Investigating Polypharmacology through Targeting Known Human Neutrophil Elastase Inhibitors to Proteinase 3.

Using a combination of multisite λ-dynamics (MSλD) together with in vitro IC50 assays, we evaluated the polypharmacological potential of a scaffold currently in clinical trials for inhibition of human neutrophil elastase (HNE), targeting cardiopulmonary disease, for efficacious inhibition of Proteinase 3 (PR3), a related neutrophil serine proteinase. The affinities we observe suggest that the dihydropyrimidinone scaffold can serve as a suitable starting point for the establishment of polypharmacologically targeting both enzymes and enhancing the potential for treatments addressing diseases like chronic obstructive pulmonary disease.

Structural determinants and cellular environment define processed actin as the sole substrate of the N-terminal acetyltransferase NAA80.

N-terminal (Nt) acetylation is a major protein modification catalyzed by N-terminal acetyltransferases (NATs). Methionine acidic N termini, including actin, are cotranslationally Nt acetylated by NatB in all eukaryotes, but animal actins containing acidic N termini, are additionally posttranslationally Nt acetylated by NAA80. Actin Nt acetylation was found to regulate cytoskeletal dynamics and motility, thus making NAA80 a potential target for cell migration regulation. In this work, we developed potent and selective bisubstrate inhibitors for NAA80 and determined the crystal structure of NAA80 in complex with such an inhibitor, revealing that NAA80 adopts a fold similar to other NAT enzymes but with a more open substrate binding region. Furthermore, in contrast to most other NATs, the substrate specificity of NAA80 is mainly derived through interactions between the enzyme and the acidic amino acids at positions 2 and 3 of the actin substrate and not residues 1 and 2. A yeast model revealed that ectopic expression of NAA80 in a strain lacking NatB activity partially restored Nt acetylation of NatB substrates, including yeast actin. Thus, NAA80 holds intrinsic capacity to posttranslationally Nt acetylate NatB-type substrates in vivo. In sum, the presence of a dominant cotranslational NatB in all eukaryotes, the specific posttranslational actin methionine removal in animals, and finally, the unique structural features of NAA80 leave only the processed actins as in vivo substrates of NAA80. Together, this study reveals the molecular and cellular basis of NAA80 Nt acetylation and provides a scaffold for development of inhibitors for the regulation of cytoskeletal properties.

Total Synthesis of Phorbazole B.

Phorbazoles are polychlorinated heterocyclic secondary metabolites isolated from a marine sponge and several of these natural products have shown inhibitory activity against cancer cells. In this work, a synthesis of the trichlorinated phorbazole B using late stage electrophilic chlorination was developed. The synthesis relied on the use of an oxazole precursor, which was protected with an iodine in the reactive 4-position, followed by complete chlorination of all pyrrole positions. Attempts to prepare phorbazole A and C, which contain a 3,4-dichlorinated pyrrole, were unsuccessful as the desired chlorination pattern on the pyrrole could not be obtained. The identities of the dichlorinated intermediates and products were determined using NMR techniques including NOESY/ROESY, 1,1-ADEQUATE and high-resolution CLIP-HSQMBC.

Influence of chain length on the activity of tripeptidomimetic antagonists for CXC chemokine receptor 4 (CXCR4).

Here we report a series of close analogues of our recently published scaffold-based tripeptidomimetic CXCR4 antagonists, containing positively charged guanidino groups in R1 and R2, and an aromatic group in R3. While contraction/elongation of the guanidine carrying side chains (R1 and R2) resulted in loss of activity, introduction of bromine in position 1 on the naphth-2-ylmethyl moiety (R3) resulted in an EC50 of 61µM (mixture of diastereoisomers) against wild-type CXCR4; thus, the antagonistic activity of these tripeptidomimetics seems to be amenable to optimization of the aromatic moiety. Moreover, for analogues carrying a naphth-2-ylmethyl substituent, we observed that a Pictet-Spengler like cyclization side reaction depended on the nature of the R1 substituent.

Design, synthesis, and biological evaluation of scaffold-based tripeptidomimetic antagonists for CXC chemokine receptor 4 (CXCR4).

Structure-activity relationship studies of the cyclopentapeptide CXCR4 antagonists (cyclo(-l-/d-Arg(1)-Arg(2)-2-Nal(3)-Gly(4)-d-Tyr(5)-)) suggest that the l-/d-Arg(1)-Arg(2)-2-Nal(3) tripeptide sequence contained within these cyclopentapeptides serves as a recognition motif for peptidic CXCR4 antagonists. Starting by dissecting the cyclopentapeptide structure and reintroducing cyclic constraints in a stepwise manner, we here report a novel class of scaffold-based tripeptidomimetic CXCR4 antagonists based on the d-Arg-Arg-2-Nal motif. Biological testing of the prototype compounds showed that they represent new peptidomimetic hits; importantly, the modular nature of the scaffold provides an interesting starting point for future ligand optimization.

Expression and purification of human neutrophil proteinase 3 from insect cells and characterization of ligand binding.

Neutrophil proteinase 3 (PR3) is an important drug target for inflammatory lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Drug discovery efforts targeting PR3 require active enzyme for in vitro characterization, such as inhibitor screening, enzymatic assays, and structural studies. Recombinant expression of active PR3 overcomes the need for enzyme supplies from human blood and in addition allows studies on the influence of mutations on enzyme activity and ligand binding. Here, we report the expression of recombinant PR3 (rPR3) using a baculovirus expression system. The purification and activation process described resulted in highly pure and active PR3. The activity of rPR3 in the presence of commercially available inhibitors was compared with human PR3 by using a fluorescence-based enzymatic assay. Purified rPR3 had comparable activity to the native human enzyme, thus being a suitable alternative for enzymatic studies in vitro. Further, we established a surface plasmon resonance-based assay to determine binding affinities and kinetics of PR3 ligands. These methods provide valuable tools for early drug discovery aiming towards treatment of lung inflammation.

Phospholipid Membrane Interactions of Model Ac-WL-X-LL-OH Peptides Investigated by Solid-State Nuclear Magnetic Resonance.

The role of aromatic amino acids in peripheral protein membrane binding has been reported to involve cation-π interactions with choline lipids. In this study, we have investigated the interactions of the model pentapeptide Ac-WL-X-LL-OH (where X = L, Y, F, or W) with the phospholipid membrane using solid-state NMR. The effect of guest residue X on the peptide-lipid interactome was complementary to the seminal report on the interfacial hydrophobicity scale by Wimley and White. We found that the phospholipids retained a lamellar phase in the presence of each of the peptides with an aromatic X residue, whereas the Leu peptide perturbed the bilayer to an extent where an additional isotropic phase was observed. The solid-state NMR 13C and 31P data provide additional information on the influence of these short peptides on the membrane that has not been previously reported. The magnitude of membrane perturbation was in the order of guest residue X = L > Y~F > W, which is consistent with the relative amino acid interfacial affinity reported by Wimley and White. Further work is, however, required to uncover the behavior of the peptide and localization in the membrane domain due to ambiguity of the 13C NMR data. We have launched efforts in this regard for the objective of better understanding the role of aromatic amino acids in peripheral membrane protein binding.

Relative spatial positions of tryptophan and cationic residues in helical membrane-active peptides determine their cytotoxicity.

The cytotoxic activity of 10 analogs of the idealized amphipathic helical 21-mer peptide (KAAKKAA)3, where three of the Ala residues at different positions have been replaced with Trp residues, has been investigated. The peptide's cytotoxic activity was found to be markedly dependent upon the position of the Trp residues within the hydrophobic sector of an idealized α-helix. The peptides with Trp residues located opposite the cationic sector displayed no antitumor activity, whereas those peptides with two or three Trp residues located adjacent to the cationic sector exhibited high cytotoxic activity when tested against three different cancer cell lines. Dye release experiments revealed that in contrast to the peptides with Trp residues located opposite the cationic sector, the peptides with Trp residues located adjacent to the cationic sector induced a strong permeabilizing activity from liposomes composed of a mixture of zwitterionic phosphatidylcholine and negatively charged phosphatidylserine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS)) (2:1) but not from liposomes composed of zwitterionic phosphatidylcholine, POPC. Fluorescence blue shift and quenching experiments revealed that Trp residues inserted deeper into the hydrophobic environment of POPC/POPS liposomes for peptides with high cytotoxic activity. Through circular dichroism studies, a correlation between the cytotoxic activity and the α-helical propensity was established. Structural studies of one inactive and two active peptides in the presence of micelles using NMR spectroscopy showed that only the active peptides adopted highly coiled to helical structures when bound to a membrane surface.

Optimized bisubstrate inhibitors for the actin N-terminal acetyltransferase NAA80.

Acetylation of protein N-termini is one of the most common protein modifications in the eukaryotic cell and is catalyzed by the N-terminal acetyltransferase family of enzymes. The N-terminal acetyltransferase NAA80 is expressed in the animal kingdom and was recently found to specifically N-terminally acetylate actin, which is the main component of the microfilament system. This unique animal cell actin processing is essential for the maintenance of cell integrity and motility. Actin is the only known substrate of NAA80, thus potent inhibitors of NAA80 could prove as important tool compounds to study the crucial roles of actin and how NAA80 regulates this by N-terminal acetylation. Herein we describe a systematic study toward optimizing the peptide part of a bisubstrate-based NAA80 inhibitor comprising of coenzyme A conjugated onto the N-terminus of a tetrapeptide amide via an acetyl linker. By testing various combinations of Asp and Glu which are found at the N-termini of ß- and γ-actin, respectively, CoA-Ac-EDDI-NH2 was identified as the best inhibitor with an IC50 value of 120 nM.

Comparison of Five Near-Infrared Fluorescent Folate Conjugates in an Ovarian Cancer Model.

PURPOSE: Fluorescence imaging (FLI) using targeted near-infrared (NIR) conjugates aids the detection of tumour lesions pre- and intraoperatively. The optimisation of tumour visualisation and contrast is essential and can be achieved through high tumour-specificity and low background signal. However, the choice of fluorophore is recognised to alter biodistribution and clearance of conjugates and is therefore a determining factor in the specificity of target binding. Although ZW800-1, IRDye® 800CW and ICG are the most commonly employed NIR fluorophores in clinical settings, the fluorophore with optimal in vivo characteristics has yet to be determined. Therefore, we aimed to characterise the impact the choice of fluorophore has on the biodistribution, specificity and contrast, by comparing five different NIR fluorophores conjugated to folate, in an ovarian cancer model. PROCEDURES: ZW800-1, ZW800-1 Forte, IRDye® 800CW, ICG-OSu and an in-house synthesised Cy7 derivative were conjugated to folate through an ethylenediamine linker resulting in conjugates 1-5, respectively. The optical properties of all conjugates were determined by spectroscopy, the specificity was assessed in vitro by flow cytometry and FLI, and the biodistribution was studied in vivo and ex vivo in a subcutaneous Skov-3 ovarian cancer model. RESULTS: We demonstrated time- and receptor-dependent binding of folate conjugates in vitro and in vivo. Healthy tissue clearance characteristics and tumour-specific signal varied between conjugates 1-5. ZW800-1 Forte (2) revealed the highest contrast in folate receptor alpha (FRα)-positive xenografts and showed statistically significant target specificity. While conjugates 1, 2 and 3 are renally cleared, hepatobiliary excretion and no or very low accumulation in tumours was observed for 4 and 5. CONCLUSIONS: The choice of fluorophore has a significant impact on the biodistribution and tumour contrast. ZW800-1 Forte (2) exhibited the best properties of those tested, with significant specific fluorescence signal.

Identification of a Phage Display-Derived Peptide Interacting with the N-Terminal Region of Factor VII Activating Protease (FSAP) Enables Characterization of Zymogen Activation.

Factor VII Activating protease (FSAP) has a protective effect in diverse disease conditions as inferred from studies in FSAP-/- mice and humans deficient in FSAP activity due to single-nucleotide polymorphism. The zymogen form of FSAP in plasma is activated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear whether this activation mechanism is specific and amenable to manipulation. Using a phage display approach, we have identified a Cys-constrained 11 amino acid peptide, NNKC9/41, that activates pro-FSAP in plasma. The synthetic linear peptide has a propensity to cyclize through the terminal Cys groups, of which the antiparallel cyclic dimer, but not the monocyclic peptide, is the active component. Other commonly found zymogens in the plasma, related to the hemostasis system, were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30-35) in an intrinsically disordered stretch in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41, and this was reversed by MA-FSAP-38C7, demonstrating the utility of this peptide. Peptide NNKC9/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP, elucidate its biological role, and provide a starting point for the pharmacological manipulation of FSAP activity.

An Experimental Toolbox for Structure-Based Hit Discovery for P.†aeruginosa FabF, a Promising Target for Antibiotics.

FabF (3-oxoacyl-[acyl-carrier-protein] synthase 2), which catalyses the rate limiting condensation reaction in the fatty acid synthesis II pathway, is an attractive target for new antibiotics. Here, we focus on FabF from P. aeruginosa (PaFabF) as antibiotics against this pathogen are urgently needed. To facilitate exploration of this target we have set up an experimental toolbox consisting of binding assays using bio-layer interferometry (BLI) as well as saturation transfer difference (STD) and WaterLOGSY NMR in addition to robust conditions for structure determination. The suitability of the toolbox to support structure-based design of FabF inhibitors was demonstrated through the validation of hits obtained from virtual screening. Screening a library of almost 5 million compounds resulted in 6 compounds for which binding into the malonyl-binding site of FabF was shown. For one of the hits, the crystal structure in complex with PaFabF was determined. Based on the obtained binding mode, analogues were designed and synthesised, but affinity could not be improved. This work has laid the foundation for structure-based exploration of PaFabF.

Repurposing 18F-FMISO as a PET tracer for translational imaging of nitroreductase-based gene directed enzyme prodrug therapy.

Nitroreductases (NTR) are a family of bacterial enzymes used in gene directed enzyme prodrug therapy (GDEPT) that selectively activate prodrugs containing aromatic nitro groups to exert cytotoxic effects following gene transduction in tumours. The clinical development of NTR-based GDEPT has, in part, been hampered by the lack of translational imaging modalities to assess gene transduction and drug cytotoxicity, non-invasively. This study presents translational preclinical PET imaging to validate and report NTR activity using the clinically approved radiotracer, 18F-FMISO, as substrate for the NTR enzyme. Methods: The efficacy with which 18F-FMISO could be used to report NfsB NTR activity in vivo was investigated using the MDA-MB-231 mammary carcinoma xenograft model. For validation, subcutaneous xenografts of cells constitutively expressing NTR were imaged using 18F-FMISO PET/CT and fluorescence imaging with CytoCy5S, a validated fluorescent NTR substrate. Further, examination of the non-invasive functionality of 18F-FMISO PET/CT in reporting NfsB NTR activity in vivo was assessed in metastatic orthotopic NfsB NTR expressing xenografts and metastasis confirmed by bioluminescence imaging. 18F-FMISO biodistribution was acquired ex vivo by an automatic gamma counter measuring radiotracer retention to confirm in vivo results. To assess the functional imaging of NTR-based GDEPT with 18F-FMISO, PET/CT was performed to assess both gene transduction and cytotoxicity effects of prodrug therapy (CB1954) in subcutaneous models. Results:18F-FMISO retention was detected in NTR+ subcutaneous xenografts, displaying significantly higher PET contrast than NTR- xenografts (p < 0.0001). Substantial 18F-FMISO retention was evident in metastases of orthotopic xenografts (p < 0.05). Accordingly, higher 18F-FMISO biodistribution was prevalent ex vivo in NTR+ xenografts. 18F-FMISO NfsB NTR PET/CT imaging proved useful for monitoring in vivo NTR transduction and the cytotoxic effect of prodrug therapy. Conclusions:18F-FMISO NfsB NTR PET/CT imaging offered significant contrast between NTR+ and NTR- tumours and effective resolution of metastatic progression. Furthermore, 18F-FMISO NfsB NTR PET/CT imaging proved efficient in monitoring the two steps of GDEPT, in vivo NfsB NTR transduction and response to CB1954 prodrug therapy. These results support the repurposing of 18F-FMISO as a readily implementable PET imaging probe to be employed as companion diagnostic test for NTR-based GDEPT systems.

Kinase Chemodiversity from the Arctic: The Breitfussins.

In this work, we demonstrate that the indole-oxazole-pyrrole framework of the breitfussin family of natural products is a promising scaffold for kinase inhibition. Six new halogenated natural products, breitfussin C-H (3 - 8) were isolated and characterized from the Arctic, marine hydrozoan Thuiaria breitfussi. The structures of two of the new natural products were also confirmed by total synthesis. Two of the breitfussins (3 and 4) were found to selectively inhibit the survival of several cancer cell lines, with the lowest IC50 value of 340 nM measured against the drug-resistant triple negative breast cancer cell line MDA-MB-468, while leaving the majority of the tested cell lines not or significantly less affected. When tested against panels of protein kinases, 3 gave IC50 and Kd values as low as 200 and 390 nM against the PIM1 and DRAK1 kinases, respectively. The activity was confirmed to be mediated through ATP competitive binding in the ATP binding pocket of the kinases. Furthermore, evaluation of potential off-target and toxicological effects, as well as relevant in vitro ADME parameters for 3 revealed that the breitfussin scaffold holds promise for the development of selective kinase inhibitors.

LTX-315: a first-in-class oncolytic peptide that reprograms the tumor microenvironment.

The oncolytic peptide LTX-315, which has been de novo designed based on structure-activity relationship studies of host defense peptides, has the ability to kill human cancer cells and induce specific anticancer immune response when injected locally into tumors established in immunocompetent mice. The oncolytic effect of LTX-315 involves perturbation of plasma membrane and the mitochondria with subsequent release of danger-associated molecular pattern molecules, which highlights the ability of LTX-315 to induce complete regression and protective immune responses. Treatment with LTX-315 reprograms the tumor microenvironment by decreasing the local abundance of immunosuppressive cells and by increasing the frequency of effector T cells.

Oncolytic peptide LTX-315 induces an immune-mediated abscopal effect in a rat sarcoma model.

LTX 315 is an oncolytic peptide with potent immunological properties. In the present study, we demonstrate that intratumoral treatment with LTX-315 resulted in a complete regression and systemic immune response in a rat fibrosarcoma model. The treatment was T-cell dependent, and also resulted in an abscopal effect as demonstrated by the regression of distal non-treated lesions. Significant infiltration of CD8+ T cells was observed in both treated and non-treated lesions, as shown by immunohistochemical and flow cytometric analysis. LTX-315 rapidly killed the cells in vitro with a lytic mode of action followed by the subsequent release of Danger-Associated Molecular Pattern (DAMP) molecules such as HMGB1, ATP and Cytochrome c. Together, our data demonstrate that LTX-315 represents a new approach to cancer immunotherapy, which has the potential as a novel immunotherapeutic agent.

Crystal Structure of the Golgi-Associated Human Nα-Acetyltransferase 60 Reveals the Molecular Determinants for Substrate-Specific Acetylation.

N-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for the function of Naa60, we developed an Naa60 bisubstrate CoA-peptide conjugate inhibitor, determined its X-ray structure when bound to CoA and inhibitor, and carried out biochemical experiments. We show that Naa60 adapts an overall fold similar to that of the catalytic subunits of ribosome-associated NATs, but with the addition of two novel elongated loops that play important roles in substrate-specific binding. One of these loops mediates a dimer to monomer transition upon substrate-specific binding. Naa60 employs a catalytic mechanism most similar to Naa50. Collectively, these data reveal the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions.

Discovery of a 9-mer Cationic Peptide (LTX-315) as a Potential First in Class Oncolytic Peptide.

Oncolytic immunotherapies represent a new promising strategy in the treatment of cancer. In our efforts to develop oncolytic peptides, we identified a series of chemically modified 9-mer cationic peptides that were highly effective against both drug-resistant and drug-sensitive cancer cells and with lower toxicity toward normal cells. Among these peptides, LTX-315 displayed superior anticancer activity and was selected as a lead candidate. This peptide showed relative high plasma protein binding abilities and a human plasma half-life of 160 min, resulting in formation of nontoxic metabolites. In addition, the lead candidate demonstrated relatively low ability to inhibit CYP450 enzymes. Collectively these data indicated that this peptide has potential to be developed as a new anticancer agent for intratumoral administration and is currently being evaluated in a phase I/IIa study.

Progress toward rationally designed small-molecule peptide and peptidomimetic CXCR4 antagonists.

Over the last 5 years, X-ray structures of CXCR4 in complex with three different ligands (the small-molecule antagonist IT1t, the polypeptide antagonist CVX15 and the viral chemokine antagonist vMIP-II) have been released. In addition to the inherent scientific value of these specific X-ray structures, they provide a reliable structural foundation for studies of the molecular interactions between CXCR4 and its key peptide ligands (CXCL12 and HIV-1 gp120), and serve as valuable templates for further development of small-molecule CXCR4 antagonists with therapeutic potential. We here review recent computational studies of the molecular interactions between CXCR4 and its peptide ligands - based on the X-ray structures of CXCR4 - and the current status of small-molecule peptide and peptidomimetic CXCR4 antagonists.

Probing the Molecular Interactions between CXC Chemokine Receptor 4 (CXCR4) and an Arginine-Based Tripeptidomimetic Antagonist (KRH-1636).

We here report an experimentally verified binding mode for the known tripeptidomimetic CXCR4 antagonist KRH-1636 (1). A limited SAR study based on the three functionalities of 1 was first conducted, followed by site-directed mutagenesis studies. The receptor mapping showed that both the potency and affinity of 1 were dependent on the transmembrane residues His(113), Asp(171), Asp(262), and His(281) and also suggested the involvement of Tyr(45) and Gln(200) (potency) and Tyr(116) and Glu(288) (affinity). Molecular docking of 1 to an X-ray structure of CXCR4 showed that the l-Arg guanidino group of 1 forms polar interactions with His(113) and Asp(171) and the (pyridin-2-ylmethyl)amino moiety is anchored by Asp(262) and His(281), whereas the naphthalene ring is tightly packed in a hydrophobic subpocket formed by the aromatic side chains of Trp(94), Tyr(45), and Tyr(116). The detailed picture of ligand-receptor interactions provided here will assist in structure-based design and further development of small-molecule peptidomimetic CXCR4 antagonists.