Search

Functionalization of C-H bonds is a key challenge in medicinal chemistry, particularly for fragment-based drug discovery (FBDD) where such transformations require execution in the presence of polar functionality necessary for protein binding. Recent work has shown the effectiveness of Bayesian optimization (BO) for the self-optimization of chemical reactions; however, in all previous cases these algorithmic procedures have started with no prior information about the reaction of interest. In this work, we explore the use of multitask Bayesian optimization (MTBO) in several in silico case studies by leveraging reaction data collected from historical optimization campaigns to accelerate the optimization of new reactions. This methodology was then translated to real-world, medicinal chemistry applications in the yield optimization of several pharmaceutical intermediates using an autonomous flow-based reactor platform. The use of the MTBO algorithm was shown to be successful in determining optimal conditions of unseen experimental C-H activation reactions with differing substrates, demonstrating an efficient optimization strategy with large potential cost reductions when compared to industry-standard process optimization techniques. Our findings highlight the effectiveness of the methodology as an enabling tool in medicinal chemistry workflows, representing a step-change in the utilization of data and machine learning with the goal of accelerated reaction optimization.

XIAP antagonism measurements in HEK293-XIAP-Caspase-9 xenografts

XIAP antagonism in A375 cells in the presence or absence of TNF-α

Preparation of 2-chloro-1-{6-[(4-fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1H,2H,3H-pyrrolo[3,2-b]pyridin-1-yl}ethan-1-one

Fold induction in secreted cytokine concentrations in PBMC supernatants after IAP antagonist treatment

Because of their roles in the evasion of apoptosis, inhibitor of apoptosis proteins (IAP) are considered attractive targets for anticancer therapy. Antagonists of these proteins have the potential to switch prosurvival signaling pathways in cancer cells toward cell death. Various SMAC-peptidomimetics with inherent cIAP selectivity have been tested clinically and demonstrated minimal single-agent efficacy. ASTX660 is a potent, non-peptidomimetic antagonist of cIAP1/2 and XIAP, discovered using fragment-based drug design. The antagonism of XIAP and cIAP1 by ASTX660 was demonstrated on purified proteins, cells, and in vivo in xenograft models. The compound binds to the isolated BIR3 domains of both XIAP and cIAP1 with nanomolar potencies. In cells and xenograft tissue, direct antagonism of XIAP was demonstrated by measuring its displacement from caspase-9 or SMAC. Compound-induced proteasomal degradation of cIAP1 and 2, resulting in downstream effects of NIK stabilization and activation of noncanonical NF-κB signaling, demonstrated cIAP1/2 antagonism. Treatment with ASTX660 led to TNFα-dependent induction of apoptosis in various cancer cell lines in vitro, whereas dosing in mice bearing breast and melanoma tumor xenografts inhibited tumor growth. ASTX660 is currently being tested in a phase I–II clinical trial (NCT02503423), and we propose that its antagonism of cIAP1/2 and XIAP may offer improved efficacy over first-generation antagonists that are more cIAP1/2 selective. Mol Cancer Ther; 17(7); 1381–91. ©2018 AACR.

Comparison of XIAP activity, cIAP1 activity and selectivity of clinical IAP antagonists

IncuCyte images from neutralization of TNF-α activity with anti-TNF-α experiment in MDA-MB-231 cells

Additional data from ASTX669 in vivo xenograft studies

Functionalization of C–H bonds is a key challenge in medicinal chemistry, particularly for fragment-based drug discovery (FBDD) where such transformations need to be executed in the presence of polar functionality necessary for fragment-protein binding. New technologies such as high-throughput experimentation and self-optimization have the potential to revolutionize synthetic approaches to challenging target molecules in FBDD. Recent work has shown the effectiveness of Bayesian optimization (BO) for the self-optimization of chemical reactions, however, in all previous cases these algorithmic procedures have started with no prior information about the reaction of interest. In this work, we explore the use of multi-task Bayesian optimization (MTBO) in several in silico case studies by leveraging reaction data collected during related historical optimization campaigns to accelerate the optimization of new reactions - this was performed for Suzuki-Miyaura and C–N couplings. This methodology was then translated to real-world, medicinal chemistry applications in the yield optimization of several pharmaceutical intermediates using an autonomous flow-based reactor platform. The use of the MTBO algorithm was shown to be successful in determining optimal conditions (both continuous and categorical variables) of unseen experimental C–H activation reactions with differing substrates, demonstrating up to a 98 % cost reduction when compared to industry-standard process optimization techniques. Our findings highlight the effectiveness of the methodology as an enabling tool in medicinal chemistry workflows, where efficient utilization of precious starting materials is particularly important. This work represents a step-change in the utilization of previously obtained reaction data and machine learning with the ultimate goal of accelerated reaction optimization.

Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

We have analysed 131 fragment-to-lead (F2L) examples targeting a wide variety of protein families published by academic and industrial laboratories between 2015–2019. Our assessment of X-ray structural data identifies the most common polar functional groups involved in fragment-protein binding are: N–H (hydrogen bond donors on aromatic and aliphatic N–H, amides and anilines; totalling 35%), aromatic nitrogen atoms (hydrogen bond acceptors; totalling 23%), and carbonyl oxygen group atoms (hydrogen bond acceptors on amides, ureas and ketones; totalling 22%). Furthermore, the elaboration of each fragment into its corresponding lead is analysed to identify the nominal synthetic growth vectors. In ∼80% of cases, growth originates from an aromatic or aliphatic carbon on the fragment and more than 50% of the total bonds formed are carbon–carbon bonds. This analysis reveals that growth from carbocentric vectors is key and therefore robust C–H functionalisation methods that tolerate the innate polar functionality on fragments could transform fragment-based drug discovery (FBDD). As a further resource to the community, we have provided the full data of our analysis as well as an online overlay page of the X-ray structures of the fragment hit and leads: https://astx.com/interactive/F2L-2021/, An in depth meta analysis of 131 fragment-to-lead case-studies has shown the importance of synthetic methods that allow carbon-centred synthetic elaboration in the presence of polar pharmacophores.

Inhibitor of apoptosis proteins (IAPs) are promising anticancer targets, given their roles in the evasion of apoptosis. Several peptidomimetic IAP antagonists, with inherent selectivity for cellular IAP (cIAP) over X-linked IAP (XIAP), have been tested in the clinic. A fragment screening approach followed by structure-based optimization has previously been reported that resulted in a low-nanomolar cIAP1 and XIAP antagonist lead molecule with a more balanced cIAP–XIAP profile. We now report the further structure-guided optimization of the lead, with a view to improving the metabolic stability and cardiac safety profile, to give the nonpeptidomimetic antagonist clinical candidate 27 (ASTX660), currently being tested in a phase 1/2 clinical trial (NCT02503423).

Because of their roles in the evasion of apoptosis, inhibitor of apoptosis proteins (IAP) are considered attractive targets for anticancer therapy. Antagonists of these proteins have the potential to switch prosurvival signaling pathways in cancer cells toward cell death. Various SMAC-peptidomimetics with inherent cIAP selectivity have been tested clinically and demonstrated minimal single-agent efficacy. ASTX660 is a potent, non-peptidomimetic antagonist of cIAP1/2 and XIAP, discovered using fragment-based drug design. The antagonism of XIAP and cIAP1 by ASTX660 was demonstrated on purified proteins, cells, and in vivo in xenograft models. The compound binds to the isolated BIR3 domains of both XIAP and cIAP1 with nanomolar potencies. In cells and xenograft tissue, direct antagonism of XIAP was demonstrated by measuring its displacement from caspase-9 or SMAC. Compound-induced proteasomal degradation of cIAP1 and 2, resulting in downstream effects of NIK stabilization and activation of noncanonical NF-κB signaling, demonstrated cIAP1/2 antagonism. Treatment with ASTX660 led to TNFα-dependent induction of apoptosis in various cancer cell lines in vitro, whereas dosing in mice bearing breast and melanoma tumor xenografts inhibited tumor growth. ASTX660 is currently being tested in a phase I–II clinical trial (NCT02503423), and we propose that its antagonism of cIAP1/2 and XIAP may offer improved efficacy over first-generation antagonists that are more cIAP1/2 selective. Mol Cancer Ther; 17(7); 1381–91. ©2018 AACR.

XIAP and cIAP1 are members of the inhibitor of apoptosis protein (IAP) family and are key regulators of anti-apoptotic and pro-survival signaling pathways. Overexpression of IAPs occurs in various cancers and has been associated with tumor progression and resistance to treatment. Structure-based drug design (SBDD) guided by structural information from X-ray crystallography, computational studies, and NMR solution conformational analysis was successfully applied to a fragment-derived lead resulting in AT-IAP, a potent, orally bioavailable, dual antagonist of XIAP and cIAP1 and a structurally novel chemical probe for IAP biology.

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signalling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Recent advances have shown that genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signalling and inhibits proliferation of RTK-driven cancer cell lines. SHP2 is now understood to act upstream of RAS and plays a role in KRAS-driven cancers, an area of research which is rapidly growing. Considering that RTK deregulation often leads to a wide range of cancers and the newly appreciated role of SHP2 in KRAS-driven cancers, SHP2 inhibitors are therefore a promising therapeutic approach. SHP2 contains two N-terminal tandem SH2 domains (N-SH2, C-SH2), a catalytic phosphatase domain and a C-terminal tail. SHP2 switches between “open” active and “closed” inactive forms due to autoinhibitory interactions between the N-SH2 domain and the phosphatase domain. Historically, phosphatases were deemed undruggable as there had been no advancements with active site inhibitors. We hypothesised that fragment screening would be highly applicable and amenable to this target to enable alternative means of inhibition through identification of allosteric binding sites. Here we describe the first reported fragment screen against SHP2. Using our fragment-based PyramidTM approach, screening was carried out on two constructs of SHP2; a closed autoinhibited C-terminal truncated form (phosphatase and both SH2 domains), as well as the phosphatase-only domain. A combination of screening methods such as X-ray crystallography and NMR were employed to identify fragment hits at multiple sites on SHP2, including the tunnel-like allosteric site reported by Chen et al, 2016. Initial fragment hits had affinities for SHP2 in the range of 1mM as measured by ITC. Binding of these hits was improved using structure-guided design to generate compounds which inhibit SHP2 phosphatase activity and are promising starting points for further optimization. Citation Format: Philip J. Day, Valerio Berdini, Juan Castro, Gianni Chessari, Thomas G. Davies, James E. Day, Satoshi Fukaya, Chris Hamlett, Keisha Hearn, Steve Hiscock, Rhian Holvey, Satoru Ito, Yasuo Kodama, Kenichi Matsuo, Yoko Nakatsuru, Nick Palmer, Amanda Price, Tadashi Shimamura, Jeffrey D.St. Denis, Nicola G. Wallis, Glyn Williams, Christopher N. Johnson. Fragment-based drug discovery to identify small molecule allosteric inhibitors of SHP2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1039.

Inhibitor of apoptosis proteins (IAPs) are promising anticancer targets, given their roles in the evasion of apoptosis. Several peptidomimetic IAP antagonists, with inherent selectivity for cellular IAP (cIAP) over X-linked IAP (XIAP), have been tested in the clinic. A fragment screening approach followed by structure-based optimization has previously been reported that resulted in a low-nanomolar cIAP1 and XIAP antagonist lead molecule with a more balanced cIAP-XIAP profile. We now report the further structure-guided optimization of the lead, with a view to improving the metabolic stability and cardiac safety profile, to give the nonpeptidomimetic antagonist clinical candidate 27 (ASTX660), currently being tested in a phase 1/2 clinical trial (NCT02503423).

Here we report the discovery of a highly conserved novel binding site located at the interface between the protease and helicase domains of the Hepatitis C Virus (HCV) NS3 protein. Using a chemical lead, identified by fragment screening and structure-guided design, we demonstrate that this site has a regulatory function on the protease activity via an allosteric mechanism. We propose that compounds binding at this allosteric site inhibit the function of the NS3 protein by stabilising an inactive conformation and thus represent a new class of direct acting antiviral agents.

In response to cellular stress, the p53 tumor suppressor is activated to modulate cell cycle progression, DNA repair, and cell death. The activity of p53 is tightly regulated by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Inhibition of the MDM2-p53 interaction in tumors carrying wild-type p53 can therefore reactivate p53 and elicit an anti-cancer effect. Small molecule inhibitors of the MDM2-p53 interaction remains a promising strategy for cancer therapy and a number of these compounds are in clinical development. An isoindolinone series, identified by the Northern Institute for Cancer Research (NICR), has been used as a starting point for the development of potent MDM2-p53 inhibitors. Structure based drug design was applied during lead optimisation to gain potency whilst also focusing on stabilizing the main metabolically labile position and reducing lipophilicity. This approach led to potent compounds with EC50 Citation Format: Lynsey Fazal, Maria Ahn, Luke Bevan, Ildiko Buck, Juan Castro, Gianni Chessari, Ben Cons, Keisha Hearn, Steven Howard, Chris Johnson, Judith Reeks, Emiliano Tamanini, Neil Thompson, Hugh Walton, Pamela Williams, Ruth H. Bawn, Tim J. Blackburn, Celine Cano, Sarah J. Cully, Bernard Golding, Roger Griffin, Karen Haggerty, Ian Hardcastle, Herbie Newell, Martin Noble, Huw Thomas, Elaine Willmore, Yan Zhao, Steve Wedge. Development of a potent class of small molecule inhibitors of the MDM2-p53 protein-protein interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1652.

We investigated the cellular response to two MDM2-p53 antagonists (RG7388 and Cpd 1) in a panel of 19 tumor cell lines and in patient-derived chronic lymphocytic leukemia (CLL) cells. RG7388 (Idasanutlin) is an MDM2-p53 antagonist developed by Roche and Cpd1 a novel potent and selective MDM2-p53 antagonist developed as part of an ongoing Alliance between Newcastle University, Cancer Research UK and Astex Pharmaceuticals. RG7388 and Cpd1 potently inhibited the proliferation of many cell lines, with MDM2-amplified SJSA-1 osteosarcoma cells and cells from haematological malignancies being the most sensitive (e.g. GI50 values in the acute myeloid leukemia cell line MOLM13 were 33 + 16 nM for RG7388 and 8 + 1 nM for Cpd 1). We also examined induction of apoptosis by measuring caspase 3/7 activation (24 h treatment). SJSA-1 cells showed a 20-fold increase in caspase activation and Molm13 cells a 6-fold increase (measured at 300nM with RG7388). However, cell lines from solid tumours such as the colorectal carcinoma HCT116 or the hepatocellular carcinoma HepG2 did not show any induction of apoptosis, even at concentrations up to 1µM RG3788. In primary CLL cells, Cpd 1 reduced cell viability (48 h) with an average LC50 of 131 + 46 nM (n = 4), compared to 400 + 55 nM for RG7388 (n =23). We also quantified mRNA levels in 25 primary CLL samples and found that RG7388 induced a predominantly pro-apoptotic gene signature: following 6h treatment, 1µM RG7388 resulted in an average 8-fold induction of PUMA and 3.5-fold increase in BAX compared to a 1.6-fold induction of CDKN1A (p21). Our data confirm that targeting the MDM2-p53 interaction is an effective strategy to inhibit growth and viability of tumor cells, and that some cells, including those expressing high levels of MDM2 or those derived from hematological malignancies, display a striking apoptotic response. Citation Format: Elaine Willmore, Yan Zhao, Carmela Ciardullo, Laura Woodhouse, Huw D. Thomas, Maria Ahn, Lynsey Fazal, Martin E. Noble, Ian Hardcastle, Steven Howard, Gianni Chessari, John Lunec, Steve Wedge. The anti-proliferative and pro-apoptotic effect of MDM2-p53 antagonists evaluated in human tumor cells lines and chronic lymphocytic leukemia patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1870.

The field of fragment-based drug discovery (FBDD) has developed significantly over the past 10 years and is now recognized as a tangible alternative to more traditional methods of hit identification, such as high throughput screening (HTS). The number of commercial and academic groups actively engaged in fragment-based research has increased, and as a consequence, there has been continued development and refinement of techniques and methods. From its inception, the fragment-based approach had two central tenets that were critical to its success and that have set it apart from HTS and other hit identification techniques. The first is the concept that chemical space can be more efficiently probed by screening collections of small fragments rather than libraries of larger molecules. The number of potential fragments with up to 12 heavy atoms (not including threeand four-membered ring structures) has been estimated at 10, whereas the number of potential druglike molecules with up to 30 heavy atoms is estimated at more than 10. Therefore, a much greater proportion of “fragment-like” chemical space can feasibly be screened in FBDD compared to “druglike” chemical space covered in a HTS where molecular size is much larger. The second idea is that, because by definition fragment molecules are small in size (typically less than 250 Da), they should typically bind with lower affinity to their target protein (micromolar to millimolar range) compared with druglike molecules that can form many more interactions (nanomolar to micromolar range) but that the binding efficiency per atom is at least as high as for larger hit molecules. Implicitly, the screening techniques employed in FBDD must be correspondingly much more sensitive than a HTS bioassay. Generally, sensitive biophysical techniques are employed to detect these weak binding events and to characterize the fragment interactions with the target active site. Nuclear magnetic resonance (NMR) and protein X-ray crystallography have been used extensively in fragment-based research because these techniques are highly sensitive in detecting low affinity fragment binding and also give information about the fragmentprotein interactions being formed. There have been a number of recently published general review articles that have discussed the various aspects of the FBDD field. In addition, there are now two books on the subject. In this journal in 2004, Erlanson et al. summarized the major developments in FBDD since the original publication by Fesik and co-workers of the “SAR by NMR” approach in the late 1990s. Particular note was given to the biophysical methods employed to screen for fragment binding and the merits and drawbacks of each of these techniques, along with the approaches that can be used to optimize fragments into lead molecules. Herein, the trends and developments over the past 4 years will be outlined and some selected examples that are illustrative of the approaches being utilized by those active in the field examined. Additionally, this review will look in some detail at representative protein-ligand complexes observed between fragment-sized molecules and their protein targets from the Protein Data Bank (PDB). Finally, some conclusions will be drawn from these data and the future of FBDD discussed.

This paper describes an application of fragment screening to the aspartyl protease enzyme, beta-secretase (BACE-1), using high throughput X-ray crystallography. Three distinct chemotypes were identified by X-ray crystallography as binding to the catalytic aspartates either via an aminoheterocycle (such as 2-aminoquinoline), a piperidine, or an aliphatic hydroxyl group. The fragment hits were weak inhibitors of BACE-1 in the millimolar range but were of interest because most of them displayed relatively good ligand efficiencies. The aminoheterocycles exhibited a novel recognition motif that has not been seen before with aspartic proteases. Virtual screening around this motif identified an aminopyridine with increased potency and attractive growth points for further elaboration using structure-based drug design. The companion paper illustrates how sub-micromolar inhibitors were developed starting from this fragment.

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.

Crowned calix[8]arenes are obtained by direct alkylation of p-tert-butylcalix[8]arene (1) with poly(ethylene glycol) ditosylates in the presence of various bases. K2CO3 promotes the preferential formation of 1,3-calix[8]crowns. Cs2CO3 mainly gives the 1,5-isomers, which are selectively obtained in high yields when shorter chains are used (1,5-crown-2, 88%; 1,5-crown-3, 78%). NaH affords the 1,4-isomers in yields up to 46%, often as the sole crown derivative, besides unreacted 1. 1,2-Calix[8]crowns are also obtained in appreciable amount in some instances. The observed regioselectivity is rationalized in terms of preferential formation of specific anions in dependence of the base strength. Dynamic NMR and modeling studies prove that the polyether chain, depending on its bridging mode, may significantly reduce the available space for the through the annulus passages leading to derivatives conformationally blocked (on the NMR time scale).

Inhibitors of the chaperone Hsp90 are potentially useful as chemotherapeutic agents in cancer. This paper describes an application of fragment screening to Hsp90 using a combination of NMR and high throughput X-ray crystallography. The screening identified an aminopyrimidine with affinity in the high micromolar range and subsequent structure-based design allowed its optimization into a low nanomolar series with good ligand efficiency. A phenolic chemotype was also identified in fragment screening and was found to bind with affinity close to 1 mM. This fragment was optimized using structure based design into a resorcinol lead which has subnanomolar affinity for Hsp90, excellent cell potency, and good ligand efficiency. This fragment to lead campaign improved affinity for Hsp90 by over 1,000,000-fold with the addition of only six heavy atoms. The companion paper (DOI: 10.1021/jm100060b) describes how the resorcinol lead was optimized into a compound that is now in clinical trials for the treatment of cancer.

Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) are currently generating significant interest in clinical development as potential treatments for cancer. In a preceding publication (DOI: 10.1021/jm100059d ) we describe Astex's approach to screening fragments against Hsp90 and the subsequent optimization of two hits into leads with inhibitory activities in the low nanomolar range. This paper describes the structure guided optimization of the 2,4-dihydroxybenzamide lead molecule 1 and details some of the drug discovery strategies employed in the identification of AT13387 (35), which has progressed through preclinical development and is currently being tested in man.

Solid phase organic chemistry coupled with combinatorial methods promises to increase dramatically the diversity and number of small molecules available for medical and biological applications. However, optimizing the reaction conditions can be a time consuming step, especially since analytical tools to monitor reaction progress and detect impurities for solid phase chemistry are less developed than for solution chemistry. The use of high resolution magic angle spinning (HRMAS) NMR is described here as such an analytical tool. Whereas initial applications of molecular identification using deuterated organic solvents to swell the resins presented a significant gain in time over the cleave-and- analysis methods, the introduction of a differential diffusion filter has made immediate recording of spectra possible without any sample treatment. The applications of HRMAS NMR to different solid supports that are used in combinatorial chemistry will be described in terms of rapidity, robustness and sensitivity.

As recently as ten years ago few scientists had heard of fragment screening, let alone considered low molecular weight fragments (MW

The field of fragment-based drug discovery (FBDD) has developed significantly over the past 10 years and is now recognized as a tangible alternative to more traditional methods of hit identification, such as high throughput screening (HTS). The number of commercial and academic groups actively engaged in fragment-based research has increased, and as a consequence, there has been continued development and refinement of techniques and methods. From its inception, the fragment-based approach had two central tenets that were critical to its success and that have set it apart from HTS and other hit identification techniques. The first is the concept that chemical space can be more efficiently probed by screening collections of small fragments rather than libraries of larger molecules. The number of potential fragments with up to 12 heavy atoms (not including threeand four-membered ring structures) has been estimated at 10, whereas the number of potential druglike molecules with up to 30 heavy atoms is estimated at more than 10. Therefore, a much greater proportion of “fragment-like” chemical space can feasibly be screened in FBDD compared to “druglike” chemical space covered in a HTS where molecular size is much larger. The second idea is that, because by definition fragment molecules are small in size (typically less than 250 Da), they should typically bind with lower affinity to their target protein (micromolar to millimolar range) compared with druglike molecules that can form many more interactions (nanomolar to micromolar range) but that the binding efficiency per atom is at least as high as for larger hit molecules. Implicitly, the screening techniques employed in FBDD must be correspondingly much more sensitive than a HTS bioassay. Generally, sensitive biophysical techniques are employed to detect these weak binding events and to characterize the fragment interactions with the target active site. Nuclear magnetic resonance (NMR) and protein X-ray crystallography have been used extensively in fragment-based research because these techniques are highly sensitive in detecting low affinity fragment binding and also give information about the fragmentprotein interactions being formed. There have been a number of recently published general review articles that have discussed the various aspects of the FBDD field. In addition, there are now two books on the subject. In this journal in 2004, Erlanson et al. summarized the major developments in FBDD since the original publication by Fesik and co-workers of the “SAR by NMR” approach in the late 1990s. Particular note was given to the biophysical methods employed to screen for fragment binding and the merits and drawbacks of each of these techniques, along with the approaches that can be used to optimize fragments into lead molecules. Herein, the trends and developments over the past 4 years will be outlined and some selected examples that are illustrative of the approaches being utilized by those active in the field examined. Additionally, this review will look in some detail at representative protein-ligand complexes observed between fragment-sized molecules and their protein targets from the Protein Data Bank (PDB). Finally, some conclusions will be drawn from these data and the future of FBDD discussed.

Publisher Summary This chapter discusses the structural biology and anticancer drug design. The use of structural biology techniques, such as NMR and X-ray crystallography, are now widely accepted to be powerful tools in structure-based drug design. The discovery and development of a new drug is a multi-step process, which starts with the detailed analysis of the disease to be investigated. The next step is the identification of the key biological molecules that are involved in the disease process, whereby modulation of these molecules, usually proteins, will likely have a therapeutic effect. Particularly in the case of X-ray crystallography, the advent of improved equipment, software, and techniques has resulted in a dramatic improvement in the speed with which structural data can be obtained. Structural biology methods enable the generation of a three-dimensional model of a protein molecule by various techniques, allowing researchers to gain powerful insights into the structure and mechanisms by which proteins and larger protein complexes operate. Structural biology now has an impact throughout the drug discovery process, from hit identification through to late-stage lead optimization. The structure-guided design of AT7519 provides a good example of this being put into practice for an oncology target.

Fragment-based lead discovery has been applied to urokinase-type plasminogen activator (uPA). The (R)-enantiomer of the orally active drug mexiletine 5 (a fragment hit from X-ray crystallographic screening) was the chemical starting point. Structure-aided design led to elaborated inhibitors that retained the key interactions of (R)-5 while gaining extra potency by simultaneously occupying neighboring regions of the active site. Subsequent optimization led to 15, a potent, selective, and orally bioavailable inhibitor of uPA.

Fragment-based lead generation has led to the discovery of a novel series of cyclic amidine-based inhibitors of beta-secretase (BACE-1). Initial fragment hits with an isocytosine core having millimolar potency were identified via NMR affinity screening. Structure-guided evolution of these fragments using X-ray crystallography together with potency determination using surface plasmon resonance and functional enzyme inhibition assays afforded micromolar inhibitors. Similarity searching around the isocytosine core led to the identification of a related series of inhibitors, the dihydroisocytosines. By leveraging the knowledge of the ligand-BACE-1 recognition features generated from the isocytosines, the dihydroisocytosines were efficiently optimized to submicromolar potency. Compound 29, with an IC50 of 80 nM, a ligand efficiency of 0.37, and cellular activity of 470 nM, emerged as the lead structure for future optimization.

Fragment-based lead discovery has been successfully applied to the aspartyl protease enzyme beta-secretase (BACE-1). Fragment hits that contained an aminopyridine motif binding to the two catalytic aspartic acid residues in the active site of the enzyme were the chemical starting points. Structure-based design approaches have led to identification of low micromolar lead compounds that retain these interactions and additionally occupy adjacent hydrophobic pockets of the active site. These leads form two subseries, for which compounds 4 (IC50 = 25 microM) and 6c (IC50 = 24 microM) are representative. In the latter series, further optimization has led to 8a (IC50 = 690 nM).

A procedure for analyzing and classifying publicly available crystal structures has been developed. It has been used to identify high-resolution protein-ligand complexes that can be assessed by reconstructing the electron density for the ligand using the deposited structure factors. The complexes have been clustered according to the protein sequences, and clusters have been discarded if they do not represent proteins thought to be of direct interest to the pharmaceutical or agrochemical industry. Rules have been used to exclude complexes containing non-drug-like ligands. One complex from each cluster has been selected where a structure of sufficient quality was available. The final Astex diverse set contains 85 diverse, relevant protein-ligand complexes, which have been prepared in a format suitable for docking and are to be made freely available to the entire research community (http://www.ccdc.cam.ac.uk). The performance of the docking program GOLD against the new set is assessed using a variety of protocols. Relatively unbiased protocols give success rates of approximately 80% for redocking into native structures, but it is possible to get success rates of over 90% with some protocols.

The screening of fragments is an alternative approach to high-throughput screening for the identification of leads for therapeutic targets. Fragment hits have been discovered using X-ray crystallographic screening of protein crystals of the serine protease enzyme thrombin. The fragment library was designed to avoid any well-precedented, strongly basic functionality. Screening hits included a novel ligand (3), which binds exclusively to the S2-S4 pocket, in addition to smaller fragments which bind to the S1 pocket. The structure of these protein-ligand complexes are presented. A chemistry strategy to link two such fragments together and to synthesize larger drug-sized compounds resulted in the efficient identification of hybrid inhibitors with nanomolar potency (e.g., 7, IC50 = 3.7 nM). These potent ligands occupy the same area of the active site as previously described peptidic inhibitors, while having very different chemical architecture.

We implemented a novel approach to score water mediation and displacement in the protein-ligand docking program GOLD. The method allows water molecules to switch on and off and to rotate around their three principal axes. A constant penalty, sigma(p), representing the loss of rigid-body entropy, is added for water molecules that are switched on, hence rewarding water displacement. We tested the methodology in an extensive validation study. First, sigma(p) is optimized against a training set of 58 protein-ligand complexes. For this training set, our algorithm correctly predicts water mediation/displacement in approximately 92% of the cases. We observed small improvements in the quality of the predicted binding modes for water-mediated complexes. In the second part of this work, an entirely independent set of 225 complexes is used. For this test set, our algorithm correctly predicts water mediation/displacement in approximately 93% of the cases. Improvements in binding mode quality were observed for individual water-mediated complexes.

The synthesis and evaluation for telomerase-inhibitory and quadruplex DNA binding properties of three related series of rationally designed trisubstituted acridine derivatives are described. These are substituted on the acridine ring at the 2,6,9; 2,7,9; and 3,6,9 positions. The ability of several of the most potent compounds to interact with and stabilize an intramolecular G-quadruplex DNA was evaluated by surface plasmon resonance methods, and affinities were found to correlate with potency in a telomerase assay. The interactions of a number of compounds with a parallel quadruplex DNA structure were simulated by molecular modeling methods. The calculated interaction energies were compared with telomerase activity and showed generally consistent correlations between quadruplex affinity and telomerase inhibition. These data support a model for the action of these compounds that involves the stabilization of intermediate quadruplex structures that inhibit the elongation of telomeric DNA by telomerase in tumor cells.

The binding of two symmetric bis-benzimidazole compounds, 2,2-bis-[4′-(3″-dimethylamino-1″-propyloxy)phenyl]-5,5-bi-1H-benzimidazole and its piperidinpropylphenyl analog, to the minor groove of DNA, have been studied by DNA footprinting, surface plasmon resonance (SPR) methods and molecular dynamics simulations in explicit solvent. The footprinting and SPR methods find that the former compound has enhanced affinity and selectivity for AT sequences in DNA. The molecular modeling studies have suggested that, due to the presence of the oxygen atom in each side chain of the former compound, a water molecule is immobilized and effectively bridges between side chain and DNA base edges via hydrogen bonding interactions. This additional contribution to ligand–DNA interactions would be expected to result in enhanced DNA affinity, as is observed.

Experimental measurements of edge-to-face aromatic interactions have been used to test a series of molecular mechanics force fields. The experimental data were determined for a range of differently substituted aromatic rings using chemical double mutant cycles on hydrogen-bonded zipper complexes. These complexes were truncated for the purposes of the molecular mechanics calculations so that problems of conformational searching and the optimisation of large structures could be avoided. Double-mutant cycles were then carried out in silico using these truncated systems. Comparison of the experimental aromatic interaction energies and the X-ray crystal structures of these truncated complexes with the calculated data show that conventional molecular mechanics force fields (MM2, MM3, AMBER and OPLS) do not perform well. However, the XED force field which explicitly represents electron anisotropy as an expansion of point charges around each atom reproduces the trends in interaction energy and the three-dimensional structures exceedingly well. Collapsing the XED charges onto atom centres or the use of semi-empirical atom-centred charges within the XED force field gives poor results. Thus the success of XED is not related to the methods used to assign the atomic charge distribution but can be directly attributed to the use of off-atom centre charges.

Despite the use of high resolution magic angle spinning NMR, the NMR linewidth of anchored molecules on the commonly used Merrifield solid phase resins remains larger than that of the corresponding molecules in solution. We investigate the different mechanisms that might be at the origin of this line broadening. Experimentally, we use the CPMG method to determine the (15)N relaxation times of a tethered tripeptide and show that the slow resin dynamics significantly contributes to the transverse relaxation.

Experimental measurements of edge-to-face aromatic interactions have been used to test a series of molecular mechanics force fields. The experimental data were determined for a range of differently substituted aromatic rings using chemical double mutant cycles on hydrogen-bonded zipper complexes. These complexes were truncated for the purposes of the molecular mechanics calculations so that problems of conformational searching and the optimisation of large structures could be avoided. Double-mutant cycles were then carried out in silico using these truncated systems. Comparison of the experimental aromatic interaction energies and the X-ray crystal structures of these truncated complexes with the calculated data show that conventional molecular mechanics force fields (MM2, MM3, AMBER and OPLS) do not perform well. However, the XED force field which explicitly represents electron anisotropy as an expansion of point charges around each atom reproduces the trends in interaction energy and the three-dimensional structures exceedingly well. Collapsing the XED charges onto atom centres or the use of semi-empirical atom-centred charges within the XED force field gives poor results. Thus the success of XED is not related to the methods used to assign the atomic charge distribution but can be directly attributed to the use of off-atom centre charges.