RESUMO
Multispecific biologics are an emerging class of drugs, in which antibodies and/or proteins designed to bind pharmacological targets are covalently linked or expressed as fusion proteins to increase both therapeutic efficacy and safety. Epitope mapping on the target proteins provides key information to improve the affinity and also to monitor the manufacturing process and drug stability. Solid-state NMR has been here used to identify the pattern of the residues of the programmed cell death ligand 1 (PD-L1) ectodomain that are involved in the interaction with a new multispecific biological drug. This is possible because the large size and the intrinsic flexibility of the complexes are not limiting factors for solid-state NMR.
Assuntos
Produtos Biológicos , Anticorpos , Mapeamento de Epitopos , Espectroscopia de Ressonância Magnética , Proteínas/químicaRESUMO
Biocompatible hydrogels for tissue regeneration/replacement and drug release with specific architectures can be obtained by three-dimensional bioprinting techniques. The preservation of the higher order structure of the proteins embedded in the hydrogels as drugs or modulators is critical for their biological activity. Solution nuclear magnetic resonance (NMR) experiments are currently used to investigate the higher order structure of biotherapeutics in comparability, similarity, and stability studies. However, the size of pores in the gel, protein-matrix interactions, and the size of the embedded proteins often prevent the use of this methodology. The recent advancements of solid-state NMR allow for the comparison of the higher order structure of the matrix-embedded and free isotopically enriched proteins, allowing for the evaluation of the functionality of the material in several steps of hydrogel development. Moreover, the structural information at atomic detail on the matrix-protein interactions paves the way for a structure-based design of these biomaterials.
Assuntos
Bioimpressão , Liberação Controlada de Fármacos , Hidrogéis , Impressão Tridimensional , Engenharia Tecidual , Alicerces TeciduaisRESUMO
HMGB1 is a ubiquitous non-histone protein, which biological effects depend on its expression and subcellular location. Inside the nucleus, HMGB1 is engaged in many DNA events such as DNA repair, transcription and telomere maintenance. HMGB1 has been reported to bind preferentially to bent DNA as well as to noncanonical DNA structures like 4-way junctions and, more recently, to G-quadruplexes. These are four-stranded conformations of nucleic acids involved in important cellular processes, including telomere maintenance. In this frame, G-quadruplex recognition by specific proteins represents a key event to modulate physiological or pathological pathways. Herein, to get insights into the telomeric G-quadruplex DNA recognition by HMGB1, we performed detailed biophysical studies complemented with biological analyses. The obtained results provided information about the molecular determinants for the interaction and showed that the structural variability of human telomeric G-quadruplex DNA may have significant implications in HMGB1 recognition. The biological data identified HMGB1 as a telomere-associated protein in both telomerase-positive and -negative tumor cells and showed that HMGB1 gene silencing in such cells induces telomere DNA damage foci. Altogether, these findings provide a deeper understanding of telomeric G-quadruplex recognition by HMGB1 and suggest that this protein could actually represent a new target for cancer therapy.
Assuntos
Quadruplex G , Proteína HMGB1/genética , Conformação de Ácido Nucleico , Telômero/genética , DNA/química , DNA/genética , Escherichia coli/genética , Proteína HMGB1/química , Humanos , Telomerase/química , Telomerase/genética , Telômero/químicaRESUMO
Paramagnetic restraints have been used in biomolecular NMR for the last three decades to elucidate and refine biomolecular structures, but also to characterize protein-ligand interactions. A common technique to generate such restraints in proteins, which do not naturally contain a (paramagnetic) metal, consists in the attachment to the protein of a lanthanide-binding-tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry of the complex (conformational exchanges and coordination) and the chemical inertness of the ligand. Here we describe a photo-catalyzed thiol-ene reaction for the cysteine-selective paramagnetic tagging of proteins. As a model, we designed an LBT with a vinyl-pyridine moiety which was used to attach our tag to the protein GB1 in fast and irreversible fashion. Our tag T1 yields magnetic susceptibility tensors of significant size with different lanthanides and has been characterized using NMR and relaxometry measurements.
Assuntos
Proteínas/química , Compostos de Sulfidrila/química , Catálise , Cisteína/química , Elementos da Série dos Lantanídeos/química , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Processos Fotoquímicos , Piridinas/químicaRESUMO
Cyclocarbonylative Sonogashira reactions of ortho-ethynylbenzamides have been investigated. The process is carried out under CO pressure, in the presence of a very small amount of PdCl2(PPh3)2 (0.4 mol %) as a catalytic precursor and without the need for a Cu salt as the co-catalyst. 2-Ethynylbenzamide reacted successfully with iodoarenes bearing electron-withdrawing and electron-donating groups, giving rise to different classes of compounds depending on the solvent used. On the contrary, N-(4-chlorophenyl)-2-ethynylbenzamide afforded exclusively polyfunctionalized isoindolinones with high stereoselectivity toward (E) isomers.
RESUMO
FlowNMR has the aim of continuously monitoring processes that occur in conditions that are not compatible with being carried out within a closed tube. However, it is sample intensive and not suitable for samples, such as proteins or living cells, that are often available in limited volumes and possibly low concentrations. We here propose a dialysis-based modification of a commercial flowNMR setup that allows for recycling the medium while confining the sample (proteins and cells) within the active volume of the tube. This approach is demonstrated in the specific cases of in-cell NMR and protein-based ligand studies.
Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Reatores Biológicos , Espectroscopia de Ressonância Magnética/métodos , Técnicas de Cultura Celular por Lotes/instrumentação , Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica II/metabolismo , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética/instrumentação , Ligação Proteica , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/metabolismoRESUMO
Solid state NMR (SSNMR) has earned a substantial success in the characterization of paramagnetic systems over the last decades. Nowadays, the resolution and sensitivity of solid state NMR in biological molecules has improved significantly and these advancements can be translated into the study of paramagnetic biomolecules. However, the electronic properties of different metal centers affect the quality of their SSNMR spectra differently, and not all systems turn out to be equally easy to approach by this technique. In this review we will try to give an overview of the properties of different paramagnetic centers and how they can be used to increase the chances of experimental success.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Metaloproteínas/química , Metais/química , Ressonância Magnética Nuclear Biomolecular/métodos , Compostos Organometálicos/química , Cobre/química , Ferro/química , Modelos Moleculares , Níquel/química , Conformação ProteicaRESUMO
Partial symmetry, i.e., the presence of more than one molecule in the asymmetric unit of a crystal, is a relatively rare phenomenon in small-molecule crystallography, but is quite common in protein crystallography, where it is typically known as non-crystallographic symmetry (NCS). Several papers in literature propose molecular determinants such as crystal contacts, thermal factors, or TLS parameters as an explanation for the phenomenon of intrinsic asymmetry among molecules that are in principle equivalent. Nevertheless, are all of the above determinants the cause or are they rather the effect? In the general frame of the NCS often observed in crystals of biomolecules, this paper deals with nickel(II)-substituted human carbonic anhydrase(II) (hCAII) and its SAD structure determination at the nickel edge. The structure revealed two non-equivalent molecules in the asymmetric unit, the presence of a secondary nickel-binding site at the N-terminus of both molecules (which had never been found before in the nickel-substituted enzyme) and two different coordination geometries of the active site nickel (hexa-coordinated in one molecule and mainly penta-coordinated in the other). The above-mentioned standard molecular crystallographic determinants of this asymmetry are analyzed and presented in detail for this particular case. From these considerations, we speculate on the existence of a fundamental, although yet unknown, common cause for the partial symmetry that is so often encountered in X-ray structures of biomolecules.
Assuntos
Anidrase Carbônica II/química , Níquel/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
Resonance assignment and structural characterization of pharmacologically relevant proteins promise to improve understanding and safety of these proteins by rational design. However, the PEG coating that is used to evade the immune system also causes these molecules to "evade" the standard structural biology methodologies. We here demonstrate that it is possible to obtain the resonance assignment and a reliable structural model of large PEGylated proteins through an integrated approach encompassing NMR and X-ray crystallography.
Assuntos
Asparaginase , Asparaginase/química , Asparaginase/metabolismo , Materiais Revestidos Biocompatíveis , Espectroscopia de Ressonância Magnética/métodos , Polietilenoglicóis , Multimerização ProteicaRESUMO
The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.
Assuntos
Proteína Semelhante a ELAV 1/química , Fenantrenos/química , Fenantrenos/farmacologia , Regiões 3' não Traduzidas , Elementos Ricos em Adenilato e Uridilato , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Furanos , Humanos , Espectroscopia de Ressonância Magnética , Camundongos Mutantes Neurológicos , Simulação de Dinâmica Molecular , Fenantrenos/metabolismo , Mutação Puntual , Conformação Proteica , Domínios Proteicos , Quinonas , RNA Mensageiro/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Paramagnetic NMR data can be profitably incorporated in structural refinement protocols of metalloproteins or metal-substituted proteins, mostly as distance or angle restraints. However, they could in principle provide much more information, because the magnetic susceptibility of a paramagnetic metal ion is largely determined by its coordination sphere. This information can in turn be used to evaluate changes occurring in the coordination sphere of the metal when ligands (e.g.: inhibitors) are bound to the protein. This gives an experimental handle on the molecular structure in the vicinity of the metal which falls in the so-called blind sphere. The magnetic susceptibility anisotropy tensors of cobalt(II) and nickel(II) ions bound to human carbonic anhydrase II in free and inhibited forms have been determined. The change of the magnetic susceptibility anisotropy is directly linked to the binding mode of different ligands in the active site of the enzyme. Indication about the metal coordination sphere in the presence of an inhibitor in pharmaceutically relevant proteins could be important in the design of selective drugs with a structure-based approach.
Assuntos
Anidrase Carbônica II/metabolismo , Cobalto/metabolismo , Espectroscopia de Ressonância Magnética/estatística & dados numéricos , Metaloproteínas/metabolismo , Níquel/metabolismo , Anisotropia , Anidrase Carbônica II/química , Domínio Catalítico , Cobalto/química , Complexos de Coordenação/química , Furosemida/química , Humanos , Ligantes , Metaloproteínas/química , Modelos Teóricos , Estrutura Molecular , Níquel/química , Ligação ProteicaRESUMO
Protein glycosylation is the most complex post-translational modification process. More than 50 % of human cells proteins are glycosylated, whereas bacteria such as E. coli do not have this modification machinery. Indeed, the carbohydrate residues in natural proteins affect their folding, immunogenicity, and stability toward proteases, besides controlling biological properties and activities. It is therefore important to introduce such structural modification in bioengineered proteins lacking the presence of carbohydrate residues. This is not trivial as it requires reagents and conditions compatible with the protein's stability and reactivity. This work reports on the introduction of lactose moieties in two natural proteins, namely ubiquitin (Ub) and l-asparaginaseâ II (ANSII). The synthetic route employed is based on the sulfur(VI) fluoride exchange (SuFEx) coupling of a lactose tethered arylfluorosulfate (Lact-Ar-OSO2 F) with the ϵ-NH2 group of lysine residues of the proteins. This metal-free click SuFEx reaction relies on the properties of the fluorosulfate employed, which is easily prepared in multigram scale from available precursors and reacts chemoselectively with the ϵ-NH2 group of lysine residues under mild conditions. Thus, iterative couplings of Lact-Ar-OSO2 F to Ub and ANSII, afforded multiple glycosylations of these proteins so that up to three and four Lact-Ar-OSO2 groups were introduced in Ub and ANSII, respectively, via the formation of a sulfamoyl (OSO2 -NH) linkage.
RESUMO
Active bioinspired materials are appealing biotechnological targets, and their study is gaining momentum. These materials, which comprise of an inorganic matrix and one or more biomolecules, are extremely variable and therefore may result difficult to characterize in their intimate structure. In this work we have prepared a hydroxyapatite-l-asparaginase composite, with the perspective of using it in acute leukemia treatment. We demonstrate that the use of electron microscopy and powder X-ray diffraction, combined with the atomic-resolution information coming from solid-state NMR, allows us to understand the topology of the material and how the different components interplay to obtain an active composite.
Assuntos
Asparaginase/química , Materiais Biocompatíveis/síntese química , Durapatita/química , Proteínas de Escherichia coli/química , Engenharia de Proteínas , Sequência de Aminoácidos , Asparaginase/genética , Asparaginase/ultraestrutura , Linhagem Celular Tumoral , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Humanos , Espectroscopia de Ressonância Magnética , Teste de Materiais , Microscopia Eletrônica de Varredura , Estabilidade Proteica , Propriedades de Superfície , Difração de Raios XRESUMO
Carbohydrate-based vaccines are among the safest and most effective vaccines and represent potent tools for prevention of life-threatening bacterial infectious diseases, like meningitis and pneumonia. The chemical conjugation of a weak antigen to protein as a source of T-cell epitopes generates a glycoconjugate vaccine that results more immunogenic. Several methods have been used so far to characterize the resulting polysaccharide-protein conjugates. However, a reduced number of methodologies has been proposed for measuring the degree of saccharide conjugation at the possible protein sites. Here we show that detailed information on large proteins conjugated with large polysaccharides can be achieved by a combination of solution and solid-state NMR spectroscopy. As a test case, a large protein assembly, l-asparaginase II, has been conjugated with Neisseria meningitidis serogroup C capsular polysaccharide and the pattern and degree of conjugation were determined.
RESUMO
PEGylated proteins are widely used in biomedicine but, in spite of their importance, no atomic-level information is available since they are generally resistant to structural characterization approaches. PEGylated proteins are shown here to yield highly resolved solid-state NMR spectra, which allows assessment of the structural integrity of proteins when PEGylated for therapeutic or diagnostic use.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Polietilenoglicóis/química , Proteínas/químicaRESUMO
BACKGROUND: Aggregation of α-synuclein (α-syn) is a prominent feature of Parkinson's disease (PD) and other synucleinopathies. Currently, α-syn seed amplification assays (SAAs) using cerebrospinal fluid (CSF) represent the most promising diagnostic tools for synucleinopathies. However, CSF itself contains several compounds that can modulate the aggregation of α-syn in a patient-dependent manner, potentially undermining unoptimized α-syn SAAs and preventing seed quantification. METHODS: In this study, we characterized the inhibitory effect of CSF milieu on detection of α-syn aggregates by means of CSF fractionation, mass spectrometry, immunoassays, transmission electron microscopy, solution nuclear magnetic resonance spectroscopy, a highly accurate and standardized diagnostic SAA, and different in vitro aggregation conditions to evaluate spontaneous aggregation of α-syn. RESULTS: We found the high-molecular weight fraction of CSF (> 100,000 Da) to be highly inhibitory on α-syn aggregation and identified lipoproteins to be the main drivers of this effect. Direct interaction between lipoproteins and monomeric α-syn was not detected by solution nuclear magnetic resonance spectroscopy, on the other hand we observed lipoprotein-α-syn complexes by transmission electron microscopy. These observations are compatible with hypothesizing an interaction between lipoproteins and oligomeric/proto-fibrillary α-syn intermediates. We observed significantly slower amplification of α-syn seeds in PD CSF when lipoproteins were added to the reaction mix of diagnostic SAA. Additionally, we observed a decreased inhibition capacity of CSF on α-syn aggregation after immunodepleting ApoA1 and ApoE. Finally, we observed that CSF ApoA1 and ApoE levels significantly correlated with SAA kinetic parameters in n = 31 SAA-negative control CSF samples spiked with preformed α-syn aggregates. CONCLUSIONS: Our results describe a novel interaction between lipoproteins and α-syn aggregates that inhibits the formation of α-syn fibrils and could have relevant implications. Indeed, the donor-specific inhibition of CSF on α-syn aggregation explains the lack of quantitative results from analysis of SAA-derived kinetic parameters to date. Furthermore, our data show that lipoproteins are the main inhibitory components of CSF, suggesting that lipoprotein concentration measurements could be incorporated into data analysis models to eliminate the confounding effects of CSF milieu on α-syn quantification efforts.
Assuntos
Doença de Parkinson , Sinucleinopatias , Humanos , alfa-Sinucleína/química , Doença de Parkinson/diagnóstico , LipoproteínasRESUMO
Structural data on the SARS-CoV-2 main protease in complex with a zinc-containing organic inhibitor are already present in the literature and gave hints on the presence of a zinc binding site involving the catalytically relevant cysteine and histidine residues. In this paper, the structural basis of ionic zinc binding to the SARS-CoV-2 main protease has been elucidated by X-ray crystallography. The zinc binding affinity and its ability to inhibit the SARS-CoV-2 main protease have been investigated. These findings provide solid ground for the design of potent and selective metal-conjugated inhibitors of the SARS-CoV-2 main protease.
Assuntos
Proteases 3C de Coronavírus/antagonistas & inibidores , SARS-CoV-2/enzimologia , Sítios de Ligação , COVID-19/virologia , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Cristalografia por Raios X , Humanos , Conformação Proteica , Zinco/metabolismoRESUMO
Glioblastoma Multiforme (GBM) is a highly invasive primary brain tumour characterized by chemo- and radio-resistance and poor overall survival. GBM can present an aberrant functionality of p53, caused by the overexpression of the murine double minute 2 protein (MDM2) and its analogue MDM4, which may influence the response to conventional therapies. Moreover, tumour resistance/invasiveness has been recently attributed to an overexpression of the chemokine receptor CXCR4, identified as a pivotal mediator of glioma neovascularization. Notably, CXCR4 and MDM2-4 cooperate in promoting tumour invasion and progression. Although CXCR4 actively promotes MDM2 activation leading to p53 inactivation, MDM2-4 knockdown induces the downregulation of CXCR4 gene transcription. Our study aimed to assess if the CXCR4 signal blockade could enhance glioma cells' sensitivity to the inhibition of the p53-MDMs axis. Rationally designed inhibitors of MDM2/4 were combined with the CXCR4 antagonist, AMD3100, in human GBM cells and GBM stem-like cells (neurospheres), which are crucial for tumour recurrence and chemotherapy resistance. The dual MDM2/4 inhibitor RS3594 and the CXCR4 antagonist AMD3100 reduced GBM cell invasiveness and migration in single-agent treatment and mainly in combination. AMD3100 sensitized GBM cells to the antiproliferative activity of RS3594. It is noteworthy that these two compounds present synergic effects on cancer stem components: RS3594 inhibited the growth and formation of neurospheres, AMD3100 induced differentiation of neurospheres while enhancing RS3594 effectiveness preventing their proliferation/clonogenicity. These results confirm that blocking CXCR4/MDM2/4 represents a valuable strategy to reduce GBM proliferation and invasiveness, acting on the stem cell component too.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzilaminas/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Proteínas de Ciclo Celular/antagonistas & inibidores , Ciclamos/farmacologia , Glioblastoma/tratamento farmacológico , Indóis/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores CXCR4/antagonistas & inibidores , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Glioblastoma/enzimologia , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Invasividade Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Neurogênese/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , Esferoides Celulares , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The inhibition of the PD-1/PD-L1 axis by monoclonal antibodies has achieved remarkable success in treating a growing number of cancers. However, a novel class of small organic molecules, with BMS-202 (1) as the lead, is emerging as direct PD-L1 inhibitors. Herein, we report a series of 2,4,6-tri- and 2,4-disubstituted 1,3,5-triazines, which were synthesized and assayed for their PD-L1 binding by NMR and homogeneous time-resolved fluorescence. Among them, compound 10 demonstrated to strongly bind with the PD-L1 protein and challenged it in a co-culture of PD-L1 expressing cancer cells (PC9 and HCC827 cells) and peripheral blood mononuclear cells enhanced antitumor immune activity of the latter. Compound 10 significantly increased interferon γ release and apoptotic induction of cancer cells, with low cytotoxicity in healthy cells when compared to 1, thus paving the way for subsequent preclinical optimization and medical applications.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias/imunologia , Neoplasias/patologia , Bibliotecas de Moléculas Pequenas/farmacologia , Triazinas/farmacologia , Varredura Diferencial de Calorimetria , Linhagem Celular Tumoral , Técnicas de Cocultura , Humanos , Inibidores de Checkpoint Imunológico/química , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Triazinas/químicaRESUMO
Protein-protein interactions (PPIs) contribute to the onset and/or progression of several diseases, especially cancer, and this discovery has paved the way for considering disruption of the PPIs as an attractive anti-tumor strategy. In this regard, simple and efficient biophysical methods for detecting the interaction of the inhibitors with the protein counterpart are still in high demand. Herein, we describe a convenient NMR method for the screening of putative PPI inhibitors based on the use of "hot peptides" (HOPPI-NMR). As a case study, HOPPI-NMR was successful applied to the well-known p53/MDM2 system. Our outcomes highlight the main advantages of the method, including the use of a small amount of unlabeled proteins, the minimization of the risk of protein aggregation, and the ability to identify weak binders. The last leaves open the possibility for application of HOPPI-NMR in tandem with fragment-based drug discovery as a valid strategy for the identification of novel chemotypes acting as PPI inhibitors.