Carlos R. Reis

DR4-selective tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) variants obtained by structure-based design

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer agent that selectively induces apoptosis in a variety of cancer cells by interacting with death receptors DR4 and DR5. TRAIL can also bind to decoy receptors (DcR1, DcR2, and osteoprotegerin receptor) that cannot induce apoptosis. Different tumor types respond either to DR4 or to DR5 activation, and chemotherapeutic drugs can increase the expression of DR4 or DR5 in cancer cells. Thus, DR4 or DR5 receptor-specific TRAIL variants would permit new and tumor-selective therapies. Previous success in generating a DR5-selective TRAIL mutant using computer-assisted protein design prompted us to make a DR4-selective TRAIL variant. Technically, the design of DR4 receptor-selective TRAIL variants is considerably more challenging compared with DR5 receptor-selective variants, because of the lack of a crystal structure of the TRAIL-DR4 complex. A single amino acid substitution of Asp at residue position 218 of TRAIL to His or Tyr was predicted to have a favorable effect on DR4 binding specificity. Surface plasmon resonance-based receptor binding tests showed a lowered DR5 affinity in concert with increased DR4 specificity for the designed variants, D218H and D218Y. Binding to DcR1, DcR2, and osteoprotegerin was also decreased. Cell line assays confirmed that the variants could not induce apoptosis in DR5-responsive Jurkat and A2780 cells but were able to induce apoptosis in DR4-responsive EM-2 and ML-1 cells.

Rapid and efficient cancer cell killing mediated by high-affinity death receptor homotrimerizing TRAIL variants.

The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.

Crosstalk between Akt/GSK3β signaling and dynamin-1 regulates clathrin-mediated endocytosis.

Clathrin‐mediated endocytosis (CME) regulates signaling from the plasma membrane. Analysis of clathrin‐coated pit (CCP) dynamics led us to propose the existence of a rate‐limiting, regulatory step(s) that monitor the fidelity of early stages in CCP maturation. Here we show that nascent endocytic vesicles formed in mutant cells displaying rapid, dysregulated CME are defective in early endosomal trafficking, maturation and acidification, confirming the importance of this “checkpoint.” Dysregulated CME also alters EGF receptor signaling and leads to constitutive activation of the protein kinase Akt. Dynamin‐1, which was thought to be neuron specific, is activated by the Akt/GSK3β signaling cascade in non‐neuronal cells to trigger rapid, dysregulated CME. Acute activation of dynamin‐1 in RPE cells by inhibition of GSK3β accelerates CME, alters CCP dynamics and, unexpectedly, increases the rate of CCP initiation. CRISPR‐Cas9n‐mediated knockout and reconstitution studies establish that dynamin‐1 is activated by Akt/GSK3β signaling in H1299 non‐small lung cancer cells. These findings provide direct evidence for an isoform‐specific role for dynamin in regulating CME and reveal a feed‐forward pathway that could link signaling from cell surface receptors to the regulation of CME.

Engineering methylaspartate ammonia lyase for the asymmetric synthesis of unnatural amino acids

The redesign of enzymes to produce catalysts for a predefined transformation remains a major challenge in protein engineering. Here, we describe the structure-based engineering of methylaspartate ammonia lyase (which in nature catalyses the conversion of 3-methylaspartate to ammonia and 2-methylfumarate) to accept a variety of substituted amines and fumarates and catalyse the asymmetric synthesis of aspartic acid derivatives. We obtained two single-active-site mutants, one exhibiting a wide nucleophile scope including structurally diverse linear and cyclic alkylamines and one with broad electrophile scope including fumarate derivatives with alkyl, aryl, alkoxy, aryloxy, alkylthio and arylthio substituents at the C2 position. Both mutants have an enlarged active site that accommodates the new substrates while retaining the high stereo- and regioselectivity of the wild-type enzyme. As an example, we demonstrate a highly enantio- and diastereoselective synthesis of threo-3-benzyloxyaspartate (an important inhibitor of neuronal excitatory glutamate transporters in the brain). Substituted aspartic acids are highly valuable as tools for biological research and as chiral building blocks for pharmaceuticals. Here, engineering of the enzyme methylaspartate ammonia lyase to accept a large variety of substituted amines and fumarates and catalyse the asymmetric synthesis of aspartic acid derivatives is described.

Enhancement of Antitumor Properties of rhTRAIL by Affinity Increase toward Its Death Receptors

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent and selective inducer of apoptosis in various tumor types, raising enthusiasm for TRAIL as a potential anticancer agent. TRAIL-induced apoptosis is mediated by death receptors 4 (DR4) and DR5. The design of rhTRAIL variants either with improved affinity or selectivity toward one or both death-inducing receptors is thought to enhance the therapeutical potential of TRAIL. Here we demonstrate that a single amino acid mutation at the position of glycine 131 to lysine or arginine in wild-type rhTRAIL significantly improved the affinity of rhTRAIL toward its death receptors, with the highest affinity increase observed for the DR4 receptor. These variants were able to induce higher in vitro levels of apoptosis in cancer cells responsive to only DR4 or to both death receptors and could therefore increase the potential use of rhTRAIL as an anticancer therapeutic agent.

Reducing virulence of the human pathogen Burkholderia by altering the substrate specificity of the quorum-quenching acylase PvdQ

Significance Resistance toward commonly used antibiotics is becoming a serious issue in the fight against bacterial pathogens. One promising strategy lies in the interference of bacterial quorum sensing by the hydrolysis of the signaling molecules. In this study, we present a structure-aided computational design approach to alter the substrate specificity of the quorum-quenching acylase PvdQ. Introduction of two point mutations in residues lining the active site led to a switch in substrate specificity, rendering the enzyme highly active toward C8-HSL and thereby reducing virulence caused by Burkholderia cenocepacia. Thus, this work not only provides a structural insight into the substrate specificity of quorum-quenching acylases but also indicates their potential in the fight against specific bacterial pathogens. The use of enzymes to interfere with quorum sensing represents an attractive strategy to fight bacterial infections. We used PvdQ, an effective quorum-quenching enzyme from Pseudomonas aeruginosa, as a template to generate an acylase able to effectively hydrolyze C8-HSL, the major communication molecule produced by the Burkholderia species. We discovered that the combination of two single mutations leading to variant PvdQLα146W,Fβ24Y conferred high activity toward C8-HSL. Exogenous addition of PvdQLα146W,Fβ24Y dramatically decreased the amount of C8-HSL present in Burkholderia cenocepacia cultures and inhibited a quorum sensing-associated phenotype. The efficacy of this PvdQ variant to combat infections in vivo was further confirmed by its ability to rescue Galleria mellonella larvae upon infection, demonstrating its potential as an effective agent toward Burkholderia infections. Kinetic analysis of the enzymatic activities toward 3-oxo-C12-L-HSL and C8-L-HSL corroborated a substrate switch. This work demonstrates the effectiveness of quorum-quenching acylases as potential novel antimicrobial drugs. In addition, we demonstrate that their substrate range can be easily switched, thereby paving the way to selectively target only specific bacterial species inside a complex microbial community.

Decoy receptors block TRAIL sensitivity at a supracellular level: the role of stromal cells in controlling tumour TRAIL sensitivity.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand cytokine known for its cytotoxic activity against malignantly transformed cells. TRAIL induces cell death through binding to death receptors DR4 and DR5. The inhibitory decoy receptors (DcR1 and DcR2) co-expressed with death receptor 4 (DR4)/DR5 on the same cell can block the transmission of the apoptotic signal. Here, we show that DcRs also regulate TRAIL sensitivity at a supracellular level and thus represent a mechanism by which the microenvironment can diminish tumour TRAIL sensitivity. Mathematical modelling and layered or spheroid stroma–extracellular matrix–tumour cultures were used to model the tumour microenvironment. By engineering TRAIL to escape binding by DcRs, we found that DcRs do not only act in a cell-autonomous or cis-regulatory manner, but also exert trans-cellular regulation originating from stromal cells and affect tumour cells, highlighting the potent inhibitory effect of DcRs in the tumour tissue and the necessity of selective targeting of the two death-inducing TRAIL receptors to maximise efficacy.

Alteration of the Diastereoselectivity of 3-Methylaspartate Ammonia Lyase by Using Structure-Based Mutagenesis

3‐Methylaspartate ammonia‐lyase (MAL) catalyzes the reversible amination of mesaconate to give both (2S,3S)‐3‐methylaspartic acid and (2S,3R)‐3‐methylaspartic acid as products. The deamination mechanism of MAL is likely to involve general base catalysis, in which a catalytic base abstracts the C3 proton of the respective stereoisomer to generate an enolate anion intermediate that is stabilized by coordination to the essential active‐site MgII ion. The crystal structure of MAL in complex with (2S,3S)‐3‐methylaspartic acid suggests that Lys331 is the only candidate in the vicinity that can function as a general base catalyst. The structure of the complex further suggests that two other residues, His194 and Gln329, are responsible for binding the C4 carboxylate group of (2S,3S)‐3‐methylaspartic acid, and hence are likely candidates to assist the MgII ion in stabilizing the enolate anion intermediate. In this study, the importance of Lys331, His194, and Gln329 for the activity and stereoselectivity of MAL was investigated by site‐directed mutagenesis. His194 and Gln329 were replaced with either an alanine or arginine, whereas Lys331 was mutated to a glycine, alanine, glutamine, arginine, or histidine. The properties of the mutant proteins were investigated by circular dichroism (CD) spectroscopy, kinetic analysis, and 1H NMR spectroscopy. The CD spectra of all mutants were comparable to that of wild‐type MAL, and this indicates that these mutations did not result in any major conformational changes. Kinetic studies demonstrated that the mutations have a profound effect on the values of kcat and kcat/KM; this implicates Lys331, His194 and Gln329 as mechanistically important. The 1H NMR spectra of the amination and deamination reactions catalyzed by the mutant enzymes K331A, H194A, and Q329A showed that these mutants have strongly enhanced diastereoselectivities. In the amination direction, they catalyze the conversion of mesaconate to yield only (2S,3S)‐3‐methylaspartic acid, with no detectable formation of (2S,3R)‐3‐methylaspartic acid. The results are discussed in terms of a mechanism in which Lys331, His194, and Gln329 are involved in positioning the substrate and in formation and stabilization of the enolate anion intermediate.

Targeting AML through DR4 with a novel variant of rhTRAIL

Despite progress in the treatment of acute myelogenous leukaemia (AML) the outcome often remains poor. Tumour necrosis factor related apoptosis‐inducing ligand (TRAIL) is a promising therapeutic agent in many different types of tumours, but AML cells are relatively insensitive to TRAIL‐induced apoptosis. Here we show that TRAIL‐induced apoptosis in AML cells is predominantly mediated by death receptor 4 (DR4) and not DR5. Therefore, we constructed a variant of TRAIL (rhTRAIL‐C3) that is a strong inducer of DR4‐mediated apoptosis. TRAIL‐C3 demonstrated much stronger pro‐apoptotic activity than wild‐type (WT) TRAIL in a panel of AML cell lines as well as in primary AML blasts. The higher pro‐apoptotic potential was further enhanced when the TRAIL mutant was used in combination with BMS‐345541, a selective inhibitor of inhibitor‐κB kinases. It illustrates that combination of this TRAIL variant with chemotherapeutics or other targeted agents can kill AML with high efficacy. This may represent a major advantage over the currently used therapies that have serious toxic side effects. The high efficacy of rhTRAIL‐C3 containing therapies may enable the use of lower drug doses to reduce the toxic side effects and improve patient outcome. Our findings suggest that the rational design of TRAIL variants that target DR4 potentiate the death‐inducing activity of TRAIL and offer a novel therapeutic strategy for the treatment of AML.

Antibody-free LC-MS/MS quantification of rhTRAIL in human and mouse serum

The major challenge in targeted protein quantification by LC-MS/MS in serum lies in the complexity of the biological matrix with regard to the wide diversity of proteins and their extremely large dynamic concentration range. In this study, an LC-MS/MS method was developed for the simultaneous quantification of the 60-kDa biopharmaceutical proteins recombinant human tumor necrosis factor-related apoptosis-inducing ligand wild type (rhTRAIL(WT)) and its death receptor 4 (DR4)-specific variant rhTRAIL(4C7) in human and mouse serum. Selective enrichment of TRAIL was accomplished by immobilized metal affinity chromatography (IMAC), which was followed by tryptic digestion of the enriched sample and quantification of a suitable signature peptide. For absolute quantification, (15)N-metabolically labeled internal standards of rhTRAIL(WT) and rhTRAIL(4C7) were used. Since the signature peptides that provided the highest sensitivity and allowed discrimination between rhTRAIL(WT) and rhTRAIL(4C7) contained methionine residues, we oxidized these quantitatively to their sulfoxides by the addition of 0.25% (w/w) hydrogen peroxide. The final method has a lower limit of quantification of 20 ng/mL (ca. 350 pM) and was fully validated according to current international guidelines for bioanalysis. To show the applicability of the LC-MS/MS method for pharmacokinetic studies, we quantified rhTRAIL(WT) and rhTRAIL(4C7) simultaneously in serum from mice injected intraperitoneally at a dose of 5 mg/kg for each protein. This is the first time that two variants of rhTRAIL differing by only a few amino acids have been analyzed simultaneously in serum, an approach that is not possible by conventional enzyme-linked immuno-sorbent assay (ELISA) analysis.