Fragment-based Screening Approach Research Articles

Fragment-based virtual screening approach and molecular dynamics simulation studies for identification of BACE1 inhibitor leads

Traditional structure-based virtual screening method to identify drug-like small molecules for BACE1 is so far unsuccessful. Location of BACE1, poor Blood Brain Barrier permeability and P-glycoprotein (Pgp) susceptibility of the inhibitors make it even more difficult. Fragment-based drug design method is suitable for efficient optimization of initial hit molecules for target like BACE1. We have developed a fragment-based virtual screening approach to identify/optimize the fragment molecules as a starting point. This method combines the shape, electrostatic, and pharmacophoric features of known fragment molecules, bound to protein conjugate crystal structure, and aims to identify both chemically and energetically feasible small fragment ligands that bind to BACE1 active site. The two top-ranked fragment hits were subjected for a 53 ns MD simulation. Principle component analysis and free energy landscape analysis reveal that the new ligands show the characteristic features of established BACE1 inhibitors. The potent method employed in this study may serve for the development of potential lead molecules for BACE1-directed Alzheimer’s disease therapeutics.

FRET Studies of Quinolone-Based Bitopic Ligands and Their Structural Analogues at the Muscarinic M1 Receptor.

Aiming to design partial agonists as well as allosteric modulators for the M1 muscarinic acetylcholine (M1AChR) receptor, two different series of bipharmacophoric ligands and their structural analogues were designed and synthesized. The hybrids were composed of the benzyl quinolone carboxylic acid (BQCA)-derived subtype selective allosteric modulator 3 and the orthosteric building block 4-((4,5-dihydroisoxazol-3-yl)oxy)-N,N-dimethylbut-2-yn-1-amine (base of iperoxo) 1 or the endogenous ligand 2-(dimethylamino)ethyl acetate (base of acetylcholine) 2, respectively. The two pharmacophores were linked via alkylene chains of different lengths (C4, C6, C8, and C10). Furthermore, the corresponding structural analogues of 1 and 2 and of modified BQCA 3 with varying alkyl chain length between C2 and C10 were investigated. Fluorescence resonance energy transfer (FRET) measurements in a living single cell system were investigated in order to understand how these compounds interact with a G protein-coupled receptor (GPCR) on a molecular level and how the single moieties contribute to ligand receptor interaction. The characterization of the modified orthosteric ligands indicated that a linker attached to an orthoster rapidly attenuates the receptor response. Linker length elongation increases the receptor response of bitopic ligands, until reaching a maximum, followed by a gradual decrease. The optimal linker length was found to be six methylene groups at the M1AChR. A new conformational change is described that is not of inverse agonistic origin for long linker bitopic ligands and was further investigated by exceptional fragment-based screening approaches.

Abstract 2180: Drug-like inhibitors of SGK1: Discovery and optimization of low molecular weight fragment leads

Abstract Triple negative (ER, PR and HER2/neu negative) breast cancers (TNBC) constitute 15-24% of breast cancers, but account for a disproportionate share of mortality. TNBC tumors are more aggressive, lack a targeted therapy, and are prone to relapse and metastasis after cytotoxic drug treatments, the only drug therapy currently available. Serum and glucocorticoid-regulated kinase 1 (SGK1) promotes the growth, invasiveness and chemo-resistance of cancer cells and is overexpressed in 48% of breast cancers, yet is not detected in normal breast tissue. In particular, SGK1 is overexpressed in TNBC cells and knockdown of SGK1 blocks the proliferation and invasiveness of TNBC cells. Thus, SGK1 is an attractive targeted therapy for TNBC. In the SGK1 inhibitors that have been reported previously, poor physicochemical properties have prevented these inhibitors from being active in vivo and advancing to the clinic. To discover drug-like inhibitors of SGK1, we used our technology platform (Leap-To-Lead™) and a fragment-based screening approach to find SGK1 inhibitor scaffolds. Low molecular weight inhibitors like ours are more readily optimized for potency while retaining drug-like properties. From this screen, we identified 11 scaffolds as novel inhibitors of SGK1. Four scaffold series had clear structure activity relationships (SAR). To illustrate the potential of these scaffolds, we carried out a limited synthetic expansion around one scaffold and improved potency more than 600-fold (SGK1 IC50 = 1 μM; LE = 0.4). Several compounds dose-dependently inhibited the proliferation of a TNBC cell line (MDA-MB-231) and inhibited the phosphorylation of the biomarker protein N-Myc downstream regulated 1 (NDRG-1). Unlike previously reported SGK1 inhibitors, our fragment lead compounds show excellent cellular penetration in Caco-2 assays. In summary, a fragment-based screening approach was used to identify four novel scaffolds with clear SAR trends. A single scaffold series was further optimized and shows anti-proliferative activity and biomarker modulation in a TNBC cell line. These SGK1 inhibitor series represent excellent starting points for further SAR studies and development of a novel preclinical candidate. Citation Format: James Zapf, Todd Meyer, Warren Wade, Laura Lingardo, Ayse Batova, Gordon Alton, Peter Pallai. Drug-like inhibitors of SGK1: Discovery and optimization of low molecular weight fragment leads. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2180.

Abstract A113: A fragment-based approach towards the identification of small molecule inhibitors of fascin 1

Abstract Fascin 1 is an actin-bundling protein that is dramatically overexpressed in a variety of invasive tumors and thought to have a critical role in cancer cell metastasis. However, as a drug target it is highly challenging due to its mechanism of protein-protein interaction and the lack of knowledge around the critical actin-binding sites. Using a fragment-based approach, biophysical assay screening and X-ray crystallography, we have been able to identify and optimize novel fascin 1 inhibitors. Furthermore we have developed robust and reproducible biochemical binding and bundling assays which have allowed us to develop ligands with submicromolar affinity. Fascin 1 cross-links filamentous actin (F-actin) into parallel bundles that are involved in the formation of dynamic cellular protrusions (such as lamellipodia and filopodia) used during cell migration. It also contributes to the formation of actin-rich finger-like protrusions, termed invadopodia, that tumor cell lines use to degrade the tumor extracellular matrix (ECM). Fascin 1 expression is low or absent in normal epithelia but is dramatically increased in a variety of tumor types including bladder, colon, lung, ovarian and pancreas. Its increased expression is associated with the most aggressive and metastatic tumor types and has been shown to be an independent prognostic indicator of poor clinical outcome. Fascin knockdown has been shown to reduce tumor cell invasion both in vitro and in vivo, suggesting fascin as a valid target for small molecule inhibitors. We have utilized a fragment-based screening approach incorporating surface plasmon resonance (SPR) to identify small molecules that bind to fascin 1. Our fragment library (∼1000 compounds) yielded 53 hit compounds that were confirmed by dose-response. Using these hits, X-ray co-crystallography identified four independent ligand binding sites within fascin. We have concentrated our efforts on one site, a deeply enclosed pocket between fascin domains 1 and 2. A number of fragment hits and commercial analogues were found to bind in this pocket and open a channel towards the surface of the protein. Subsequent virtual screening studies identified BDP-00010834, a compound which binds in this pocket with an SPR Kd of 29.7μM. Moreover, BDP-00010834 shows activity (IC50 = 50μM) in a functional biochemical screening assay that measures fascin-mediated F-actin bundling. Structure-based optimization together with X-ray co-crystallography has generated compounds with a 25- to 50-fold increase in both binding affinity and functional activity over this screening hit. Best-in-series compounds include BDP-00011163 (Kd = 1.2μM, IC50 = 0.8μM) and BDP-00011165 (Kd = 0.6μM, IC50 = 1.1μM). Citation Format: Daniel Croft, Stuart Francis, Alexander Schuettelkopf, Charles Parry, Gillian Goodwin, Angelo Pugliese, Laura McDonald, Maeve Clarke, Andrew Pannifer, Jen Lonsdale, Christopher Gray, Justin Bower, Heather McKinnon, Laura Machesky, Martin Drysdale. A fragment-based approach towards the identification of small molecule inhibitors of fascin 1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A113.

Fragment-Based Drug Discovery Targeting Inhibitor of Apoptosis Proteins: Discovery of a Non-Alanine Lead Series with Dual Activity Against cIAP1 and XIAP.

Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptosis and pro-survival signaling pathways whose deregulation is often associated with tumor genesis and tumor growth. IAPs have been proposed as targets for anticancer therapy, and a number of peptidomimetic IAP antagonists have entered clinical trials. Using our fragment-based screening approach, we identified nonpeptidic fragments binding with millimolar affinities to both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP). Structure-based hit optimization together with an analysis of protein-ligand electrostatic potential complementarity allowed us to significantly increase binding affinity of the starting hits. Subsequent optimization gave a potent nonalanine IAP antagonist structurally distinct from all IAP antagonists previously reported. The lead compound had activity in cell-based assays and in a mouse xenograft efficacy model and represents a highly promising start point for further optimization.

Fragment-Based Discovery of a Small Molecule Inhibitor of Bruton's Tyrosine Kinase.

The discovery and optimization of a series of 4-aminocinnoline-3-carboxamide inhibitors of Bruton's tyrosine kinase are reported. A fragment-based screening approach incorporating X-ray co-crystallography was used to identify a cinnoline fragment and characterize its binding mode in the ATP binding site of Btk. Optimization of the fragment hit resulted in the identification of a lead compound which reduced paw swelling in a dose- and exposure-dependent fashion in a rat model of collagen-induced arthritis.

Discovery of novel P1 groups for coagulation factor VIIa inhibition using fragment-based screening.

A multidisciplinary, fragment-based screening approach involving protein ensemble docking and biochemical and NMR assays is described. This approach led to the discovery of several structurally diverse, neutral surrogates for cationic factor VIIa P1 groups, which are generally associated with poor pharmacokinetic (PK) properties. Among the novel factor VIIa inhibitory fragments identified were aryl halides, lactams, and heterocycles. Crystallographic structures for several bound fragments were obtained, leading to the successful design of a potent factor VIIa inhibitor with a neutral lactam P1 and improved permeability.

Abstract LB-16: Discovery of novel SGK1 inhibitors by a fragment-based approach: Generating patentable, chemically tractable and ligand efficient leads

Abstract Triple negative (ER, PR and HER2/neu negative) breast cancer (TNBC) can have an unfavorable prognosis compared to other breast cancers. TNBC is more difficult to treat since most targeted breast cancer therapies are directed at one of the three receptors. Aggressive combinatorial therapies are usually required and the cytotoxic agents used in these regimens frequently loose effectiveness with time. Serum and glucocorticoid-regulated kinase 1 (SGK1) (a serine/threonine protein kinase), promotes the growth and invasiveness of breast cancer cells. SGK1 is a key protein regulated by the glucocorticoid receptor (GR). In TNBC tumors, high expression/activity in the GR/SGK axis correlates with shorter survival times and resistance to chemotherapy. The small numbers of SGK1 inhibitors reported in the literature have resulted from screening against other kinases (such as CHK1/azaindoles) and thus are not selective and lack patentable chemical diversity. We describe the identification of novel SGK1 inhibitors using a fragment-based library screening approach. The BioBlocks Leap-To-Lead platform contains a library of novel fragment-like compounds that span a range of polarities and sizes without compromising on flexibility. For example, this 275 compound library has an average molecule weight of 160 Da; AlogP, 1.1; hydrogen bond donors, 1.1; hydrogen bond acceptors, 2.2, and rotatable bonds, 0.7 yet still contains 114 unique rings. The library was screened against fully activated human SGK1 in an ADP-Glo assay using physiologically relevant peptide substrate and an optimal ATP concentration to also identify non-classical kinase inhibitor mode binders. The fragment library was screened at 2 mM and hits were profiled in IC50 dose response. From these, several novel chemical matter hit compounds were selected that had ligand efficiency values greater than 0.3 and spanned IC50 values from 75 µM to 2000 µM. In summary, a fragment-based screening approach was used to identify several novel scaffolds that represent both classical and non-classical kinase inhibitor binding modes as hit-to-lead starting points for the optimization of novel, potent and selective SGK1 inhibitors. Citation Format: Gordon Alton, Warren Wade, Laura Lingardo, Ayse Batova, Tom Smart, Chris Buhr, James W. Zapf, Peter Pallai. Discovery of novel SGK1 inhibitors by a fragment-based approach: Generating patentable, chemically tractable and ligand efficient leads. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-16. doi:10.1158/1538-7445.AM2014-LB-16

Abstract SY05-01: Fragment-based drug discovery: Less is more

Abstract Over the past decade, fragment-based drug discovery (FBDD) has become increasingly widely used across industry and academia, as highlighted recently (1). This is likely driven by the successful identification of leads for targets considered intractable using conventional screening strategies, in addition to delivering leads with improved physicochemical properties for more tractable targets such as kinases (2). The fundamental concept of fragment screening is to use simpler molecules so that the chemical space can be sampled much more efficiently than is possible when using molecules of greater complexity (3). One consequence of screening smaller, simpler fragments is that their affinity is expected to be relatively low (>1 mM), given the limited numbers of potential interactions that they can make with the protein, and the detection of low-potency fragment hits presents substantial technical challenges. However, although weak in potency, fragment hits make high-quality interactions with the protein as they must overcome a substantial entropic barrier to binding, relative to their size, and their binding thereby frequently occurs with high efficiencies. We now have experience of over 30 high-throughput crystallographic and biophysical fragment screens against a broad range of protein classes, and our results support the view that less complex molecules give a higher hit rate. This rich dataset of protein-fragment interactions has allowed continuous optimization of our fragment library, to maximize both success in fragment screening campaigns, and synthetic efficiency during fragment development. In addition, the ability to routinely produce multiple iterations of fast-turnaround crystallography has proven invaluable in guiding medicinal chemistry optimization, allowing parsimonious building of affinity, which provides drug candidates with enhanced ligand efficiency likely to minimize attrition in development. The success of the approach will be illustrated by the fragment-based discovery of novel dual inhibitors of protein-protein interactions involving XIAP and cIAP1. XIAP and cIAP1 are members of the inhibitor of apoptosis protein (IAP) family, a class of proteins that can down-regulate the apoptosis process by inhibiting caspases and other apoptotic factors (4). A defining feature of these proteins is the presence of three Baculoviral IAP Repeat (BIR) domains in their sequence. SMAC (second mitochondrial activator of caspases) is released by mitochondria in response to apoptotic stimuli and deactivates the function of IAPs through binding of its N-terminal region (AVPI) to the IAP-BIR domains. This event disrupts protein-protein interactions between IAPs and caspases and promotes apoptosis. It has recently been demonstrated that dual antagonism of both XIAP and cIAP1 is required to achieve a strong apoptotic response (5). Several companies and academic groups have active programs developing SMAC peptidomimetic compounds for the treatment of cancer. These compounds are based on the key alanine motif in AVPI which engenders cIAP1 selectivity (IC50 values for AVPI vs XIAP-BIR3 and cIAP1-BIR3 are 0.3 uM and 0.016 uM respectively). Our fragment-based screening approach, PyramidTM, enabled us to identify a non-alanine fragment which binds with similar affinity for both XIAP and cIAP1. Hit optimisation using a structure-based approach led to the discovery of a dual XIAP and cIAP1 antagonist with potent in vivo pharmacodynamic activity when delivered orally in a mouse xenograft model. High concentrations of the compound were measured in tumor and plasma over a 24 h period which ensured potent antagonism of both XIAP and cIAP1. Induction of apoptosis biomarkers (cleaved PARP, cleaved caspase-3) and strong inhibition of tumor growth were also observed for this new class of inhibitors.

Fragment-screening at the BESSY II MX-beamlines of the Helmholtz-Zentrum Berlin

"Within the last decade, the fragment-based screening approach has been matured to a reliable and powerful instrument of pharmaceutical drug discovery. The success of fragment screening strongly depends on the quality of the chosen fragment library (100-200 Da), the quality of the target protein diffraction as well as the possibility to use high throughput methods for the screen application. A thorough crystallographic analysis of many protein-fragment complex structures and their binding modes has the perspective to result in the development of new potential lead structures and to map the interaction landscape of protein surfaces. Recently we started the development of a dedicated experimental facility for high throughput fragment screening at the BESSY II storage ring. The in house data processing pipeline ""XDSAPP"" [1] has been developed to speed up the data evaluation of large amounts of diffraction data. We have assembled a fragment library of 96 compounds and have validated this library against two target proteins. These first results suggest that our library is capable of identifying binding partners at a hit rate of close to 10%. This library together with a fully automated beam line [2] will be made accessible to users, thus enabling fragment-screening experiments on a much broader basis. "

Synthesis, screening and docking of small heterocycles as Glycogen Phosphorylase inhibitors

A series of morpholine substituted amino acids (phenylalanine, leucine, lysine and glutamic acid) was synthesized. A fragment-based screening approach was then used to evaluate a series of small heterocycles, including morpholine, oxazoline, dihydro-1,3-oxazine, tetrahydro-1,3-oxazepine, thiazoline, tetrahydro-1,3-pyrimidine, tetrahydro-1,3-diazepine and hexahydro-1H-benzimidazole, as potential inhibitors of Glycogen Phosphorylase a. Thiazoline 7 displayed an improved potency (IC50 of 25 μM) and had good LE and LELP values, as compared to heterocycles 1, 5, 9–13 and 19 (IC50 values of 1.1 mM–23.9 mM). A docking study using the crystal structure of human liver Glycogen Phosphorylase, provided insight into the interactions of heterocycles 5, 7, 9–13 and 19 with Glycogen Phosphorylase.

A fragment-based approach to identifying S-adenosyl-l-methionine -competitive inhibitors of catechol O-methyl transferase (COMT).

Catechol O-methyl transferase belongs to the diverse family of S-adenosyl-l-methionine transferases. It is a target involved in the treatment of Parkinson's disease. Here we present a fragment-based screening approach to discover noncatechol derived COMT inhibitors which bind at the SAM binding pocket. We describe the identification and characterization of a series of highly ligand efficient SAM competitive bisaryl fragments (LE = 0.33-0.58). We also present the first SAM-competitive small-molecule COMT co-complex crystal structure.

Protein Crystallography and Fragment-Based Drug Design

Crystallography is a major tool for structure-driven drug design, as it allows knowledge of the 3D structure of protein targets and protein-ligand complexes. However, the route for crystal structure determination involves many steps, some of which may hamper its high-throughput use. Recent efforts have produced significant advances in experimental and computational tools and protocols. They include automatic crystallization tools, faster data collection devices, more efficient phasing methods and improved ligand-fitting procedures. The timescales of drug-discovery processes have been also reduced by using a fragment-based screening approach. Herein, the achievements in protein crystallography over the last 5 years are reviewed, and advantages and disadvantages of the fragment-based approaches to drug discovery that make use of x-ray crystallography as a primary screening method are examined. In particular, in some detail, five recent case studies pertaining to the development of new hits or leads in relevant therapeutic areas, such as cancer, immune response, inflammation, metabolic syndrome and neurology are described.

An in silico virtual screening study for the design of norovirus inhibitors: fragment-based molecular docking and binding free energy calculations

Gastrointestinal infections caused by noroviruses may be prevented by the inhibition of their binding to histo-blood group carbohydrate antigens. A fragment-based virtual screening approach was used, employing docking followed by molecular dynamics simulations in order to enable binding free energy calculations using the linear interaction energy method. The resulting structures, composed of high-affinity fragments, can be a good starting point for lead optimizations and four molecules that pass both REOS and SYLVIA filters, which can remove known toxic features and assess the synthetic accessibility, respectively, are proposed as inhibitors.

Identification of Purple Acid Phosphatase Inhibitors by Fragment‐Based Screening: Promising New Leads for Osteoporosis Therapeutics

Purple acid phosphatases are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters and anhydrides under acidic conditions. In humans, elevated purple acid phosphatases levels in sera are correlated with the progression of osteoporosis and metabolic bone malignancies, making this enzyme a target for the development of new chemotherapeutics to treat bone-related illnesses. To date, little progress has been achieved towards the design of specific and potent inhibitors of this enzyme that have drug-like properties. Here, we have undertaken a fragment-based screening approach using a 500-compound library identifying three inhibitors of purple acid phosphatases with K(i) values in the 30-60 μm range. Ligand efficiency values are 0.39-0.44 kcal/mol per heavy atom. X-ray crystal structures of these compounds in complex with a plant purple acid phosphatases (2.3-2.7 Å resolution) have been determined and show that all bind in the active site within contact of the binuclear centre. For one of these compounds, the phenyl ring is positioned within 3.5 Å of the binuclear centre. Docking simulations indicate that the three compounds fit into the active site of human purple acid phosphatases. These studies open the way to the design of more potent and selective inhibitors of purple acid phosphatases that can be tested as anti-osteoporotic drug leads.

Thieno[3,2-d]pyrimidin-4(3H)-one derivatives as PDK1 inhibitors discovered by fragment-based screening

Ligand efficient fragments binding to PDK1 were identified by an NMR fragment-based screening approach. Computational modeling of the fragments bound to the active site led to the design and synthesis of a series of novel 6,7-disubstituted thienopyrimidin-4-one compounds, with low micromolar inhibitory activity against PDK1 in a biochemical enzyme assay.

Abstract 2315: Pharmacodynamic and antitumor activity of fragment-derived inhibitors of MetAP2 in tumor xenografts

Abstract Background: Methionine aminopeptidase (MetAP) 2 is one of the two eukaryotic enzymes responsible for cleaving the N-terminal methionine from newly synthesized polypeptides to allow further post-translational modifications such as myristoylation to take place. The enzyme is the target of the anti-angiogenic natural product, fumagillin and so is believed to play a role in angiogenesis. Fumagillin analogues have shown activity in several disease models including oncology, inflammation and obesity indicating MetAP2 is a promising target in a number of indications. The semi-synthetic fumagillin analogue, TNP470, has shown activity in a Phase I/II cancer trial, suggesting that MetAP2 is a good oncology target. Here we describe the discovery, optimization and anti-tumor activity of fragment-derived MetAP2 inhibitors. Results: We identified fragment hits to MetAP2 using our fragment-based screening approach, Pyramid™. These were optimized by structure-based drug design to novel, potent lead compounds with sub-100 nM potency against the isolated MetAP2 enzyme. The two most advanced compounds inhibited proliferation of HUVECs with potencies of 130 nM and 300 nM. Levels of the MetAP2 substrate, Met-14-3-3, were shown to increase on treatment of HUVECs with these compounds indicating that Met-14-3-3 was not being processed and hence MetAP2 was being inhibited. The compounds also inhibited HUVEC tubule formation demonstrating their anti-angiogenic properties. The two lead compounds were further tested in vivo. Both compounds were well tolerated at doses up to 200 mg/kg bid. Levels of Met-14-3-3 were seen to increase in the thymus and spleen (where high expression of MetAP2 has been reported) of mice treated with the compounds, indicating again that MetAP2 was being inhibited in these tissues. Compound 1 was tested in a mouse HCT116 xenograft model. Mice were subcutaneously inoculated with HCT116 cells and oral dosing at 200 mg/kg bid started one day later. Tumor growth was inhibited in treated mice compared with control and growth inhibition was greater in Compound 1 treated mice (T/C 46%) compared with mice treated with 30 mg/kg TNP470 subcutaneously q2d (T/C 61%). Conclusions: The compounds described here are novel small-molecule inhibitors of MetAP2. Their promising tumor growth inhibitory properties merit their testing in further tumor models and potentially other indications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2315. doi:1538-7445.AM2012-2315

Abstract 2018: Discovery of potent dual inhibitors of both XIAP and cIAP1 using fragment based drug discovery

Abstract XIAP and cIAP1 are members of the inhibitor of apoptosis (IAP) protein family. Both proteins have the ability to attenuate apoptosis induced through intrinsic and extrinsic stimuli via inhibition of caspase-3, -7, -8 and -9. The defining feature of both XIAP and cIAP1 is the presence in their protein sequence of 3 Baculoviral IAP Repeat (BIR) domains, which are necessary for their antiapoptotic activity. The mitochondrial protein SMAC uses its N-terminal region (AVPI) to interact with BIR domains and deactivate the antiapoptotic function of IAPs. Several companies and academic groups have active programs developing SMAC peptidomimetic compounds based on the AVPI motif. In general, those compounds have the tendency to be cIAP1 selective like their tetrapeptide progenitor (AVPI IC50 values for XIAP-BIR3 and cIAP1-BIR3 are 0.3 uM and 0.016 uM respectively). Using our fragment-based screening approach, PyramidTM, we identified a non-peptidomimetic chemotype which binds with similar potency to the BIR3 domain of both XIAP and cIAP1. Hit optimisation using a structure based approach led to the discovery of potent true dual XIAP and cIAP1 antagonists with good in vivo physico-chemical profile and no P450 or hERG liabilities. Dual XIAP/cIAP1 inhibitors have potential for more effective apoptosis and less toxicity associated with cytokine production. Compounds were initially characterised in fluorescence polarisation binding assays using XIAP-BIR3 or cIAP1-BIR3 domains. Robust induction of apoptosis was observed in two sensitive breast cancer cell lines (EC50s well below 0.1 uM in EVSA-T and MDA-MB-231); whilst HCT116 cells (colon cancer) were insensitive (unless exogenous TNF-α was added). This in vitro cell line killing was demonstrated to correlate closely with cIAP1 antagonism and hence a parallel cell assay was established to measure XIAP antagonism. An engineered HEK293 cell line was stably co-transfected with full length FLAG-tagged human XIAP cDNA and full length (untagged) human caspase-9 cDNA. Inhibition of caspase-9 binding to XIAP was measured in immunoprecipitation assays. This gave us a sensitive read-out for XIAP antagonism in cells which could be plotted against the most sensitive cell killing read-out (from the EVSA-T cell line) to establish relative XIAP vs cIAP1 selectivities and to select dual antagonists of both IAPs. Potent compounds (HEK293-EC50 <0.01 uM and EVSA-T-EC50 <0.01 uM) were further characterised in PKPD studies in mice bearing MDA-MB-231 xenografts. Compounds with good oral exposure achieved high concentration in tumor over 24h periods which ensured excellent inhibition of both XIAP and cIAP1 with consequent reduction of cIAP1 levels and induction of apoptosis markers (PARP, Caspase-3). Finally, dual XIAP/cIAP1 inhibitors have been investigated in xenograft models (melanoma, breast and colorectal cancer) and have achieved significant efficacy at tolerated doses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2018. doi:1538-7445.AM2012-2018

Riboswitches: Discovery of Drugs That Target Bacterial Gene-Regulatory RNAs

Riboswitches are messenger RNA (mRNA) domains that regulate gene function in response to the intracellular concentration of a variety of metabolites and second messengers. They control essential genes in many pathogenic bacteria, thus representing an inviting new class of biomolecular target for the development of antibiotics and chemical-biological tools. In this Account, we briefly review the discovery of riboswitches in the first years of the 21st century and their ensuing characterization over the past decade. We then discuss the progress achieved so far in using riboswitches as a focus for drug discovery, considering both the value of past serendipity and the particular challenges that confront current researchers. Five mechanisms of gene regulation have been determined for riboswitches. Most bacterial riboswitches modulate either transcription termination or translation initiation in response to ligand binding. All known examples of eukaryotic riboswitches, and some bacterial riboswitches, control gene expression by alternative splicing. The glmS riboswitch, which is widespread in Gram-positive bacteria, is a catalytic RNA activated by ligand binding: its self-cleavage destabilizes the mRNA of which it is part. Finally, one example of a trans-acting riboswitch is known. Three-dimensional structures have been determined for representatives of 13 structurally distinct riboswitch classes, providing atomic-level insight into their mechanisms of ligand recognition. While cellular and viral RNAs have attracted widespread interest as potential drug targets, riboswitches show special promise due to the diversity of small-molecule recognition strategies that are on display in their ligand-binding pockets. Moreover, riboswitches have evolved to recognize small-molecule ligands, which is unique among known structured RNA domains. Structural and biochemical advances in the study of riboswitches provide an impetus for the development of methods for the discovery of novel riboswitch activators and inhibitors. Recent rational drug design efforts focused on select riboswitch classes have yielded a small number of candidate antibiotic compounds, including one active in a mouse model of Staphylococcus aureus infection. The development of high-throughput methods suitable for riboswitch-specific drug discovery is ongoing. A fragment-based screening approach employing equilibrium dialysis that may be generically useful has demonstrated early success. Riboswitch-mediated gene regulation is widely employed by bacteria; however, only the thiamine pyrophosphate-responsive riboswitch has thus far been found in eukaryotes. Thus, riboswitches are particularly attractive as targets for antibacterials. Indeed, antimicrobials with previously unknown mechanisms have been found to function by binding riboswitches and causing aberrant gene expression.

Fragment-based screening by X-ray crystallography, MS and isothermal titration calorimetry to identify PNMT (phenylethanolamine N-methyltransferase) inhibitors

CNS (central nervous system) adrenaline (epinephrine) is implicated in a wide range of physiological and pathological conditions. PNMT (phenylethanolamine N-methyltransferase) catalyses the final step in the biosynthesis of adrenaline, the conversion of noradrenaline (norepinephrine) to adrenaline by methylation. To help elucidate the role of CNS adrenaline, and to develop potential drug leads, potent, selective and CNS-active inhibitors are required. The fragment screening approach has advantages over other lead discovery methods including high hit rates, more efficient hits and the ability to sample chemical diversity more easily. In the present study we applied fragment-based screening approaches to the enzyme PNMT. We used crystallography as the primary screen and identified 12 hits from a small commercial library of 384 drug-like fragments. The hits include nine chemicals with two fused rings and three single-ring chemical systems. Eight of the hits come from three chemical classes: benzimidazoles (a known class of PNMT inhibitor), purines and quinolines. Nine of the hits have measurable binding affinities (~5-700 μM) as determined by isothermal titration calorimetry and all nine have ligand efficiencies of 0.39 kcal/mol per heavy atom or better (1 kcal≈4.184 kJ). We synthesized five elaborated benzimidazole compounds and characterized their binding to PNMT, showing for the first time how this class of inhibitors interact with the noradrenaline-binding site. Finally, we performed a pilot study with PNMT for fragment-based screening by MS showing that this approach could be used as a fast and efficient first-pass screening method prior to characterization of binding mode and affinity of hits.