Plasticity of S2-S4 specificity pockets of executioner caspase-7 revealed by structural and kinetic analysis

Many protein substrates of caspases are cleaved at noncanonical sites in comparison to the recognition motifs reported for the three caspase subgroups. To provide insight into the specificity and aid in the design of drugs to control cell death, crystal structures of caspase-7 were determined in complexes with six peptide analogs (Ac-DMQD-Cho, Ac-DQMD-Cho, Ac-DNLD-Cho, Ac-IEPD-Cho, Ac-ESMD-Cho, Ac-WEHD-Cho) that span the major recognition motifs of the three subgroups. The crystal structures show that the S2 pocket of caspase-7 can accommodate diverse residues. Glu is not required at the P3 position because Ac-DMQD-Cho, Ac-DQMD-Cho and Ac-DNLD-Cho with varied P3 residues are almost as potent as the canonical Ac-DEVD-Cho. P4 Asp was present in the better inhibitors of caspase-7. However, the S4 pocket of executioner caspase-7 has alternate regions for binding of small branched aliphatic or polar residues similar to those of initiator caspase-8. The observed plasticity of the caspase subsites agrees very well with the reported cleavage of many proteins at noncanonical sites. The results imply that factors other than the P4-P1 sequence, such as exosites, contribute to the in vivo substrate specificity of caspases. The novel peptide binding site identified on the molecular surface of the current structures is suggested to be an exosite of caspase-7. These results should be considered in the design of selective small molecule inhibitors of this pharmacologically important protease.     

Structural basis of specificity of a peptidyl urokinase inhibitor, upain-1

Urokinase-type plasminogen activator (uPA) plays a crucial role in the regulation of plasminogen activation, tumor cell adhesion and migration. The inhibition of uPA activity is a promising mechanism for anti-cancer therapy. A cyclic peptidyl inhibitor, upain-1, CSWRGLENHRMC, was identified recently as a competitive and highly specific uPA inhibitor. We determined the crystal structure of uPA in complex with upain-1 at 2.15 A. The structure reveals that the cyclic peptide adopts a rigid conformation stabilized by a disulfide bond (residues 1-12) and three tight beta turns (residues 3-6, 6-9, 9-12). The Glu7 residue of upain-1 forms hydrogen bonds with the main chain nitrogen atoms of residues 4, 5, and 6 of upain-1, and is also critical for maintaining the active conformation of upain-1. The Arg4 of upain-1 is inserted into the uPA's specific S1 pocket. The Ser2 residue of upain-1 locates close to the S1beta pocket of uPA. The Gly5 and Glu7 residues of upain-1 occupy the S2 pocket and the oxyanion hole of uPA, respectively. Furthermore, the Asn8 residue of upain-1 binds to the 37- and 60-loops of uPA and renders the specificity of upain-1 for uPA. Based on this structure, a new pharmacophore for the design of highly specific uPA inhibitors was proposed.     

Ordering the Cytochrome c–initiated Caspase Cascade: Hierarchical Activation of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9–dependent Manner

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1–mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c–inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.     

Crystallographic structure of the human leukocyte antigen DRA, DRB3*0101: models of a directional alloimmune response and autoimmunity

We describe structural studies of the human leukocyte antigen DR52a, HLA-DRA/DRB3*0101, in complex with an N-terminal human platelet integrin alphaII(B)betaIII glycoprotein peptide which contains a Leu/Pro dimorphism. The 33:Leu dimorphism is the epitope for the T cell directed response in neonatal alloimmune thrombocytopenia and post-transfusion purpura in individuals with the alphaII(B)betaIII 33:Pro allele, and defines the unidirectional alloimmune response. This condition is always associated with DR52a. The crystallographic structure has been refined to 2.25 A. There are two alphabeta heterodimers to the asymmetric unit in space group P4(1)2(1)2. The molecule is characterized by two prominent hydrophobic pockets at either end of the peptide binding cleft and a deep, narrower and highly charged P4 opening underneath the beta 1 chain. Further, the peptide in the second molecule displays a sharp upward turn after pocket P9. The structure reveals the role of pockets and the distinctive basic P4 pocket, shared by DR52a and DR3, in selecting their respective binding peptide repertoire. We observe an interesting switch in a residue from the canonically assigned pocket 6 seen in prior class II structures to pocket 4. This occludes the P6 pocket helping to explain the distinctive "1-4-9" peptide binding motif. A beta57 Asp-->Val substitution abrogates the salt-bridge to alpha76 Arg and along with a hydrophobic beta37 is important in shaping the P9 pocket. DRB3*0101 and DRB1*0301 belong to an ancestral haplotype and are associated with many autoimmune diseases linked to antigen presentation, but whereas DR3 is susceptible to type 1 diabetes DR52a is not. This dichotomy is explored for clues to the disease.     

Endoplasmic reticulum stress-induced death of mouse embryonic fibroblasts requires the intrinsic pathway of apoptosis

Members of the caspase family are essential for many apoptotic programs. We studied mouse embryonic fibroblasts (MEFs) deficient in caspases 3 and 7 and in caspase 9 to determine the role of these proteases in endoplasmic reticulum (ER) stress-induced apoptosis. Both caspase 3(-/-)/caspase 7(-/-) and caspase 9(-/-) MEFs were resistant to cytotoxicity induced via ER stress and failed to exhibit apoptotic morphology. Specifically, apoptosis induced by increased intracellular calcium was shown to depend only on caspases 3 and 9, whereas apoptosis induced by disruption of ER function depended additionally on caspase 7. Caspase 3(-/-)/caspase 7(-/-) and caspase 9(-/-) MEFs also exhibited decreased loss of mitochondrial membrane potential, which correlated with altered caspase 9 processing, increased induction of procaspase 11, and decreased processing of caspase 12 in caspase 3(-/-)/caspase 7(-/-) cells. Furthermore, disruption of ER function was sufficient to induce accumulation of cleaved caspase 3 and 7 in a heavy membrane compartment, suggesting a potential mechanism for caspase 12 processing and its role as an amplifier in the death pathway. Caspase 8(-/-) MEFs were not resistant to ER stress-induced cytotoxicity, and processing of caspase 8 was not observed upon induction of ER stress. This study thus demonstrates a requirement for caspases 3 and 9 and a key role for the intrinsic pathway in ER stress-induced apoptosis.     

Delineation of the caspase-9 signaling cascade

In the intrinsic apoptosis pathway, mitochondrial disruption leads to the release of multiple apoptosis signaling molecules, triggering both caspase-dependent and -independent cell death. The release of cytochrome c induces the formation of the apoptosome, resulting in caspase-9 activation. Multiple caspases are activated downstream of caspase-9, however, the precise order of caspase activation downstream of caspase-9 in intact cells has not been completely resolved. To characterize the caspase-9 signaling cascade in intact cells, we employed chemically induced dimerization to activate caspase-9 specifically. Dimerization of caspase-9 led to rapid activation of effector caspases, including caspases-3, -6 and -7, as well as initiator caspases, including caspases-2, -8 and -10, in H9 and Jurkat cells. Knockdown of caspase-3 suppressed caspase-9-induced processing of the other caspases downstream of caspase-9. Silencing of caspase-6 partially inhibited caspase-9-mediated processing of caspases-2, -3 and -10, while silencing of caspase-7 partially inhibited caspase-9-induced processing of caspase-2, -3, -6 and -10. In contrast, deficiency in caspase-2, -8 or -10 did not significantly affect the caspase-9-induced caspase cascade. Our data provide novel insights into the ordering of a caspase signaling network downstream of caspase-9 in intact cells during apoptosis.     

Structural and kinetic analysis of caspase-3 reveals role for s5 binding site in substrate recognition

The molecular basis for the substrate specificity of human caspase-3 has been investigated using peptide analog inhibitors and substrates that vary at the P2, P3, and P5 positions. Crystal structures were determined of caspase-3 complexes with the substrate analogs at resolutions of 1.7 A to 2.3 A. Differences in the interactions of caspase-3 with the analogs are consistent with the Ki values of 1.3 nM, 6.5 nM, and 12.4 nM for Ac-DEVD-Cho, Ac-VDVAD-Cho and Ac-DMQD-Cho, respectively, and relative kcat/Km values of 100%, 37% and 17% for the corresponding peptide substrates. The bound peptide analogs show very similar interactions for the main-chain atoms and the conserved P1 Asp and P4 Asp, while interactions vary for P2 and P3. P2 lies in a hydrophobic S2 groove, consistent with the weaker inhibition of Ac-DMQD-Cho with polar P2 Gln. S3 is a surface hydrophilic site with favorable polar interactions with P3 Glu in Ac-DEVD-Cho. Ac-DMQD-Cho and Ac-VDVAD-Cho have hydrophobic P3 residues that are not optimal in the polar S3 site, consistent with their weaker inhibition. A hydrophobic S5 site was identified for caspase-3, where the side-chains of Phe250 and Phe252 interact with P5 Val of Ac-VDVAD-Cho, and enclose the substrate-binding site by conformational change. The kinetic importance of hydrophobic P5 residues was confirmed by more efficient hydrolysis of caspase-3 substrates Ac-VDVAD-pNA and Ac-LDVAD-pNA compared with Ac-DVAD-pNA. In contrast, caspase-7 showed less efficient hydrolysis of the substrates with P5 Val or Leu compared with Ac-DVAD-pNA. Caspase-3 and caspase-2 share similar hydrophobic S5 sites, while caspases 1, 7, 8 and 9 do not have structurally equivalent hydrophobic residues; these caspases are likely to differ in their selectivity for the P5 position of substrates. The distinct selectivity for P5 will help define the particular substrates and signaling pathways associated with each caspase.     

Synthesis and structure-activity investigation of novel vasopressin hypotensive peptide agonists.

We report the solid phase synthesis and vasodepressor potencies of the novel hypotensive peptide [1(-beta-mercapto-beta,beta-pentamethylene propionic acid)-2-O-ethyl-D-tyrosine, 3-arginine, 4-valine] arginine vasopressin, d(CH2)5[D-Tyr(Et)2, Arg3, Val4]AVP (A), its related Lys3 (B), Tyr-NH(9)2 (C), [Lys3, Tyr-NH(9)2 (D) analogs and in a preliminary structure-activity study of positions 2-4 and 7-9, 24 analogs (1-24) of A-C. Peptides 1-6, 9-14 have the following single substituents at positions 2, 3, 4, 8 and 9 in (A): 1, D-Tyr(Me)2; 2, L-Tyr(Et)2; 3, Orn3; 4, N-Me-Arg3; 5, Glu3; 6, Arg4; 9, D-Arg8; 10, Eda9; 11, Arg-NH(9)2; 12, Ala-NH(9)2; 13, desGly9; 14, desGly-NH(9)2. Peptides 15 and 16 are analogs of B which possess the following single modifications: 15, Arg-NH(9)2; 16, desGly9. Peptides 7 and 8 are analogs of (C) with the following single modification: 7, Gln4; 8, Lys8. Peptides 17-24 are analogs of A possessing the following multiple modifications: 17, [Sar7, Eda9]; 18, [Arg7, Eda9]; 19, [Arg7, Eda9<--Tyr10]; 20, [Arg4, Arg-NH(9)2]; 21, [Ile4, desGly9]; 22, [Arg4, desGly9]l; 23, [Arg7, desGly9]; 24, [Arg7, Lys8, desGly9]. All 24 new peptides were evaluated for agonistic and antagonistic activities in in vivo antidiuretic (V2-receptor), vasopressor (V1a-receptor) and in in vitro (no Mg2+) oxytocic (OT-receptor) assays and like the parent peptides (A-D) (Chan et al. Br. J. Pharmacol. 1998; 125: 803-811) were found to exhibit no or negligible activities in these assays. Vasodepressor potencies were determined in anesthetized male rats with baseline mean arterial blood pressure maintained at 110-120 mmHg. The effective dose (ED), in microg 100 g(-1) i.v., required to produce a vasodepressor response of 5 cm2, area under the vasodepressor response curve (AUC) during the 5-min period following the injection of the test peptide, was determined. Therefore, the EDs measure the relative vasodepressor potencies of the hypotensive peptides. The following ED values were obtained for A-D and for peptides 1-24: A, 4.66; B, 5.75; C, 10.56; D, 11.60; 1, approximately 20; 2, approximately 30; 3, 6.78; 4, non-detectable (ND); 5, ND; 6, approximately 32; 7, ND; 8, 8.67; 9, ND; 10, 2.43; 11, 3.54; 12, 10.57; 13, 4.81; 14, ND; 15, 4.47; 16, 9.78; 17, 5.72; 18, 1.10; 19, 1.05; 20, 10.41; 21, 9.13; 22, approximately 33; 23, 3.01; 24, 1.71. A is clearly the most potent of the four original hypotensive peptides A-D. These data provide insights to which modification of A enhance, retain or abolish hypotensive potencies. Six of the new hypotensive peptides are significantly more potent than A. These are peptides 10, 11, 18, 19, 23 and 24. Peptide 19, a radioiodinatable ligand, is ten times more potent than C or D. The Gln4 modification of C and the N-Me-Arg3, Glu3, D-Arg8 and desGly-NH(9)2 modifications of A abolished hypotensive potency. By contrast, the Eda9, Arg-NH(9)2, [Sar7, Eda9], [Arg7, Eda9<- -Tyr10], [Arg7, desGly9], [Arg7, Lys8, desGly9] modifications of A all led to enhancements of hypotensive potency. This initial structure-activity exploration provides useful clues to the design of (a) more potent vasodepressor peptides and (b) high affinity radioiodinatable ligands for the putative AVP vasodilating receptor. Some of the peptides here may be of value as pharmacological tools for studies on the complex cardiovascular actions of AVP and may lead to the development of a new class of anti-hypertensive agents.     

Molecular mechanism of apoptosis: prediction of three-dimensional structure of caspase-6 and its interactions by homology modeling

Caspases, the intracellular cysteine proteinases, play a central role in the process of programmed cell death. Caspases induce apoptosis through a highly integrated and regulated biological, biochemical, and genetic mechanism. Although proper execution of apoptosis is fundamental for cell growth artificial caspase inhibition can be considered in certain degenerative diseases. This realization has attracted attention towards caspases as likely targets for pharmaceutical intervention. Here we analyze the structure of caspase-6 and also predict the possible glycosylation, phosphorylation, and myristoylation sites as very little is known about the functional role of these post translational modifications in the caspase family. These studies are expected to improve our understanding of associations of caspases with other molecules and the possible role played in apoptosis. The predicted tertiary structure of caspase-6 as well as the enzyme complexed with its inhibitor (tetra-peptide aldehyde Ac-IETD-CHO) shows similar binding feature as seen in other caspases. Cys/His catalytic dyad for caspase-6 and -8 show possible involvement of a third component, i.e., Pro29 and Arg258 in caspase-6 and caspase-8, respectively. Changes in the length and nature of loop between alpha5 and beta9, involved in defining the S4 subsite, result in modification of P4 (Ile) site. These interactions provide detail of inhibitor binding on structural level and also help in designing mutants for structure-function studies of these enzymes.     

Development of cell death-based method for the selectivity screening of caspase-1 inhibitors

Caspase-1 selective inhibitors are novel therapeutic agents for inflammatory diseases. Selectivity assays for caspases can be initiated with purified enzyme, making these assays very costly and time consuming. Therefore, there is a need to develop a fast and reliable cell-based assay, which can be used for the selectivity screening of multiple caspases in a biologically relevant context in a single assay. In this study, we have developed an assay in which DNA fragmentation, a hallmark of apoptosis, of Jurkat cell line was examined post induction with etoposide in the presence or absence of inhibitors of caspases 1, 3, 8, 9 and pan-caspase inhibitors. We observed that caspases-3, -8, -9 and pan caspase inhibitors resulted in significant inhibition of etoposide-induced DNA fragmentation. However, caspase-1 specific inhibitor failed to prevent DNA fragmentation, suggesting that either caspases belonging to caspase-1 family (1, 4 and 5) are not present in the Jurkat cells or might not be involved in the etoposide-induced DNA fragmentation. Since the inhibition of caspases 3, 8 and 9 is accompanied by the down regulation of the activity of a cascade of caspases (caspases 2, 6, 7, 9 and 10), selectivity of caspase-I inhibitors can be ascertained for the above panel (caspases 2, 6, 7, 8, 9 and 10) of caspases from this single assay.     

The proteolytic procaspase activation network: an in vitro analysis

In general, apoptotic stimuli lead to activation of caspases. Once activated, a caspase can induce intracellular signaling pathways involving proteolytic activation of other caspase family members. We report the in vitro processing of eight murine procaspases by their enzymatically active counterparts. Caspase-8 processed all procaspases examined. Caspase-1 and -11 processed the effector caspases procaspase-3 and -7, and to a lesser extent procaspase-6. However, vice versa, none of the caspase-1-like procaspases was activated by the effector caspases. This suggests that the caspase-1 subfamily members either act upstream of the apoptosis effector caspases or else are part of a totally separate activation pathway. Procaspase-2 was maturated by caspase-8 and -3, and to a lesser extent by caspase-7, while the active caspase-2 did not process any of the procaspases examined, except its own precursor. Hence, caspase-2 might not be able to initiate a wide proteolytic signaling cascade. Additionally, cleavage data reveal not only proteolytic amplification between caspase-3 and -8, caspase-6 and -3, and caspase-6 and -7, but also positive feedback loops involving multiple activated caspases. Our results suggest the existence of a hierarchic proteolytic procaspase activation network, which would lead to a dramatic increase in multiple caspase activities once key caspases are activated. The proteolytic procaspase activation network might allow that different apoptotic stimuli result in specific cleavage of substrates responsible for typical processes at the cell membrane, the cytosol, the organelles, and the nucleus, which characterize a cell dying by apoptosis.     

Sox11 Reduces Caspase-6 Cleavage and Activity

The apoptotic cascade is an orchestrated event, whose final stages are mediated by effector caspases. Regulatory binding proteins have been identified for caspases such as caspase-3, -7, -8, and -9. Many of these proteins belong to the inhibitor of apoptosis (IAP) family. By contrast, caspase-6 is not believed to be influenced by IAPs, and little is known about its regulation. We therefore performed a yeast-two-hybrid screen using a constitutively inactive form of caspase-6 for bait in order to identify novel regulators of caspase-6 activity. Sox11 was identified as a potential caspase-6 interacting protein. Sox11 was capable of dramatically reducing caspase-6 activity, as well as preventing caspase-6 self- cleavage. Several regions, including amino acids 117–214 and 362–395 within sox11 as well as a nuclear localization signal (NLS) all contributed to the reduction in caspase-6 activity. Furthermore, sox11 was also capable of decreasing other effector caspase activity but not initiator caspases -8 and -9. The ability of sox11 to reduce effector caspase activity was also reflected in its capacity to reduce cell death following toxic insult. Interestingly, other sox proteins also had the ability to reduce caspase-6 activity but to a lesser extent than sox11.     

The N-terminal internal region of BLM is required for the formation of dots/rod-like structures which are associated with SUMO-1

Chemotherapeutic drug-induced apoptosis of human malignant glioma cells involves the death receptor-independent activation of caspases other than caspases 3 or 8 (Glaser et al., Oncogene 18, 5044-5053, 1999). Here, we report that caspases 1, 2, 3, 7, 8, and 9 are constitutively expressed in most human malignant glioma cell lines. Cytotoxic drug-induced apoptosisinvolves delayed activation of caspases 2, 7, and 9, but not 8 and 3, and is blocked by a broad spectrum caspase inhibitor, zVAD-fmk. Cytochrome c release from mitochondria precedes caspase activation during drug-induced apoptosis and is unaffected by zVAD-fmk or ectopic expression of the viral caspase inhibitor, crm-A. In contrast, ectopic expression of BCL-X(L) prevents drug-induced cytochrome c release, caspase activation and cell death. Thus, cancer chemotherapy targets the mitochondrial, caspase-dependent death pathway in human malignant glioma cells.     

Activity and expression of different members of the caspase family in the rat corpus luteum during pregnancy and postpartum

Studies were designed to examine the expression and activity of four caspases that contribute to the initial (caspases-2, -8, and -9) and final (caspase-3) events in apoptosis in the rat corpus luteum (CL) during pregnancy (days 7, 17, 19, and 21 of gestation), postpartum (days 1 and 4), and after injection (0, 8, 16, 24, and 36 h) of the physiological luteolysin PGF2alpha. In addition, the temporal relationship of caspase expression/activity relative to steroid production and luteal regression was evaluated. During pregnancy, the activity of all four caspases was significantly greater on day 19, before a decline in CL progesterone (P) and CYP11A1 levels at day 21 of gestation. The levels of the caspase-3 active fragment (p17, measured by Western blot) also increased at days 19 and 21 of pregnancy. Immunohistochemical analyses detected specific staining for the caspases in luteal cells (large and small) as well as in endothelial cells. However, the percentage of apoptotic cells did not increase in the CL until postpartum. Following PGF2alpha injection, there was a significant decrease in CL P by 24 h, although the activity of all four caspases did not increase until 36 h posttreatment. The active p17 fragment of caspase-3 also significantly increased at 36 h post-PGF2alpha. These results suggest that an increase in the activity of caspases-2, -8, -9, and -3 is associated with the early events of natural luteolysis at the end of pregnancy. Also, the exogenous administration of the luteolysin PGF2alpha may regulate members of the caspase family.     

Differential expression of rat brain caspase family proteins during development and aging.

It is well recognized that caspases are essential effector molecules for carrying out apoptosis in eukaryotic cells. The expression of rat brain caspase family proteins (caspase-2, -3, -6, -7, -8, -9, 10) in development and aging was assessed using immunochemical detection. All of these caspases were expressed in the rat brain. Immunoblot analysis of brain extracts from embryonic day 19 (E19) to postnatal 96-week-old rats indicated that cytosolic caspase-3, -7, -8, and -10 were highly expressed at E19, and decreased after birth. In contrast, cytosolic caspase-2, -6, and -9 were constitutively expressed from the early stages to 96 weeks of age. These results show that the expression of rat brain caspase family proteins is differentially regulated during the development and aging.     

Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways

Caspases orchestrate the controlled demise of a cell after an apoptotic signal through specific protease activity and cleavage of many substrates altering protein function and ensuring apoptosis proceeds efficiently. Comparing a variety of substrates of each apoptotic caspase (2, 3, 6, 7, 8, 9 and 10) showed that the cleavage sites had a general motif, sometimes specific for one caspase, but other times specific for several caspases. Using commercially available short peptide-based substrates and inhibitors the promiscuity for different cleavage motifs was indicated, with caspase-3 able to cleave most substrates more efficiently than those caspases to which the substrates are reportedly specific. In a cell-free system, immunodepletion of caspases before or after cytochrome c-dependent activation of the apoptosome indicated that the majority of activity on synthetic substrates was dependent on caspase-3, with minor roles played by caspases-6 and -7. Putative inhibitors of individual caspases were able to abolish all cytochrome c-induced caspase activity in a cell-free system and inhibit apoptosis in whole cells through the extrinsic and intrinsic pathways, raising issues regarding the use of such inhibitors to define relevant caspases and pathways. Finally, caspase activity in cells lacking caspase-9 displayed substrate cleavage activity of a putative caspase-9-specific substrate underlining the lack of selectivity of peptide-based substrates and inhibitors of caspases.     

Hydrophobic benzoic acids as inhibitors of influenza neuraminidase

Neuraminidase (NA) plays a critical role in the life cycle of influenza virus and is a target for new therapeutic agents. A new benzoic acid inhibitor (11) containing a lipophilic side chain at C-3 and a guanidine at C-5 was synthesized. The X-ray structure of 4-(N-acetylamino)-5-guanidino-3-(3-pentyloxy)benzoic acid in complex with NA revealed that the lipophilic side chain binds in a newly created hydrophobic pocket formed by the movement of Glu 278 to interact with Arg 226, whereas the guanidine of 11 interacts in a negatively charged pocket created by Asp 152, Glu 120 and Glu 229. Compound 11 was highly selective for type A (H2N2) influenza NA (IC50 1 microM) over type B (B/Lee/40) influenza NA (IC50 500 microM).     

The intermediate S1' pocket of the endometase/matrilysin-2 active site revealed by enzyme inhibition kinetic studies, protein sequence analyses, and homology modeling

Human matrix metalloproteinase-26 (MMP-26/endometase/matrilysin-2) is a newly identified MMP and its structure has not been reported. The enzyme active site S1' pocket in MMPs is a well defined substrate P1' amino acid residue-binding site with variable depth. To explore MMP-26 active site structure-activity, a series of new potent mercaptosulfide MMP inhibitors (MMPIs) with Leu or homophenylalanine (Homophe) side chains at the P1' site were selected. The Homephe side chain is designed to probe deep S1' pocket MMPs. These inhibitors were tested against MMP-26 and several MMPs with known x-ray crystal structures to distinguish shallow, intermediate, and deep S1' pocket characteristics. MMP-26 has an inhibition profile most similar to those of MMPs with intermediate S1' pockets. Investigations with hydroxamate MMPIs, including those designed for deep pocket MMPs, also indicated the presence of an intermediate pocket. Protein sequence analysis and homology modeling further verified that MMP-26 has an intermediate S1' pocket formed by Leu-204, His-208, and Tyr-230. Moreover, residue 233 may influence the depth of an MMP S1' pocket. The residue at the equivalent position of MMP-26 residue 233 is hydrophilic in intermediate-pocket MMPs (e.g. MMP-2, -8, and -9) and hydrophobic in deep-pocket MMPs (e.g. MMP-3, -12, and -14). MMP-26 contains a His-233 that renders the S1' pocket to an intermediate size. This study suggests that MMPIs, protein sequence analyses, and molecular modeling are useful tools to understand structure-activity relationships and provides new insight for rational inhibitor design that may distinguish MMPs with deep versus intermediate S1' pockets.     

Conformational similarity in the activation of caspase-3 and -7 revealed by the unliganded and inhibited structures of caspase-7

Caspase-mediated apoptosis has important roles in normal cell differentiation and aging and in many diseases including cancer, neuromuscular disorders and neurodegenerative diseases. Therefore, modulation of caspase activity and conformational states is of therapeutic importance. We report crystal structures of a new unliganded conformation of caspase-7 and the inhibited caspase-7 with the tetrapeptide Ac-YVAD-Cho. Different conformational states and mechanisms for substrate recognition have been proposed based on unliganded structures of the redundant apoptotic executioner caspase-3 and -7. The current study shows that the executioner caspase-3 and -7 have similar conformations for the unliganded active site as well as the inhibitor-bound active site. The new unliganded caspase-7 structure exhibits the tyrosine flipping mechanism in which the Tyr230 has rotated to block entry to the S2 binding site similar to the active site conformation of unliganded caspase-3. The inhibited structure of caspase-7/YVAD shows that the P4 Tyr binds the S4 region specific to polar residues at the expense of a main chain hydrogen bond between the P4 amide and carbonyl oxygen of caspase-7 Gln 276, which is similar to the caspase-3 complex. This new knowledge of the structures and conformational states of unliganded and inhibited caspases will be important for the design of drugs to modulate caspase activity and apoptosis.     

Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates

Traditional combinatorial peptidyl substrate library approaches generally utilize natural amino acids, limiting the usefulness of this tool in generating selective substrates for proteases that share similar substrate specificity profiles. To address this limitation, we synthesized a Hybrid Combinatorial Substrate Library (HyCoSuL) with the general formula of Ac-P4-P3-P2-Asp-ACC, testing the approach on a family of closely related proteases – the human caspases. The power of this library for caspase discrimination extends far beyond traditional PS-SCL approach, as in addition to 19 natural amino acids we also used 110 diverse unnatural amino acids that can more extensively explore the chemical space represented by caspase-active sites. Using this approach we identified and employed peptide-based substrates that provided excellent discrimination between individual caspases, allowing us to simultaneously resolve the individual contribution of the apical caspase-9 and the executioner caspase-3 and caspase-7 in the development of cytochrome-c-dependent apoptosis for the first time.Apoptosis, the most well-understood form of programmed cell death, is a highly regulated process controlled and executed by proteolytic enzymes called caspases. The apoptotic process is somewhat hierarchical and caspases can be assigned as initiators (2, 8, 9, and 10) and executioners (3, 6, and 7).^{1, 2, 3} Apoptosis can be triggered extrinsically via ligation of a death receptor by its cognate ligands, leading to the activation of caspases 8 and 10, or intrinsically following the release of cytochrome c from mitochondria with formation of a caspase 9 activation complex known as the apoptosome.^{3, 4, 5} Mechanistically, caspases display a near absolute preference for aspartate at the P1 position of their substrates. In addition, they require a minimum substrate length of four amino acids N-terminal of the scissile bond. Thornberry et al.^{6, 7} used a combinatorial library of fluorogenic substrates to profile nine human caspases at the P4–P2 region, demonstrating that the caspases tended to have specificity profiles that enabled grouping based on substrate preferences.^{6, 7} This work provided a great insight into caspase recognition patterns and opened the door for others to pursue small molecule probes for caspase investigations.To date, various types of substrates and inhibitors have been developed and biologically evaluated against caspases.^{2, 8, 9, 10, 11} Unfortunately, most of them lack selectivity and cannot be used for selectively targeting or analyzing particular enzymes in complex biological environments.^{12, 13, 14, 15} This is entirely because of the overlapping specificities of the caspases on their preferred natural amino acid sequences. To address this problem we designed and synthesized a Hybrid Combinatorial Substrate Library (HyCoSuL) containing 19 natural amino acids (omitting cysteine) and 110 unnatural amino acids. We propose that such a large and varied set of chemical structures provides an excellent tool to investigate caspases and distinguish between them. In this work we dissected the kinetic profiles of six human apoptotic recombinant caspases through HyCoSuL screening. We then designed and synthesized new caspase substrates with the ability to discriminate these enzymes within a group. To further test the specificity and utility of the designed hybrid substrates, we performed a series of experiments in a cell-free model of apoptosis where multiple caspases are activated.