Small Molecule Fragments Research Articles

Ordering a dynamic protein via a small-molecule stabilizer.

Like many coactivators, the GACKIX domain of the master coactivator CBP/p300 recognizes transcriptional activators of diverse sequence composition via dynamic binding surfaces. The conformational dynamics of GACKIX that underlie its function also render it especially challenging for structural characterization. We have found that the ligand discovery strategy of Tethering is an effective method for identifying small-molecule fragments that stabilize the GACKIX domain, enabling for the first time the crystallographic characterization of this important motif. The 2.0 Å resolution structure of GACKIX complexed to a small molecule was further analyzed by molecular dynamics simulations, which revealed the importance of specific side-chain motions that remodel the activator binding site in order to accommodate binding partners of distinct sequence and size. More broadly, these results suggest that Tethering can be a powerful strategy for identifying small-molecule stabilizers of conformationally malleable proteins, thus facilitating their structural characterization and accelerating the discovery of small-molecule modulators.

DNA as a Platform to Program Assemblies with Emerging Functions in Chemical Biology

AbstractThe programmability of oligonucleotide recognition offers an attractive platform for directing the assembly of ligands that can interact cooperatively with a target or the assembly of reactive partner that can engage in chemical reactions. In both cases, an oligonucleotide directs the assembly, which yields a functional output. In terms of the controlled display of ligands, several examples have shown a significant increase in binding upon ligand assembly using small molecule fragments, peptides, glycans, and protein fragments. Combinatorial approaches to identifying the optimal pairing have also been reported. In terms of nucleic acidtriggered reactions, several robust chemistries have been reported, leading to the unmasking of different fluorophores or bioactive molecules as well as the synthesis of a bioactive compound in response to DNA or even cellular RNA. Herein we present the historical context of this work and summarize recent developments in this area.

Highly active MnO2 nanosheet synthesis from graphene oxide templates and their application in efficient oxidative degradation of methylene blue

In this paper, a facile one-step method towards manganese oxide nanosheets is described which uses graphene oxide as a self-sacrificial template. A redox reaction between graphene oxide nanosheets and KMnO4 results in the formation of few-layer MnO2 nanosheets due to the in situ replacement of the carbon framework by edge-sharing [MnO6] octahedra. It is also demonstrated that these MnO2 nanosheets show high activity for the oxidative degradation of methylene blue (MB) dye, in which the MB dye can be decomposed into small molecule fragments through fast demethylation, followed by aromatic ring cracking, and partial mineralization into inorganic ions in a short time. We expect that the few-layer MnO2 nanosheets may be envisaged as a new non-toxic material for the treatment of organic dye-containing wastewater, and its application for the oxidation of other organic pollutants in environmental pollution cleanup.

Differential Fragment SPR (DF-SPR) for Antimalarial Drug Screening

Malaria has been a human health concern for centuries, particularly in tropical and subtropical regions of the world. Nevertheless, our repertoire of medication to treat the disease has been very limited, and emerging resistance of the malaria parasite Plasmodium has further restricted the use of current medications. The most recent reports indicating artemisinin resistance in Cambodia are indeed alarming and underscore the critical importance of exploring novel pathways for interfering with the life cycle of the malaria parasite. Surface plasmon resonance (SPR) has been a powerful tool to study protein ligand interactions. We utilize this technique for small molecule screening (∼150 Da) to identify fragment molecules that are capable of binding near a specific protein site. We have developed a method, which we termed differential fragment SPR (DF-SPR). In the present study, we utilized our approach to identify two small molecule fragments capable of inhibiting the essential protein-protein interaction of the autophagic proteins Atg8 and Atg3 from the malaria parasite Plasmodium falciparum. These fragments provide a starting point for developing larger, drug-like molecules. Furthermore, we employed our protein-protein interaction inhibition assay to discover additional small molecule inhibitors of a site which we found specific to the Plasmodium parasite, but absent in the human homolog of Atg8. Small-molecules derived from our studies may represent useful tools for further dissecting and analyzing the autophagy pathway in Plasmodium and other apicomplexan species.

Identification of Potential Small Molecule Binding Pockets on Rho Family GTPases

Rho GTPases are conformational switches that control a wide variety of signaling pathways critical for eukaryotic cell development and proliferation. They represent attractive targets for drug design as their aberrant function and deregulated activity is associated with many human diseases including cancer. Extensive high-resolution structures (>100) and recent mutagenesis studies have laid the foundation for the design of new structure-based chemotherapeutic strategies. Although the inhibition of Rho signaling with drug-like compounds is an active area of current research, very little attention has been devoted to directly inhibiting Rho by targeting potential allosteric non-nucleotide binding sites. By avoiding the nucleotide binding site, compounds may minimize the potential for undesirable off-target interactions with other ubiquitous GTP and ATP binding proteins. Here we describe the application of molecular dynamics simulations, principal component analysis, sequence conservation analysis, and ensemble small-molecule fragment mapping to provide an extensive mapping of potential small-molecule binding pockets on Rho family members. Characterized sites include novel pockets in the vicinity of the conformationaly responsive switch regions as well as distal sites that appear to be related to the conformations of the nucleotide binding region. Furthermore the use of accelerated molecular dynamics simulation, an advanced sampling method that extends the accessible time-scale of conventional simulations, is found to enhance the characterization of novel binding sites when conformational changes are important for the protein mechanism.

Structural Characterization of Inhibitors with Selectivity against Members of a Homologous Enzyme Family

The aspartate biosynthetic pathway provides essential metabolites for many important biological functions, including the production of four essential amino acids. As this critical pathway is only present in plants and microbes, any disruptions will be fatal to these organisms. An early pathway enzyme, l-aspartate-β-semialdehyde dehydrogenase, produces a key intermediate at the first branch point of this pathway. Developing potent and selective inhibitors against several orthologs in the l-aspartate-β-semialdehyde dehydrogenase family can serve as lead compounds for antibiotic development. Kinetic studies of two small molecule fragment libraries have identified inhibitors that show good selectivity against l-aspartate-β-semialdehyde dehydrogenases from two different bacterial species, Streptococcus pneumoniae and Vibrio cholerae, despite the presence of an identical constellation of active site amino acids in this homologous enzyme family. Structural characterization of enzyme-inhibitor complexes have elucidated different modes of binding between these structurally related enzymes. This information provides the basis for a structure-guided approach to the development of more potent and more selective inhibitors.

The effect of laccase on cellulase-treated lignin in 1-n-butyl-3-methylimidazolium chloride/H2O homogeneous and H2O heterogeneous solutions

In this study, the effect of laccase earlier obtained by our laboratory on cellulase-treated lignin (CEL) in two different solution systems was further investigated. Results obtained were as follows: After laccase treatment of CEL in the heterogeneous water solution, CEL was then compared with control sample A. Ultraviolet (UV) spectra showed that the total absorbance of sample B increased at 205 and 280 nm; Fourier-transform infrared (FTIR) spectra showed that the carbonyl group obviously appeared; and C-13 nuclear magnetic resonance (NMR) spectra showed that the C-gamma signals disappeared, the C-alpha signals significantly increased, and the methoxyl content decreased. After laccase treatment of CEL in the [bmim]Cl/H2O homogeneous solution, CEL was compared with control sample C. UV spectra showed that the total absorbance of sample D decreased at 280 nm; high-performance liquid chromatography (HPLC) analysis showed that some small absorption peaks disappeared; and C-13 NMR spectra showed that the C-gamma signals obviously increased, the C-alpha signals slightly decreased, while the methoxyl content increased. Clearly, for insoluble CEL in the heterogeneous water solution, laccase partly oxidized the hydroxyl group into a carbonyl group, and thereby partly degraded CEL to increase its solubility. In contrast, for soluble CEL in the [bmim]Cl/H2O homogeneous solution, laccase primarily polymerized small molecule fragments with the CEL macromolecule, increased the methoxyl content of CEL, and thereby played a role in lignin polymerization.

Biosensor-based small molecule fragment screening with biolayer interferometry

Biosensor-based fragment screening is a valuable tool in the drug discovery process. This method is advantageous over many biochemical methods because primary hits can be distinguished from non-specific or non-ideal interactions by examining binding profiles and responses, resulting in reduced false-positive rates. Biolayer interferometry (BLI), a technique that measures changes in an interference pattern generated from visible light reflected from an optical layer and a biolayer containing proteins of interest, is a relatively new method for monitoring small molecule interactions. The BLI format is based on a disposable sensor that is immersed in 96-well or 384-well plates. BLI has been validated for small molecule detection and fragment screening with model systems and well-characterized targets where affinity constants and binding profiles are generally similar to those obtained with surface plasmon resonsance (SPR). Screens with challenging targets involved in protein-protein interactions including BCL-2, JNK1, and eIF4E were performed with a fragment library of 6,500 compounds, and hit rates were compared for these targets. For eIF4E, a protein containing a PPI site and a nucleotide binding site, results from a BLI fragment screen were compared to results obtained in biochemical HTS screens. Overlapping hits were observed for the PPI site, and hits unique to the BLI screen were identified. Hit assessments with SPR and BLI are described.

Simple One-Pot Synthesis of Disulfide Fragments for Use in Disulfide-Exchange Screening

Disulfide exchange screening is a method for evaluating the binding of small molecule fragments to proteins that have at least one accessible cysteine. While operationally simple, it does require a large library of small fragment molecules bearing disulfide-containing side chains. These specialized fragments are not available commercially and this has limited the adoption of the method. We report here a convenient one-pot procedure that enables facile preparation of disulfide screening fragments while also producing less of an environmental impact. The new synthetic method involves the initial formation of symmetric disulfides, followed by a disulfide exchange reaction in which the symmetrical dimer is converted into the final screening fragment by introduction of a solubilizing 'cap'. The method is amenable to parallel synthetic methods and can be carried out in air without the need for the specialized equipment typically required for performing organic synthesis.

Abstract 1351: Computational design and synthesis of non-peptidic inhibitors targeting the cyclin binding groove of CDK2/Cyclin A

Abstract Inhibition of CDK2/Cyclin A (CDK2/CA) at G1/S phase of cell cycle controls cell proliferation and induces selective apoptosis of cancer cells. Neutralization of E2F-1by CDK2/CA phosphorylation is required prior to S-phase exit or apoptosis is triggered. E2F-1 is deregulated in many types of cancer and inhibition of CDK2/CA leads to selective cell death in these tumors. In our present study we target the substrate recruitment site knows as cyclin binding groove (CBG) for inhibition of CDK2/CA. The CBG recognizes a consensus sequence present on cyclin interacting proteins and the endogenous tumor suppressors knows cyclin binding motif (CBM) which has been minimized to the pentapeptide, RRLIF. It has been shown that the drug likeliness of the peptide can be improved by ligation of small molecules to truncated sequences. This was achieved using a unique strategy for designing inhibitors of protein-protein interactions known as REPLACE, in which small molecule fragments were identified by in silico methods. These were then ligated onto a truncated peptide sequence in order to replace critical residues, including the N-terminal Arginine moiety important for cyclin binding. Small molecule fragments for in silico docking were selected based on molecule weight, drug like features and possession of a carboxylic acid group for the formation of the peptide link. Docking was carried out using LigandFit and the crystal structure of 1-phenyl-1H-[1,2,4]triazole N-terminally capped peptides (Ncaps) in the CBG provided insight into molecular design. The 3,5 dichloro1-phenyl-1H-[1,2,4]triazole derivative linked to Arg-Leu-Asn-pF-Phe was previously shown to be a potent inhibitor with micromolar activity. In our present study we have replaced the N-terminal Arginine using different heterocyclic isosteres capable of interactions similar to critical amino acids of the parent peptide and the triazole. In order to probe the structure-activity relationship, we synthesized pyrazole, furan, pyrrole and thiazole systems and explored various substitutions of the phenyl ring. A number of Ncaps were then ligated to the tetra peptide, RLIF using solid phase synthesis, purified by reverse phase HPLC and characterized by MS. In vitro binding and functional assays were performed in order to study the inhibitory effect of compounds on CDK2/Cyclin A prior to further evaluation in cell viability assays to determine anti-tumor effects. On the basis of the results, further high throughput docking of potential heterocyclic fragments was carried out to identify N-capping groups of varying chemical diversity for synthesis and in vitro testing. The results presented demonstrate the utility of the REPLACE method in the development of protein-protein interaction inhibitors and non-ATP competitive CDK2 inhibitors as anti-tumor therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1351. doi:10.1158/1538-7445.AM2011-1351

Abstract 3240: Development of non-ATP competitive, PLK1 selective inhibitors

Abstract Through a better understanding of the molecular mechanisms that control normal and cancer cell proliferation, new therapeutic targets that regulate the cell cycle have been identified. One of these, Polo like kinase 1 (PLK1) is a member of a conserved family of serine/threonine kinases. Its activity and cellular levels play an important role in mitotic entry, DNA-damage checkpoint activation, and centrosome duplication and maturation. Because other PLK family members such as PLK3 act as putative tumor suppressors, it is imperative that a PLK1 inhibitor be exclusive for PLK1, a potential draw back for ATP competitive inhibitors. The polo box domain (PBD) of PLK1 plays an important role in sub-cellular localization and mitotic functions of PLK1. Importantly, it has been shown that inhibiting the PBD site leads to selective PLK1 inhibition and can lead to mitotic arrest and inhibit proliferation of tumor cells. The structure-guided discovery strategy called REPLACE can be used to target protein-protein interactions otherwise considered undruggable. REPLACE uses structure activity relationships of peptide inhibitors to generate a pharmaceutically acceptable lead molecule by replacing individual amino acid residues with non-peptide fragments. REPLACE has been used to identify fragment mimetics for determinants from the endogenous phosphopeptide sequence, LLCSpTPNGL (CDC25c), known to specifically bind to the PBD of PLK1. Using this approach, FLIPs (Fragment Ligated Inhibitory Peptides) were generated and tested in a fluorescence polarization (FP) assay to determine binding potential for the PBD. Small molecule fragment alternatives for the N-terminal tripeptide (LLC) were identified through this approach and shown to possess increased binding relative to the truncated peptide. Modeling and amino acid replacement was also used to elucidate essential binding sites and specificity determinants needed by the endogenous peptide to maintain affinity and selectivity for the PBD. This was confirmed through comparisons of binding for peptide analogs in the FP assay relative to that of the endogenous peptide. As part of the development process, peptides have been examined for anti-proliferative activity in cancer cells and to determine if a PLK1 inhibitory phenotype is observed. Mitotic events were evaluated by flow cytometry and by direct visualization, utilizing cells expressing GFP-labeled histone H2B. Conditions to transduce peptides have been established and these will be used to characterize FLIPs with sufficient affinity for the PBD. By targeting the PBD of PLK1 using REPLACE, we believe that we can develop a non-ATP competitive inhibitor that is not only PLK1 specific but is less toxic to normal cells and safer than current drugs on the market. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3240. doi:10.1158/1538-7445.AM2011-3240

Turning a protein kinase on or off from a single allosteric site via disulfide trapping

There is significant interest in identifying and characterizing allosteric sites in enzymes such as protein kinases both for understanding allosteric mechanisms as well as for drug discovery. Here, we apply a site-directed technology, disulfide trapping, to interrogate structurally and functionally how an allosteric site on the Ser/Thr kinase, 3-phosphoinositide-dependent kinase 1 (PDK1)--the PDK1-interacting-fragment (PIF) pocket--is engaged by an activating peptide motif on downstream substrate kinases (PIFtides) and by small molecule fragments. By monitoring pairwise disulfide conjugation between PIFtide and PDK1 cysteine mutants, we defined the PIFtide binding orientation in the PIF pocket of PDK1 and assessed subtle relationships between PIFtide positioning and kinase activation. We also discovered a variety of small molecule fragment disulfides (< 300 Da) that could either activate or inhibit PDK1 by conjugation to the PIF pocket, thus displaying greater functional diversity than is displayed by PIFtides conjugated to the same sites. Biochemical data and three crystal structures provided insight into the mechanism of action of the best fragment activators and inhibitors. These studies show that disulfide trapping is useful for characterizing allosteric sites on kinases and that a single allosteric site on a protein kinase can be exploited for both activation and inhibition by small molecules.

DNA-templated combinatorial assembly of small molecule fragments amenable to selection/amplification cycles

The discovery of small molecule probes which selectively modulate biological pathways is a cornerstone in the development of new therapeutics. Progress in our ability to access libraries of biologically relevant small molecules in conjunction with streamlined screening technologies have also enabled a more systematic approach to chemical biology. Nevertheless, the current state of the art still requires a large infrastructure and only a small fraction of the proteome has been addressed thus far. The emergence of technologies based on nucleic acid encoding of small molecules presents a new screening paradigm. We describe a method based on DNA-templated combinatorial display of PNA-encoded drug fragments affording 62 500 combinations which can be amplified following a selection. This concept was demonstrated with a screen against a representative target, carbonic anhydrase, by iterative cycles of affinity selection, amplification of DNA template and “translation” back into selected library members. The results show a clear convergence towards combinations which, upon resynthesis as covalent adducts, proved to bind cooperatively to carbonic anhydrase.

Fragment‐based discovery of novel thymidylate synthase leads by NMR screening and group epitope mapping

Solution-state nuclear magnetic resonance (NMR) is a versatile tool for the study of binding interactions between small molecules and macromolecular targets. We applied ligand-based NMR techniques to the study of human thymidylate synthase (hTS) using known nanomolar inhibitors and a library of small molecule fragments. Screening by NMR led to the rapid identification of ligand pairs that bind in proximal sites within the cofactor-binding pocket of hTS. Screening hits were used as search criteria within commercially available sources, and a subset of catalog analogs were tested for potency by in vitro assay and binding affinity by quantitative saturation transfer difference (STD)-NMR titration. Two compounds identified by this approach possess low micromolar affinity and potency, as well as excellent binding efficiency against hTS. Relative binding orientations for both leads were modeled using AutoDock, and the most likely bound conformations were validated using experimentally derived STD-NMR binding epitope data. These ligands represent novel starting points for fragment-based drug design of non-canonical TS inhibitors, and their binding epitopes highlight important and previously unexploited interactions with conserved residues in the cofactor-binding site.

Deterioration of mechanical properties: A cause for fuel cell membrane failure

The mechanical properties of Nafion® membranes that were degraded after periods of open circuit voltage (OCV) conditions in a fuel cell were studied. Severe decreases in membrane modulus and ductility at 80 °C and 100% relative humidity were observed. Stress-at-break values for 45 and 72 h degraded membrane–electrode assemblies (MEAs) were 6 and 9 times lower, respectively, while strain-at-break was 24 and 28 times lower, respectively, than that of a control MEA. Contractile stresses developed in constrained hydrated membranes that were then dried were conducted as a function of drying time. A pre-OCV degraded MEA, when subjected to creep deformation at 10 MPa, showed surface cracks which were not present in the control MEA. The 72 h-degraded MEA exhibited wider cracks than those of the 45 h-degraded MEA sample. Spectroscopic analysis of degraded membranes showed various small molecule fragments associated with side chain degradation.

Fragment-Based Discovery of Nonpeptidic BACE-1 Inhibitors Using Tethering

BACE-1 (beta-site amyloid precursor protein cleaving enzyme), a prominent target in Alzheimer's disease drug discovery efforts, was surveyed using Tethering technology to discover small molecule fragment ligands that bind to the enzyme active site. Screens of a library of >15000 thiol-containing fragments versus a panel of BACE-1 active site cysteine mutants under redox-controlled conditions revealed several novel amine-containing fragments that could be selectively captured by subsets of the tethering sites. For one such hit class, defined by a central aminobenzylpiperidine (ABP) moiety, X-ray crystal structures of BACE mutant-disulfide conjugates revealed that the fragment bound by engaging both catalytic aspartates with hydrogen bonds. The affinities of ABP fragments were improved by structure-guided chemistry, first for conjugation as thiol-containing fragments and then for stand-alone, noncovalent inhibition of wild-type (WT) BACE-1 activity. Crystallography confirmed that the inhibitors bound in exactly the same mode as the disulfide-conjugated fragments that were originally selected from the screen. The ABP ligands represent a new type of nonpeptidic BACE-1 inhibitor motif that has not been described in the aspartyl protease literature and may serve as a starting point for the development of BACE-1-directed Alzheimer's disease therapeutics.

Activation and cleavage of the N–O bond in dinuclear mixed-metal nitrosyl systems and comparative analysis of carbon monoxide, dinitrogen, and nitric oxideactivation

The activation and scission of the N-O bond in nitric oxide using dinuclear mixed-metal species, comprising transition elements with d(3) and d(2) configurations and trisamide ligand systems, have been investigated by means of density functional calculations. The [Cr(iii)-V(iii)] system is analyzed in detail and, for comparative purposes, the [Mo(iii)-Nb(iii)], [W(iii)-Ta(iii)], and (mixed-row) [Mo(iii)-V(iii)] systems are also considered. The overall reaction and individual intermediate steps are favourable for all systems, including the case where first row (Cr and V) metals are exclusively involved, a result that has not been observed for the related dinitrogen and carbon monoxide systems. In contrast to the cleavage of dinitrogen by three-coordinate Mo amide complexes where the dinuclear intermediate possesses a linear [Mo-NN-Mo] core, the [M-NO-M'] core must undergo significant bending in order to stabilize the dinuclear species sufficiently for the reaction to proceed beyond the formation of the nitrosyl encounter complex. A comparative bonding analysis of nitric oxide, dinitrogen and carbon monoxide activation is also presented. The overall results indicate that the pi interactions are the dominant factor in the bonding across the [M-L(1)L(2)-M'] (L(1)L(2) = N-O, N-N, C-O) moiety and, consequently, the activation of the L(1)-L(2) bond. These trends arise from the fact that the energy gaps between the pi orbitals on the metal and small molecule fragments are much more favourable than for the corresponding sigma orbitals. The pi energy gaps decrease in the order [NO < N(2) < CO] and consequently, for each individual pi orbital interaction, the back donation between the metal and small molecule increases in the order [CO < N(2) < NO]. These results are in accord with previous findings suggesting that optimization of the pi interactions plays a central role in increasing the ability of these transition metal systems to activate and cleave small molecule bonds.

Novel inhibitors of anthrax edema factor

Several pathogenic bacteria produce adenylyl cyclase toxins, such as the edema factor (EF) of Bacillus anthracis. These disturb cellular metabolism by catalyzing production of excessive amounts of the regulatory molecule cAMP. Here, a structure-based method, where a 3D-pharmacophore that fit the active site of EF was constructed from fragments, was used to identify non-nucleotide inhibitors of EF. A library of small molecule fragments was docked to the EF-active site in existing crystal structures, and those with the highest HINT scores were assembled into a 3D-pharmacophore. About 10,000 compounds, from over 2.7 million compounds in the ZINC database, had a similar molecular framework. These were ranked according to their docking scores, using methodology that was shown to achieve maximum accuracy (i.e., how well the docked position matched the experimentally determined site for ATP analogues in crystal structures of the complex). Finally, 19 diverse compounds with the best AutoDock binding/docking scores were assayed in a cell-based assay for their ability to reduce cAMP secretion induced by EF. Four of the test compounds, from different structural groups, inhibited in the low micromolar range. One of these has a core structure common to phosphatase inhibitors previously identified by high-throughput assays of a diversity library. Thus, the fragment-based pharmacophore identified a small number of diverse compounds for assay, and greatly enhanced the selection process of advanced lead compounds for combinatorial design.

Computational approach to site‐directed ligand discovery

A computational approach, Systematic Conformational Search & Induced Fit (SCI&FI), to site-directed ligand discovery (Tethering) is presented. SCI&FI has the ability to predict the binding site, binding mode, and bound dynamics of small molecule fragments covalently tethered to a protein. The SCI&FI method was engineered with the ability to model induced fit conformational changes of the protein because of the binding of the tether. SCI&FI generates comprehensive picture of the binding preferences of the tether to the protein by elucidating potential binding sites of the tether and by describing regions of receptor space capable of conformational change because of the binding of the tether. The SCI&FI method provides a complementary approach to experimental tethering. Initial validation of the SCI&FI method is reported by predicting the 3D structure of two Interleukin-2 and an Interleukin-4 tethered-protein systems.

Plasma pre-treatment of liquid crystal polymer and subsequent metallization by PVD

In this paper surface modification of a liquid crystal polymer (LCP) substrate by oxygen containing plasma pre-treatment and subsequent Cu/Cr deposition by physical vapour deposition (PVD) technique is described. By pre-treatment with oxygen containing plasma more volatile reaction products are generated compared to argon plasma pre-treatment as is shown by the etching rate. The small molecule fragments generated during the pre-treatment process are analyzed by mass spectrometry. After the pre-treatment metal layers with suitable adhesion strength even after 1000 cycles of thermal shock are obtained.