Identification of novel human CC chemokine receptor 8 (CCR8) antagonists via the synthesis of naphthalene amide and sulfonamide isosteres.

The human CC chemokine receptor 8 (CCR8) has been extensively pursued as target for the treatment of various inflammatory disorders. More recently, the importance of CCR8 in the tumor microenvironment has been demonstrated, spurring the interest in CCR8 antagonism as therapeutic strategy in immuno-oncology. On a previously described naphthalene sulfonamide with CCR8 antagonistic properties, the concept of isosterism was applied, leading to the discovery of novel CCR8 antagonists with IC50 values in the nM range in both the CCL1 competition binding and CCR8 calcium mobilization assay. The excellent CCR8 antagonistic activity of the most potent congeners was rationalized by homology molecular modeling.

Structure and stability of the designer protein WRAP-T and its permutants.

[Formula: see text]-Propeller proteins are common natural disc-like pseudo-symmetric proteins that contain multiple repeats ('blades') each consisting of a 4-stranded anti-parallel [Formula: see text]-sheet. So far, 4- to 12-bladed [Formula: see text]-propellers have been discovered in nature showing large functional and sequential variation. Using computational design approaches, we created perfectly symmetric [Formula: see text]-propellers out of natural pseudo-symmetric templates. These proteins are useful tools to study protein evolution of this very diverse fold. While the 7-bladed architecture is the most common, no symmetric 7-bladed monomer has been created and characterized so far. Here we describe such a engineered protein, based on a highly symmetric natural template, and test the effects of circular permutation on its stability. Geometrical analysis of this protein and other artificial symmetrical proteins reveals no systematic constraint that could be used to help in engineering of this fold, and suggests sequence constraints unique to each [Formula: see text]-propeller sub-family.

The symmetric designer protein Pizza as a scaffold for metal coordination.

Symmetric proteins are currently of interest as they allow creation of larger assemblies and facilitate the incorporation of metal ions in the larger complexes. Recently this was demonstrated by the biomineralization of the cadmium-chloride nanocrystal via the Pizza designer protein. However, the mechanism behind this formation remained unclear. Here, we set out to investigate the mechanism driving the formation of this nanocrystal via truncation, mutation, and circular permutations. In addition, the interaction of other biologically relevant metal ions with these symmetric proteins to form larger symmetric complexes was also studied. The formation of the initial nanocrystal is shown to originate from steric strain, where His 58 induces a different rotameric conformation on His 73, thereby distorting an otherwise perfect planar ring of alternating cadmium and chlorine ions, resulting in the smallest nanocrystal. Similar highly symmetric complexes were also observed for the other biological relevant metal ions. However, the flexibility of the coordinating histidine residues allows each metal ion to adopt its preferred geometry leading to either monomeric or dimeric ß-propeller units, where the metal ions are located at the interface between both propeller units. These results demonstrate that symmetric proteins are not only interesting to generate larger assemblies, but are also the perfect scaffold to create more complex metal based assemblies. Such metal protein assemblies may then find applications in bionanotechnology or biocatalysis.

Small Differences in SUC Gene Sequences Impact Saccharomyces cerevisiae Invertase Activity and Specificity toward Fructans with Different Chain Lengths.

Saccharomyces cerevisiae (S. cerevisiae)invertase is encoded by a family of closely related SUC genes. To identify and understand the molecular basis for differences in substrate specificity, we examined 29 SUC alleles from industrialS. cerevisiaestrains and cloned alleles with small sequence differences into an invertase-negative strain. Our study showed that an F102Y substitution in Suc-enzymes lowers yeast invertase activity toward fructo-oligosaccharides (FOS) by 36% and the specificity factor by 43%. By contrast, an A409P substitution in Suc-enzymes resulted in an increased capacity of the yeast to hydrolyze FOS and Fibruline by 17 and 41%, respectively, likely because of a change in the loop conformation resulting in a wider active site. Bread dough fermentation experiments revealed that sucrose and fructan hydrolysis during fermentation is influenced by this natural variation in SUC sequences. Our research thus opens the door for the selection or engineering of yeasts and Suc-enzymes with specific activities that may ultimately allow controlling fructan hydrolysis.

Structural plasticity of a designer protein sheds light on ß-propeller protein evolution.

ß-propeller proteins are common in nature, where they are observed to adopt 4- to 10-fold internal rotational pseudo-symmetry. This size diversity can be explained by the evolutionary process of gene duplication and fusion. In this study, we investigated a distorted ß-propeller protein, an apparent intermediate between two symmetries. From this template, we created a perfectly symmetric 9-bladed ß-propeller named Cake, using computational design and ancestral sequence reconstruction. The designed repeat sequence was found to be capable of generating both 8-fold and 9-fold propellers which are highly stable. Cake variants with 2-10 identical copies of the repeat sequence were characterised by X-ray crystallography and in solution. They were found to be highly stable, and to self-assemble into 8- or 9-fold symmetrical propellers. These findings show that the ß-propeller fold allows sufficient structural plasticity to permit a given blade to assemble different forms, a transition from even to odd changes in blade number, and provide a potential explanation for the wide diversity of repeat numbers observed in natural propeller proteins. DATABASE: Structural data are available in Protein Data Bank database under the accession numbers 6TJB, 6TJC, 6TJD, 6TJE, 6TJF, 6TJG, 6TJH and 6TJI.

Palladium-catalyzed cross-coupling reactions on a bromo-naphthalene scaffold in the search for novel human CC chemokine receptor 8 (CCR8) antagonists.

The naphthalene sulfonamide scaffold is known to possess CCR8 antagonistic properties. In order to expand the structure-activity relationship study of this compound class, a variety of palladium-catalyzed cross-coupling reactions was performed on a bromo-naphthalene precursor yielding a diverse library. These compounds displayed CCR8 antagonistic properties in binding and calcium mobilization assays, with IC50 values in the 0.2 - 10 µM range. The decreased activity, when compared to the original lead compound, was rationalized by homology molecular modeling.

Development and applications of artificial symmetrical proteins.

Since the determination of the first molecular models of proteins there has been interest in creating proteins artificially, but such methods have only become widely successful in the last decade. Gradual improvements over a long period of time have now yielded numerous examples of non-natural proteins, many of which are built from repeated elements. In this review we discuss the design of such symmetrical proteins and their various applications in chemistry and medicine.

Influence of circular permutations on the structure and stability of a six-fold circular symmetric designer protein.

The ß-propeller fold is adopted by a sequentially diverse family of repeat proteins with apparent rotational symmetry. While the structure is mostly stabilized by hydrophobic interactions, an additional stabilization is provided by hydrogen bonds between the N-and C-termini, which are almost invariably part of the same ß-sheet. This feature is often referred to as the "Velcro" closure. The positioning of the termini within a blade is variable and depends on the protein family. In order to investigate the influence of this location on protein structure, folding and stability, we created different circular permutants, and a circularized version, of the designer propeller protein named Pizza. This protein is perfectly symmetrical, possessing six identical repeats. While all mutants adopt the same structure, the proteins lacking the "Velcro" closure were found to be significantly less resistant to thermal and chemical denaturation. This could explain why such proteins are rarely observed in nature. Interestingly the most common "Velcro" configuration for this protein family was not the most stable among the Pizza variants tested. The circularized version shows dramatically improved stability, which could have implications for future applications.

Hybrid assemblies of a symmetric designer protein and polyoxometalates with matching symmetry.

Novel bioinorganic hybrid materials based on proteins and inorganic clusters have enormous potential for the development of hybrid catalysts that synergistically combine properties of both materials. Here we report the creation of hybrid assemblies between a computationally designed symmetrical protein Pizza6-S and different polyoxometalates with matching symmetry: the tellurotungstic Anderson-Evans (Na6[TeW6O24]·22H2O) (TEW); Keggin (H4[SiW12O40]·6H2O) (STA); and 1 : 2 CeIII-substituted Keggin (K11[CeIII[PW11O39]2]·20H2O) (Ce-K). This results in the formation of complexes with clearly defined stoichiometries in solution. Crystal structures validate the complexes as building blocks for the formation of larger assemblies.

Design, synthesis and biological evaluation of pyrazolo[3,4-d]pyrimidine-based protein kinase D inhibitors.

The multiple roles of protein kinase D (PKD) in various cancer hallmarks have been repeatedly reported. Therefore, the search for novel PKD inhibitors and their evaluation as antitumor agents has gained considerable attention. In this work, novel pyrazolo[3,4-d]pyrimidine based pan-PKD inhibitors with structural variety at position 1 were synthesized and evaluated for biological activity. Starting from 3-IN-PP1, a known PKD inhibitor with IC50 values in the range of 94-108 nM, compound 17m was identified with an improved biochemical inhibitory activity against PKD (IC50 = 17-35 nM). Subsequent cellular assays demonstrated that 3-IN-PP1 and 17m inhibited PKD-dependent cortactin phosphorylation. Furthermore, 3-IN-PP1 displayed potent anti-proliferative activity against PANC-1 cells. Finally, a screening against different cancer cell lines demonstrated that 3-IN-PP1 is a potent and versatile antitumoral agent.

Molecular assemblies built with the artificial protein Pizza.

Recently an artificial protein named Pizza6 was reported, which possesses six identical tandem repeats and adopts a monomeric ß -propeller fold with sixfold structural symmetry. Pizza2, a truncated form that consists of a double tandem repeat, self-assembles into a trimer reconstructing the same propeller architecture as Pizza6. The ability of pizza proteins to self-assemble to form complete propellers makes them interesting building blocks to engineer larger symmetrical protein complexes such as symmetric nanoparticles. Here we have explored the self-assembly of Pizza2 fused to homo-oligomerizing peptides. In total, we engineered five different fusion proteins, of which three appeared to assemble successfully into larger complexes. Further characterization of these proteins showed one monodisperse designer protein with a structure close to the intended design. This protein was further fused to eGFP to investigate functionalization of the nanoparticle. The fusion protein was stable and could be expressed in high yield, showing that Pizza-based nanoparticles may be further decorated with functional domains.

Artificial ß-propeller protein-based hydrolases.

We developed an artificial hydrolase based on the symmetrical Pizza6 ß-propeller protein for the metal-free hydrolysis of 4-nitrophenyl acetate and butyrate. Through site-specific mutagenesis and crystallisation studies, the catalytic mechanism was investigated and found to be dependent on a threonine-histidine dyad. The mutant with additional histidine residues generated the highest kcat values, forming a His-His-Thr triad and matched previously reported metalloenzymes. The highly symmetrical ß-propeller artificial enzymes and their protein-metal complexes have potential to be utilised in bioinorganic and supramolecular chemistry, as well as being developed further into 2D/3D catalytic materials.

Computational design of symmetrical eight-bladed ß-propeller proteins.

ß-Propeller proteins form one of the largest families of protein structures, with a pseudo-symmetrical fold made up of subdomains called blades. They are not only abundant but are also involved in a wide variety of cellular processes, often by acting as a platform for the assembly of protein complexes. WD40 proteins are a subfamily of propeller proteins with no intrinsic enzymatic activity, but their stable, modular architecture and versatile surface have allowed evolution to adapt them to many vital roles. By computationally reverse-engineering the duplication, fusion and diversification events in the evolutionary history of a WD40 protein, a perfectly symmetrical homologue called Tako8 was made. If two or four blades of Tako8 are expressed as single polypeptides, they do not self-assemble to complete the eight-bladed architecture, which may be owing to the closely spaced negative charges inside the ring. A different computational approach was employed to redesign Tako8 to create Ika8, a fourfold-symmetrical protein in which neighbouring blades carry compensating charges. Ika2 and Ika4, carrying two or four blades per subunit, respectively, were found to assemble spontaneously into a complete eight-bladed ring in solution. These artificial eight-bladed rings may find applications in bionanotechnology and as models to study the folding and evolution of WD40 proteins.

Discovery of a potent protein kinase D inhibitor: insights in the binding mode of pyrazolo[3,4-d]pyrimidine analogues.

In this study, we set out to rationally optimize PKD inhibitors based on the pyrazolo[3,4-d]pyrimidine scaffold. The lead compound for this study was 1-NM-PP1, which was previously found by us and others to inhibit PKD. In our screening we identified one compound (3-IN-PP1) displaying a 10-fold increase in potency over 1-NM-PP1, opening new possibilities for specific protein kinase inhibitors for kinases that show sensitivity towards pyrazolo[3,4-d]pyrimidine derived compounds. Interestingly the observed SAR was not in complete agreement with the commonly observed binding mode where the pyrazolo[3,4-d]pyrimidine compounds are bound in a similar fashion as PKD's natural ligand ATP. Therefore we suggest an alternate binding mode where the compounds are flipped 180 degrees. This possible alternate binding mode for pyrazolo[3,4-d]pyrimidine based compounds could pave the way for a new class of specific protein kinase inhibitors for kinases sensitive towards pyrazolo[3,4-d]pyrmidines.

Computational design of a symmetrical ß-trefoil lectin with cancer cell binding activity.

Computational protein design has advanced very rapidly over the last decade, but there remain few examples of artificial proteins with direct medical applications. This study describes a new artificial ß-trefoil lectin that recognises Burkitt's lymphoma cells, and which was designed with the intention of finding a basis for novel cancer treatments or diagnostics. The new protein, called "Mitsuba", is based on the structure of the natural shellfish lectin MytiLec-1, a member of a small lectin family that uses unique sequence motifs to bind α-D-galactose. The three subdomains of MytiLec-1 each carry one galactose binding site, and the 149-residue protein forms a tight dimer in solution. Mitsuba (meaning "three-leaf" in Japanese) was created by symmetry constraining the structure of a MytiLec-1 subunit, resulting in a 150-residue sequence that contains three identical tandem repeats. Mitsuba-1 was expressed and crystallised to confirm the X-ray structure matches the predicted model. Mitsuba-1 recognises cancer cells that express globotriose (Galα(1,4)Galß(1,4)Glc) on the surface, but the cytotoxicity is abolished.

Evolution-Inspired Computational Design of Symmetric Proteins.

Monomeric proteins with a number of identical repeats creating symmetrical structures are potentially very valuable building blocks with a variety of bionanotechnological applications. As such proteins do not occur naturally, the emerging field of computational protein design serves as an excellent tool to create them from nonsymmetrical templates. Existing pseudo-symmetrical proteins are believed to have evolved from oligomeric precursors by duplication and fusion of identical repeats. Here we describe a computational workflow to reverse-engineer this evolutionary process in order to create stable proteins consisting of identical sequence repeats.

The Effect of F877L and T878A Mutations on Androgen Receptor Response to Enzalutamide.

Treatment-induced mutations in the ligand-binding domain of the androgen receptor (AR) are known to change antagonists into agonists. Recently, the F877L mutation has been described to convert enzalutamide into an agonist. This mutation was seen to co-occur in the endogenous AR allele of LNCaP cells, next to the T878A mutation. Here, we studied the effects of enzalutamide on the F877L and T878A mutants, as well as the double-mutant AR (F877L/T878A). Molecular modeling revealed favorable structural changes in the double-mutant AR that lead to a decrease in steric clashes for enzalutamide. Ligand-binding assays confirmed that the F877L mutation leads to an increase in relative binding affinity for enzalutamide, but only the combination with the T878A mutation resulted in a strong agonistic activity. This correlated with changes in coregulator recruitment and chromatin interactions. Our data show that enzalutamide is only a very weak partial agonist of the AR F877L, and a strong partial agonist of the double-mutant AR. Mol Cancer Ther; 15(7); 1702-12. ©2016 AACR.

The crystal and solution structure of YdiE from Escherichia coli.

Iron-containing porphyrins are essential for all life as electron carriers. Since iron is poorly available in an oxidizing environment, bacterial growth may be restricted by iron limitation, and this has led to the evolution of a huge variety of iron-uptake systems. Among pathogens, iron scavenging from the haemoglobin of an animal host is a common means of acquiring sufficient iron for growth. The Isd system of Staphylococcus aureus is a well studied example; the bacterium devotes considerable resources to the construction of surface proteins that deftly remove haem from haemoglobin and pass it along a chain of related proteins, eventually delivering the haem to the cytoplasm, where it can be utilized or degraded. All organisms, however, must deal with haem and related molecules, which are by their nature hydrophobic and prone to precipitate, and which tend to promote the formation of reactive oxygen species. Chaperones are an obvious solution to the problem of maintaining a pool of haem for insertion into cytochromes without allowing naked haem to cause damage. YdiE is a very small protein from Escherichia coli of only 63 residues which may play a role in haem trafficking. Here, NMR analysis and the crystal structure of the protein to high resolution are reported.

Biomineralization of a Cadmium Chloride Nanocrystal by a Designed Symmetrical Protein.

We have engineered a metal-binding site into the novel artificial ß-propeller protein Pizza. This new Pizza variant carries two nearly identical domains per polypeptide chain, and forms a trimer with three-fold symmetry. The designed single metal ion binding site lies on the symmetry axis, bonding the trimer together. Two copies of the trimer associate in the presence of cadmium chloride in solution, and very high-resolution X-ray crystallographic analysis reveals a nanocrystal of cadmium chloride, sandwiched between two trimers of the protein. This nanocrystal, containing seven cadmium ions lying in a plane and twelve interspersed chloride ions, is the smallest reported to date. Our results indicate the feasibility of using rationally designed symmetrical proteins to biomineralize nanocrystals with useful properties.

Computational design of a self-assembling symmetrical ß-propeller protein.

The modular structure of many protein families, such as ß-propeller proteins, strongly implies that duplication played an important role in their evolution, leading to highly symmetrical intermediate forms. Previous attempts to create perfectly symmetrical propeller proteins have failed, however. We have therefore developed a new and rapid computational approach to design such proteins. As a test case, we have created a sixfold symmetrical ß-propeller protein and experimentally validated the structure using X-ray crystallography. Each blade consists of 42 residues. Proteins carrying 2-10 identical blades were also expressed and purified. Two or three tandem blades assemble to recreate the highly stable sixfold symmetrical architecture, consistent with the duplication and fusion theory. The other proteins produce different monodisperse complexes, up to 42 blades (180 kDa) in size, which self-assemble according to simple symmetry rules. Our procedure is suitable for creating nano-building blocks from different protein templates of desired symmetry.