s natürlichem Substrat Ubiquitin bestimmt. Die Kristallstrukturdaten dieser Doktorarbeit in Kombination mit Enzymassays und kovalenten Docking-Studien liefern wertvolle Hinweise bezüglich der Aktivität des Enzyms, der molekularen Substraterkennung, der Flexibiliät der Proteintasche rund um das aktive Zentrum und potentielle Hotspots für die Wechselwirkung mit Liganden. Ein strukturbasiertes Wirkstoffdesign erlaubte die Optimierung des Cyano-Pyrimidin-basierten Molekülgerüstes, die zu der Entwicklung der HJR108 Verbindung führte. Es ist das erste Molekül seiner Art, das speziell dazu entworfen wurde Cdu1 zu inhibieren. Strukturen potentiell noch wirksamerer und selektiver Cdu1 Inhibitoren werden in dieser Arbeit vorgeschlagen. Diese Dissertationsschrift liefert somit wertvolle Beiträge zum Verständnis der strukturellen Grundlagen der molekularen Erkennung von Ubiquitin durch Cdu1 und Hinweise, die die Entwicklung hoch-spezifischer kovalenter Cdu1 Inhibitoren erlauben sollten.

Background: The value of liver-directed therapy (LDT) in patients with metastasic renal cell carcinoma (MRCC) is still an active field of research, particularly in the era of tyrosinkinase inhibitor (TKI) therapy. Methods: The records of 35 patients with MRCC undergoing LDT of metastasic liver lesions between 1992 and 2015 were retrospectively analyzed. Immediate postoperative TKI was given in a subgroup of patients after LDT for metastasic lesions. Uni- and multivariate models were applied to assess overall survival (OS), progression-free survival (PFS) and disease-free survival (DFS). Results: Following primary tumor (renal cell cancer) resection and LDT, respectively, median OS was better for a total of 16 patients (41 %) receiving immediate postoperative TKI with 151 and 98 months, when compared to patients without TKI therapy with 61 (p = 0.003) and 40 months (p = 0.032). Immediate postoperative TKI was associated with better median PFS (47 months versus 19 months; p = 0.023), whereas in DFS only a trend was observed (51 months versus 19 months; p = 0.110). Conclusions: LDT should be considered as a suitable additive tool in the era of TKI therapy of MRCC to the liver. In this context, postoperative TKI therapy seems to be associated with better OS and PFS, but not DFS.

The immunoproteasome, known to play an important role in MHC class I antigen processing and presentation, have been linked to neurodegenerative diseases and hematological cancers. However, the pathophysiological functions of the immunoproteasome in these diseases are still not very well established. This can be attributed mainly to the lack of appropriate molecular probes that selectively target the immunoproteasome catalytic subunits. Herein, we report the development of a small molecular inhibitor (AM) that selectively targets the major catalytic subunit, LMP2, of the immunoproteasome. We show that the compound covalently modifies the LMP2 subunit with high specificity in human prostate cancer cell. AM was also shown to selectively inhibit the chymotrypsin-like activity of LMP2 subunit. More importantly, the anti-proliferative activity of AM is more pronounced in prostate cancer cells that highly express LMP2 without inducing toxicity in normal cells. These results implicate an important role of LMP2 in regulating cell growth of malignant tumors that highly express LMP2. Subsequently, the modes of action of AM were investigated. Prostate cancer cells that highly express LMP were shown to induce G2/M cell cycle arrest and apoptosis via PARP cleavage when treated with the compound. Similar to epoxomicin, the treatment of AM induced the accumulation of poly-ubiquitination in prostate cancer cells, which indicates the inhibition of proteolysis. However, unlike epoxomicin, the treatment of AM did not appear to inhibit the activation of inflammation. In conclusion, these results suggest that the LMP2 inhibitor, AM, may induce cytotoxicity prostate cancer cells that highly express LMP2 catalytic subunit in similar modes of action as epoxomicin but it does not involve the inflammatory pathway.

Iminosugars are polyhydroxylated alkaloids, and can be viewed as sugar analogues in which the endocyclic oxygen atom has been replaced with nitrogen. These compounds are highly medically relevant and their biological activity is largely due to their inhibition of glycosidases. Several examples of the iminosugar class are currently marketed as drugs, and many more are in earlier stages of development for a variety of diseases and disorders. The most fruitful approaches to the chemical synthesis of iminosugars have utilised carbohydrate starting materials as optically pure chiral building blocks, or chirons. Most of the monosaccharides are not readily available, but the relatively few naturally abundant cheap sugars have been exploited as chirons for over a century. The availability of the rare sugars is growing with the development of a new biotechnological approach to their synthesis, known as Izumoring. This thesis is primarily concerned with the chemical synthesis of iminosugars from carbohydrate starting materials. The synthesis of unnaturally functionalised sugar polyols and their suitability as substrates for the Izumoring process is also discussed. Chapter 1 provides a brief general overview of the history, natural occurrence and therapeutic application of iminosugars. General strategies for their synthesis from carbohydrate chirons are discussed. Chapter 2 concerns divergent syntheses of several iminosugar targets from both enantiomers of glucuronolactone and their biological evaluation. A new scaleable synthesis of the natural product 1-deoxynojirimycin is presented that has since been adopted for commercial purposes, as well as an efficient strategy for the synthesis of both enantiomers of 2,5-dideoxy-2,5-imino- mannitol and their novel amino acid analogues. Access to hexosaminidase inhibiting acetamido- substituted piperidines is presented, including 2-acetamido-1,5-imino-1,2,5-trideoxy-D- galactitol, which has been found to be one of the few known potent and specific inhibitors of α- N-acetyl-galactosaminidase. This inhibitory profile may allow the compound’s use for further investigation of a strategy for cancer treatment. Chapter 3 concerns the synthesis of carbon branched pyrrolidines and their biological evaluation. A novel and highly potent α-glycosidase inhibitor has been discovered, synthesised by a strategy that utilises the benzhydryl ether as key protecting group. A mild method for the introduction of this protecting group has been shown to be general to a range of sterically congested and/or acid/base sensitive carbohydrate lactones. Chapter 4 concerns the synthesis of deoxygenated and fluorinated sugar alcohols and their successful biotechnological transformation into ketoses by the Izumoring process. Publications arising from this work are included in the Appendix.

1 ABSTRACT The success of cancer vaccines depends on factors associated with the vaccine, which define the main parameters of effective immune responses such as its size and quality, as well as on factors related with the host, represented by the immunosuppressive mechanisms that allow the tumor to escape recognition by the immune system or negatively influence the function of effector T-cells. Attenuated, non-replicating viruses are at present preferred as VACV for safety reasons. A problem may arise concerning their lack of immunogenicity. Through the deletions of non-essential genes, vaccination vectors are therefore developed based on attenuated rVACV capable of replication, which induce a strong immune response. Genes of various immunological adjuvants (e.g., genes for cytokines and costimulatory molecules) are inserted into the vectors for the purpose of eliminating the influence of the immunosuppressive mechanisms of tumors. The first part of the work describes our study of the influence of vCCI on biological properties of rVACV derived from the Prague strain. Testing of vCCI deletion and insertion mutants expressing tumor associated protein HPV16 E7 has shown that secreted vCCI attenuated the virus in vivo, which correlated with reduced levels of the corresponding CC chemokines in the blood compared...

As neoplasias mieloproliferativas (NMPs) negativas para o rearranjo t(9;22)/BCRABL1, incluindo Policitemia Vera (PV), Trombocitemia Essencial (TE) e Mielofibrose Primária (MFP), são doenças hematopoéticas clonais e estão frequentemente associadas à mutação JAK2V617F. Apesar dos avanços no conhecimento da fisiopatologia após a descoberta da mutação JAK2V617F e do desenvolvimento de inibidores da JAK2, o tratamento permanece não curativo. Sabe-se que as célulastronco mais primitivas nas NMPs são responsáveis pela iniciação da doença e que a expansão dos precursores mieloeritróides contribui para o fenótipo clínico. Dados recentes obtidos com ensaios in vitro mostram que as proteínas da família BCL2, reguladoras da apoptose mitocondrial, desempenham um papel relevante na patogênese das NMPs. Acreditamos que a expressão anômala de BCL2 nas células progenitoras hematopoéticas (CPH) das NMPs pode contribuir para a patogênese desse grupo de doenças. Avaliamos a expressão gênica, por meio de PCR em Tempo Real, da família BCL2 (genes antiapoptóticos BCL-xL e BCL2 e o pró-apoptótico BIM) nas diferentes subpopulações de progenitores hematopoéticos murinos (de um modelo condicional knockin de expressão heterozigótica condicional da Jak2V617F) e de pacientes portadores de NMPs bem como sua contribuição para o fenótipo da doença e resposta ao inibidores da JAK2 (com a droga ruxolitinibe) e/ou inibição da família BCL2 (com o inibidor de BCL2 obatoclax). Não encontramos diferença de expressão basal dos genes BCL2, BCL-xL e BIM nas células CD34+ bem como nas subpopulações de células CD34+38-/+ de pacientes com NMPs, independente da presença da mutação JAK2V617F, em relação às células CD34+ e subpopulações CD34+38-/+ dos controles (p>0.05). Nas células CD34+ de pacientes com TE encontramos aumento de expressão de BCL2 em relação às células CD34+ pacientes com MFP (p=0.03). No modelo transgênico de camundongos Jak2 wt/VF (que apresentam uma NMP semelhante à PV) e Jak2 wt/wt (controles), comparamos a expressão diferencial dos genes da família Bcl2 em precursores hematopoéticos imaturos (LSKs) e progenitores mieloides mais maduros (MPs). A expressão do BclxL em MPs de camundongos wt/VF foi maior em relação à subpopulação de células LSKs e em relação as duas subpopulações de células dos controles (p=0.0011). Não houve diferença significativa de expressão do Bcl2 nas subpopulações de células LSKs e MPs de animais wt/VF e wt/wt (p=0.12). Observou-se menor expressão de Bim em LSKs em relação às células MPs dos animais mutados (p=0.026), diferença essa não observada entre os controles Jak2 wt/wt. O tratamento isolado com inibidor de JAK2 ou de BCL2 resultou em aumento de expressão do Bim nas CPH (LSKs e MPs) de camungongos Jak2 wt/VF em relação aos animais Jak2 wt/wt. Este aumento da expressão de Bim foi ainda mais evidente após o tratamento das células com a combinação das duas drogas quando comparadas às células não tratadas ou tratadas com um dos dois inibidores, sendo maior em animais doentes do que em animais controles (p<0.0001). A análise do efeito do tratamento com os inibidores de JAK2 e BCL2 na indução de apoptose por meio de citometria de fluxo (marcação com anexina/7-AAD) revelou que as células LSKs foram mais resistentes à apoptose tardia do que as células MPs independentemente da mutação da JAK2 (p<0.05). O tratamento com obatoclax resultou em indução de apoptose diferentemente do que foi observado com o tratamento com ruxolitinibe (p=0.594) nas células MPs de animais Jak2 wt/VF. Ademais, o tratamento combinado com ruxolitinibe e obatoclax resultou no aumento da apoptose nas células MPs dos animais com fenótipo de PV (Jak2 wt/VF) em relação aos animais Jak2 wt/wt (p=0.05). Em conclusão, demonstramos que a resistência à apoptose nas NMPs ocorre desde as CPH iniciadoras da doença. Nossos resultados sugerem que a modulação da apoptose mitocondrial pode ser uma nova estratégia terapêutica para pacientes com NMP em combinação aos inibidores de JAK2, na medida em que atua tanto nas CPH que iniciam a doença como nos MPs, responsáveis pelos sinais e sintomas de mieloproliferação. / Myeloproliferative Neoplasms (MPNs) negative for t(9;22)/BCR-ABL1 rearrangement, including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF), are clonal hematopoietic diseases and are often associated with the JAK2V617F mutation. Despite advances in the pathophysiology knowledge after the discovery of the JAK2V617F mutation and the development of JAK2 inhibitors, treatment remains non-curative. It is known that MPN primitive stem cells are essential for the initiation of the disease and that the expansion of the myeloeritroid precursors contributes to the clinical phenotype. Recent data, obtained with in vitro assays, showed that BCL2 family proteins, regulators of mitochondrial apoptosis, play a relevant role in the pathogenesis of MPNs. We believe that the anomalous expression of BCL2 in hematopoietic progenitor cells (HPCs) of MPNs may contribute to their pathogenesis. We evaluated BCL2 family (antiapoptotic genes BCL-xL and BCL2 and the pro-apoptotic BIM) gene expression by real-time PCR in different subpopulations of hematopoietic progenitors from a conditional Jak2V617F knockin murine model and from patients with MPNs as well as their contribution to the disease phenotype and response to JAK2 inhibitors (with ruxolitinib) and/or to the inhibition of the BCL2 family (with the BH3-mimetic obatoclax). We found no difference in the basal expression of the BCL2, BCL-xL and BIM in CD34+ cells as well as in subpopulations of CD34+ 38-/+ cells from patients with MPNs, regardless of the presence of the JAK2V617F mutation. In CD34+ cells obtained from patients with ET, we found an increase of BCL2 expression when compared to CD34+ cells with PMF (p=0.03). In the Jak2 wt/VF transgenic mice (that develop a MPN similar to PV) and Jak2 wt/wt controls, we compared the differential expression of Bcl2 family genes in immature hematopoietic precursors (LSKs) and more mature myeloid progenitors (MPs). Expression of Bcl-xL in MPs of wt/VF mice was greater when compared to LSKs and to the two progenitor subpopulations of control cells (p=0.0011). There was no significant difference in Bcl2 expression between the subpopulations of LSKs and MPs from wt/VF and wt/wt animals (p=0.12). Lower Bim expression in LSKs than in MPs was observed in samples from JAK2-mutated animals (p=0.026). Such difference was not observed between the Jak2 wt/wt subpopulations. Treatment with JAK2 or BCL2 inhibitors alone resulted in increased Bim expression in LSKs and MPs of the Jak2 wt/VF mice when compared to Jak2 wt/wt animals. This increase in Bim expression was even more evident when these cells were treated with the combination of the two drugs as compared to single treatment with one of the two inhibitors, being higher in mutaded than control animals (p<0.0001). The analysis of apoptosis by flow cytometry (annexin / 7-AAD labeling) revealed that LSK cells were more resistant to late apoptosis than MP cells regardless of the JAK2 mutation (p<0.05). Treatment with obatoclax resulted in greater apoptosis induction than it was observed with ruxolitinib treatment (p=0.594) on MP cells of Jak2 wt/VF animals. In addition, the combined treatment with ruxolitinib and obatoclax resulted in increased apoptosis in MP cells of animals with the PV phenotype (Jak2 wt/VF) as compared to the Jak2 wt/wt animals (p=0.05). In conclusion, we demonstrated that resistance to apoptosis in MPNs occurs at the level of the hematopoietic progenitors that initiate the disease. Our results suggest that modulation of mitochondrial apoptosis may be a new therapeutic strategy for MPN patients in combination with JAK2 inhibitors, as it acts on both the disease initiating and more mature progenitors, responsible for the clinical findings of myeloproliferation.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

This master thesis considers an investigation of protective formulations (ointment, cream) containing enzyme inhibitors. Model experiments have been made on the enzyme trypsin. It is well accepted that feces and urine are an important causing factor for skin irritation (dermatitis) while using diaper. A protective formulation is a physical barrier that separates the harmful substances from the skin. It can also be an active barrier containing active substances, which can be active both towards the skin, and the substances from feces and urine. By preventing contact from these substances the skin will not be harmed, at least for a period of time. A number of different inhibitors were tested towards trypsin and they all showed good inhibition, two of the inhibitors were selected to be immobilized with the help of NHS-­activated Sepharose. Immobilization of these two inhibitors leads to a lesser extent of the risk of developing allergy and also that the possible toxic effect can be minimized.

Three protease inhibitor-like proteins (PILPs) identified from Bungarus multicinctus genome are structurally homologous with Kunitz-type proteinase inhibitor. The goal of the present study is to explore whether PILPs exhibit an inhibitory action on matrix metalloproteinase 2 (MMP-2) activity. Unlike PILP-1 and PILP-2, PILP-3 was found to inhibit MMP-2 activity as evidenced by specific substrate assay. Moreover, in vitro migration and invasion assays, and wound-healing assay showed that PILP-3 suppressed the migration and invasion of human neuroblastoma SK-N-SH cells. Pull-down assay and dot blotting-binding assay proved an interaction between PILP-3 and MMP-2. Nevertheless, PILP-3 did not affect either expression or secretion of MMP-2 in SK-N-SH cells. In terms of highly structural similarity between PILP-2 and PILP-3, two chimeric mutants in which amino acids at N-terminus and C-terminus of PILP-3 were substituted by those of PILP-2 were prepared. In contrast to N-terminus chimera, C-terminus mutant of PILP-3 was unable to inhibit MMP-2 activity and showed a reduction in binding with MMP-2. Taken together, our data suggest that PILP-3 may be a useful template for rational designing pharmaceutical agent in inhibiting MMP-2 activity.