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PDF file - 178K, Tenovin-D3 causes cell cycle arrest in SirT2 wild type but not knockout cells.

PDF file - 38K, Inhibition of purified SirT1 and SirT2 by tenovin-D3 using FdL substrates at 15 μM.

PDF file - 18K, Retention time of tenovin-D3 = 0.61 minutes.

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PDF file - 42K, MDAMB468 cells were treated with the indicated amounts of TSA for 6 hours.

PDF file - 139K, Cell lines with differing p53 status were treated with TSA for 6 hours.

PDF file - 30K, Tenovin-6 and tenovin-D3 are poor inhibitors of HDAC8 and SirT3.

While small-molecule inhibitors of class I/II histone deacetylases (HDAC) have been approved for cancer treatment, inhibitors of the sirtuins (a family of class III HDACs) still require further validation and optimization to enter clinical trials. Recent studies show that tenovin-6, a small-molecule inhibitor of sirtuins SirT1 and SirT2, reduces tumor growth in vivo and eliminates leukemic stem cells in a murine model for chronic myelogenous leukemia. Here, we describe a tenovin analogue, tenovin-D3, that preferentially inhibits sirtuin SirT2 and induces predicted phenotypes for SirT2 inhibition. Unlike tenovin-6 and in agreement with its weak effect on SirT1 (a p53 deacetylase), tenovin-D3 fails to increase p53 levels or transcription factor activity. However, tenovin-D3 promotes expression of the cell-cycle regulator and p53 target p21WAF1/CIP1 (CDKN1A) in a p53-independent manner. Structure–activity relationship studies strongly support that the ability of tenovin-D3 to inhibit SirT2 contributes to this p53-independent induction of p21. The ability of tenovin-D3 to increase p21 mRNA and protein levels is shared with class I/II HDAC inhibitors currently used in the clinic and therefore suggests that SirT2 inhibition and class I/II HDAC inhibitors have similar effects on cell-cycle progression. Mol Cancer Ther; 12(4); 352–60. ©2013 AACR.

PDF file - 163K, Supplementary Figure S1A. 1H NMR spectrum of tenovin-D3. Supplementary Figure S1B. 13C NMR spectrum of tenovin-D3.

PDF file - 55K, Cell lines with differing p53 status were treated with tenovin-D3 for 8 hours.

Graphical abstract, The monosaccharide l-Rhamnose is an important component of bacterial cell walls. The first step in the l-rhamnose biosynthetic pathway is catalysed by glucose-1-phosphate thymidylyltransferase (RmlA), which condenses glucose-1-phosphate (Glu-1-P) with deoxythymidine triphosphate (dTTP) to yield dTDP-d-glucose. In addition to the active site where catalysis of this reaction occurs, RmlA has an allosteric site that is important for its function. Building on previous reports, SAR studies have explored further the allosteric site, leading to the identification of very potent P. aeruginosa RmlA inhibitors. Modification at the C6-NH2 of the inhibitor’s pyrimidinedione core structure was tolerated. X-ray crystallographic analysis of the complexes of P. aeruginosa RmlA with the novel analogues revealed that C6-aminoalkyl substituents can be used to position a modifiable amine just outside the allosteric pocket. This opens up the possibility of linking a siderophore to this class of inhibitor with the goal of enhancing bacterial cell wall permeability.

Dispersing hydrophobin HFBII under air saturated with perfluorohexane gas limits HFBII aggregation to nanometer-sizes. Critical basic findings include an unusual co-adsorption effect caused by the fluorocarbon gas, a strong acceleration of HFBII adsorption at the air/water interface, the incorporation of perfluorohexane into the interfacial film, the suppression of the fluid-to-solid 2D phase transition exhibited by HFBII monolayers under air, and a drastic change in film elasticity of both Gibbs and Langmuir films. As a result, perfluorohexane allows the formation of homogenous populations of spherical, narrowly dispersed, exceptionally stable, and echogenic microbubbles.

Hydrophobins are natural surfactant proteins endowed with exceptional surface activity and film-forming capabilities and their use as effective “fluorine-free fluorosurfactants” has been recently reported. In order to increase their fluorophilicity further, here we report the preparation of a unique fluorous-modified hydrophobin, named F-HFBI. F-HFBI was found to be more effective than its wild-type parent protein HFBI at reducing interface tension of water at both air/water and oil/water interfaces, being particularly effective at the fluorous/water interface. F-HFBI was also found to largely retain the exceptionally good capability of forming strong and elastic films, typical of the hydrophobin family. Further studies by interface shear rheology and isothermal compression, alongside Quartz Crystal Microbalance and Atomic Force Microscopy, demonstrated the tendency of F-HFBI to form thicker films compared to the wild-type protein. These results suggest that F-HFBI may function as an effective compatibilizer for biphasic systems comprising a fluorous phase.

While small-molecule inhibitors of class I/II histone deacetylases (HDAC) have been approved for cancer treatment, inhibitors of the sirtuins (a family of class III HDACs) still require further validation and optimization to enter clinical trials. Recent studies show that tenovin-6, a small-molecule inhibitor of sirtuins SirT1 and SirT2, reduces tumor growth in vivo and eliminates leukemic stem cells in a murine model for chronic myelogenous leukemia. Here, we describe a tenovin analogue, tenovin-D3, that preferentially inhibits sirtuin SirT2 and induces predicted phenotypes for SirT2 inhibition. Unlike tenovin-6 and in agreement with its weak effect on SirT1 (a p53 deacetylase), tenovin-D3 fails to increase p53 levels or transcription factor activity. However, tenovin-D3 promotes expression of the cell-cycle regulator and p53 target p21WAF1/CIP1 (CDKN1A) in a p53-independent manner. Structure–activity relationship studies strongly support that the ability of tenovin-D3 to inhibit SirT2 contributes to this p53-independent induction of p21. The ability of tenovin-D3 to increase p21 mRNA and protein levels is shared with class I/II HDAC inhibitors currently used in the clinic and therefore suggests that SirT2 inhibition and class I/II HDAC inhibitors have similar effects on cell-cycle progression. Mol Cancer Ther; 12(4); 352–60. ©2013 AACR.

Glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the condensation of glucose-1-phosphate (G1P) with deoxy-thymidine triphosphate (dTTP) to yield dTDP-d-glucose and pyrophosphate. This is the first step in the l-rhamnose biosynthetic pathway. l-Rhamnose is an important component of the cell wall of many microorganisms, including Mycobacterium tuberculosis and Pseudomonas aeruginosa. Here we describe the first nanomolar inhibitors of P. aeruginosa RmlA. These thymine analogues were identified by high-throughput screening and subsequently optimized by a combination of protein crystallography, in silico screening, and synthetic chemistry. Some of the inhibitors show inhibitory activity against M. tuberculosis. The inhibitors do not bind at the active site of RmlA but bind at a second site remote from the active site. Despite this, the compounds act as competitive inhibitors of G1P but with high cooperativity. This novel behavior was probed by structural analysis, which suggests that the inhibitors work by preventing RmlA from undergoing the conformational change key to its ordered bi-bi mechanism.

HIV-1 maturation inhibitors are a novel class of antiretroviral compounds that consist of two structurally distinct chemical classes: betulinic acid derivatives and the pyridone-based compound PF-46396. It is currently believed that both classes act by similar modes of action to generate aberrant noninfectious particles via inhibition of CA-SP1 cleavage during Gag proteolytic processing. In this study, we utilized a series of novel analogues with decreasing similarity to PF-46396 to determine the chemical groups within PF-46396 that contribute to antiviral activity, Gag binding, and the relationship between these essential properties. A spectrum of antiviral activity (active, intermediate, and inactive) was observed across the analogue series with respect to CA-SP1 cleavage and HIV-1 (NL4-3) replication kinetics in Jurkat T cells. We demonstrate that selected inactive analogues are incorporated into wild-type (WT) immature particles and that one inactive analogue is capable of interfering with PF-46396 inhibition of CA-SP1 cleavage. Mutations that confer PF-46396 resistance can impose a defective phenotype on HIV-1 that can be rescued in a compound-dependent manner. Some inactive analogues retained the capacity to rescue PF-46396-dependent mutants (SP1-A3V, SP1-A3T, and CA-P157S), implying that they can also interact with mutant Gag. The structure-activity relationships observed in this study demonstrate that (i) the tert-butyl group is essential for antiviral activity but is not an absolute requirement for Gag binding, (ii) the trifluoromethyl group is optimal but not essential for antiviral activity, and (iii) the 2-aminoindan group is important for antiviral activity and Gag binding but is not essential, as its replacement is tolerated. IMPORTANCE Combinations of antiretroviral drugs successfully treat HIV/AIDS patients; however, drug resistance problems make the development of new mechanistic drug classes an ongoing priority. HIV-1 maturation inhibitors are novel as they target the Gag protein, specifically by inhibiting CA-SP1 proteolytic cleavage. The lack of high-resolution structural information of the CA-SP1 target in Gag has hindered our understanding of the inhibitor-binding pocket and maturation inhibitor mode of action. Therefore, we utilized analogues of the maturation inhibitor PF-46396 as chemical tools to determine the chemical components of PF-46396 that contribute to antiviral activity and Gag binding and the relationship between these essential properties. This is the first study to report structure-activity relationships of the maturation inhibitor PF-46396. PF-46396 is chemically distinct from betulinic acid-derived maturation inhibitors; therefore, our data provide a foundation of knowledge that will aid our understanding of how structurally distinct maturation inhibitors act by similar modes of action.

The tenovins are small molecule inhibitors of the NAD(+)-dependent family of protein deacetylases known as the sirtuins. There remains considerable interest in inhibitors of this enzyme family due to possible applications in both cancer and neurodegenerative disease therapy. Through the synthesis of novel tenovin analogues, further insights into the structural requirements for activity against the sirtuins in vitro are provided. In addition, the activity of one of the analogues in cells led to an improved understanding of the function of SirT1 in cells.

Amyloid supramolecular assemblies have found widespread exploitation as ordered nanomaterials in a range of applications from materials science to biotechnology. New strategies are, however, required for understanding and promoting mature fibril formation from simple monomer motifs through easy and scalable processes. Noncovalent interactions are key to forming and holding the amyloid structure together. On the other hand, the halogen bond has never been used purposefully to achieve control over amyloid self-assembly. Here we show that single atom replacement of hydrogen with iodine, a halogen-bond donor, in the human calcitonin-derived amyloidogenic fragment DFNKF results in a super-gelator peptide, which forms a strong and shape-persistent hydrogel at 30-fold lower concentration than the wild-type pentapeptide. This is remarkable for such a modest perturbation in structure. Iodination of aromatic amino acids may thus develop as a general strategy for the design of new hydrogels from unprotected peptides and without using organic solvents., Amyloid assemblies lie at the heart of many physiological functions, as well as being the cause of numerous diseases. Here, the authors subtly modify wild-type pentapeptides with halides, and discover that the new halogen bonding interactions have a remarkable influence on their physical properties.

A new and simple method is presented to fluorinate the surfaces of poorly reactive hydrophobic polymers in a more environmentally friendly way using the protein hydrophobin (HFBII) as a nanosized primer layer. In particular, HFBII, via electrostatic interactions, enables the otherwise inefficient binding of a phosphate-terminated perfluoropolyether onto polystyrene, polypropylene, and low-density polyethylene surfaces. The binding between HFBII and the perfluoropolyether depends significantly on the environmental pH, reaching the maximum stability at pH 4. Upon treatment, the polymeric surfaces mostly retain their hydrophobic character but also acquire remarkable oil repellency, which is not observed in the absence of the protein primer. The functionalization proceeds rapidly and spontaneously at room temperature in aqueous solutions without requiring energy-intensive procedures, such as plasma or irradiation treatments.

Background: Activation of wild-type p53 with the small molecule sirtuin inhibitor Tenovin-6 (Tnv-6) induces p53-dependent apoptosis in many malignant cells. In contrast, Tnv-6 reduces chronic lymphocytic leukaemia (CLL) cell viability with dysregulation of autophagy, without increasing p53-pathway activity. Methods: Here, we have investigated whether a quiescent phenotype (unique to CLL) determines the Tnv-6 response, by comparing the effects of Tnv-6 on activated and proliferating CLL. We further studied if these responses are p53-dependent. Results: Unlike quiescent cells, cell death in activated cultures treated with Tnv-6 was consistently associated with p53 upregulation. However, p53 acetylation remained unchanged, without caspase-3 cleavage or apoptosis on electron microscopy. Instead, cellular ultrastructure and protein profiles indicated autophagy inhibition, with reduced ubiquitin–proteasome activity. In specimens with mutant TP53 cultured with Tnv-6, changes in the autophagy-associated protein LC3 occurred independently of p53. Cells treated with Tnv-6 analogues lacking sirtuin inhibitory activity had attenuated LC3 lipidation compared with Tnv-6 (P⩽0.01), suggesting that autophagy dysregulation occurs predominantly through an effect on sirtuins. Conclusion: These cell cycle and p53-independent anti-leukaemic mechanisms potentially offer novel therapeutic approaches to target leukaemia-sustaining cells in CLL, including in disease with p53-pathway dysfunction. Whether targets in addition to sirtuins contribute to autophagy dysregulation by Tnv-6, requires further investigation.

The search for potent and selective sirtuin inhibitors continues as chemical tools of this type are of use in helping to assign the function of this interesting class of deacetylases. Here we describe SAR studies starting from the unselective sirtuin inhibitor tenovin-6. These studies identify a sub-micromolar inhibitor that has increased selectivity for SIRT2 over SIRT1 compared to tenovin-6. In addition, a H-1-NMR-based method is developed and used to validate further this class of sirtuin inhibitors. A thermal shift analysis of SIRT2 in the presence of tenovin-6, -43, a control tenovin and the known SIRT2 inhibitor AGK2 is also presented. Publisher PDF

For better or worse, there are considerable changes underway in the world of drug discovery. Whilst it is unclear what the future will bring, one possibility is a strategic shift in the modus operandi of the large pharmaceutical companies. One consequence of a shift away from high level investment in “in-house” research and development by pharma could be increased industrial interest in early stage research carried out in biotech, government or charity-funded research centres and academia. In many of these institutions, this type of research is often referred to as “chemical biology”. But what is chemical biology? In this chapter we take a less-trodden path to addressing this question with the overall aim of shedding some light on areas of chemical biology which are relevant to drug discovery. Chemical biology is a difficult topic to define in a few sentences since the subject is very wide ranging. Recent attempts have included an excellent review article in ChemBioChem written by some of the experts in the field (Altmann et al., 2009). A book focusing on the new frontiers in chemical biology has also recently been published (Bunnage, 2010). Interestingly, the Editorial in this book identifies “..reducing the high levels of attrition currently seen in “proof-of concept” Phase II clinical trials” as a key future challenge in drug discovery. It goes on to argue that this can only be achieved through “a much deeper knowledge of biological systems in order to identify and validate those biomolecular targets for which there is the highest possible confidence of disease-relevance in humans”. The Editor concludes by proposing that chemical biology has great potential to help address the challenges that have been summarised above. So again we ask what is chemical biology? Several excellent articles cover this issue in the New Frontiers book (Bunnage, 2010), but here we use an alternative method of probing what chemical biology really involves at the grass roots level. Our goal was to reflect on whether chemical biology, as currently practiced, really is and will be of interest to the drug discovery community in the future. Our chosen approach was to provide a review of all the chemical biology papers which were published in 2009 in the RSC journal Organic and Biomolecular Chemistry. Due to the number of papers, over 300, coverage of many of these exciting pieces of research is brief. We apologise for this and for any we may have inadvertently overlooked. However, our goal was to bring this body of work together in an organised and accessible form and to try and continue efforts to define what chemical biology involves. The chapter is split into three

Recent advances in the field of pharmacological activation of the p53 tumor suppressor are beginning to be translated into the clinic. In addition, small molecules that activate p53 through established mechanisms of action are proving invaluable tools for basic research. Here we analyze and compare the effects of nutlin-3, tenovin-6 and low doses of actinomycin-D on p53 and its main negative regulator, mdm2. We reveal striking differences in the speed at which these compounds increase p53 protein levels, with nutlin-3 having a substantial impact within minutes. We also show that nutlin-3 is very effective at increasing the synthesis of mdm2 mRNA, mdm2 being not only a modulator of p53 but also a transcriptional target. In addition, we show that nutlin-3 stabilizes mdm2's conformation and protects mdm2 from degradation. These strong effects of nutlin-3 on mdm2 correlate with a remarkable rate of recovery of p53 levels upon removal of the compound. We discuss the potential application of our results as molecular signatures to assess the on-target effects of small-molecule mdm2 inhibitors. To conclude, we discuss the implications of our observations for using small-molecule p53 activators to reduce the growth of tumors retaining wild-type p53 or to protect normal tissues against the undesired side effects of conventional chemotherapy.

The human deacetylase SIRT2 is believed to promote neurodegeneration with recent studies demonstrating that a reduction in the activity of SIRT2 can rescue alpha synuclein toxicity in Parkinson's disease models. In contrast, a second member of the sirtuin family, SIRT1, is believed to play a neuroprotective role. This dichotomy places an additional challenge in the path of sirtuin inhibitor development as a need for isozyme selectivity arises. By combining computational methods with assessment of the biological activity of novel N1-substituted cambinol analogues, further insights that are relevant to this challenge are obtained.