Discovery of an allosteric mechanism for the regulation of HCV NS3 protein function.

Here we report a highly conserved new 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 stabilizing an inactive conformation and thus represent a new class of direct-acting antiviral agents.

Fragment-Based Discovery of a Series of Allosteric-Binding Site Modulators of ß-Glucocerebrosidase.

ß-Glucocerebrosidase (GBA/GCase) mutations leading to misfolded protein cause Gaucher's disease and are a major genetic risk factor for Parkinson's disease and dementia with Lewy bodies. The identification of small molecule pharmacological chaperones that can stabilize the misfolded protein and increase delivery of degradation-prone mutant GCase to the lysosome is a strategy under active investigation. Here, we describe the first use of fragment-based drug discovery (FBDD) to identify pharmacological chaperones of GCase. The fragment hits were identified by using X-ray crystallography and biophysical techniques. This work led to the discovery of a series of compounds that bind GCase with nM potency and positively modulate GCase activity in cells.

Fragment-Based Discovery of Allosteric Inhibitors of SH2 Domain-Containing Protein Tyrosine Phosphatase-2 (SHP2).

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2. Structure-guided optimization, including several computational methods, led to the discovery of two structurally distinct series of SHP2 inhibitors binding to the previously reported allosteric tunnel binding site (Tunnel Site). One of these series was advanced to a low-nanomolar lead that inhibited tumor growth when dosed orally to mice bearing HCC827 xenografts. Furthermore, a third series of SHP2 inhibitors was discovered binding to a previously unreported site, lying at the interface of the C-terminal SH2 and catalytic domains.

Accelerated Chemical Reaction Optimization Using Multi-Task Learning.

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.

Enabling synthesis in fragment-based drug discovery (FBDD): microscale high-throughput optimisation of the medicinal chemist's toolbox reactions.

Miniaturised high-throughput experimentation (HTE) is widely employed in industrial and academic laboratories for rapid reaction optimisation using material-limited, multifactorial reaction condition screening. In fragment-based drug discovery (FBDD), common toolbox reactions such as the Suzuki-Miyaura and Buchwald-Hartwig cross couplings can be hampered by the fragment's intrinsic heteroatom-rich pharmacophore which is required for ligand-protein binding. At Astex, we are using microscale HTE to speed up reaction optimisation and prevent target down-prioritisation. By identifying catalyst/base/solvent combinations which tolerate unprotected heteroatoms we can rapidly optimise key cross-couplings and expedite route design by avoiding superfluous protecting group manipulations. However, HTE requires extensive upfront training, and this modern automated synthesis technique largely differs to the way organic chemists are traditionally trained. To make HTE accessible to all our synthetic chemists we have developed a semi-automated workflow enabled by pre-made 96-well screening kits, rapid analytical methods and in-house software development, which is empowering chemists at Astex to run HTE screens independently with minimal training.

Electrostatic Complementarity in Structure-Based Drug Design.

Optimization of electrostatic complementarity is an important strategy in structure-based drug discovery for improving the affinity of molecules against a specific protein target. In this Miniperspective we identify examples where deliberate optimization of protein-ligand electrostatic complementarity or intramolecular electrostatic interactions gave improvements in target affinity (up to 250-fold), physicochemical properties, in vitro properties, and off-target selectivity. We also look retrospectively at a series of factor Xa inhibitors that show an almost 8000-fold range in potency that can be correlated with the calculated electrostatic potential (ESP) surfaces. Recent developments using a graph-convolutional deep neural network to rapidly generate high quality ESP surfaces have the potential to make this useful tool more accessible for a wider audience within the field of medicinal chemistry.

X-ray Screening of an Electrophilic Fragment Library and Application toward the Development of a Novel ERK 1/2 Covalent Inhibitor.

Fragment-based drug discovery (FBDD) has become an established method for the identification of efficient starting points for drug discovery programs. In recent years, electrophilic fragment screening has garnered increased attention from both academia and industry to identify novel covalent hits for tool compound or drug development against challenging drug targets. Herein, we describe the design and characterization of an acrylamide-focused electrophilic fragment library and screening campaign against extracellular signal-regulated kinase 2 (ERK2) using high-throughput protein crystallography as the primary hit-finding technology. Several fragments were found to have covalently modified the adenosine triphosphate (ATP) binding pocket Cys166 residue. From these hits, 22, a covalent ATP-competitive inhibitor with improved potency (ERK2 IC50 = 7.8 µM), was developed.

C-H functionalisation tolerant to polar groups could transform fragment-based drug discovery (FBDD).

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/.

Structure-Based Design of Potent and Orally Active Isoindolinone Inhibitors of MDM2-p53 Protein-Protein Interaction.

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.

Fragment-based discovery of mexiletine derivatives as orally bioavailable inhibitors of urokinase-type plasminogen activator.

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.

ASTX660, a Novel Non-peptidomimetic Antagonist of cIAP1/2 and XIAP, Potently Induces TNFα-Dependent Apoptosis in Cancer Cell Lines and Inhibits Tumor Growth.

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.

A Fragment-Derived Clinical Candidate for Antagonism of X-Linked and Cellular Inhibitor of Apoptosis Proteins: 1-(6-[(4-Fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1 H,2 H,3 H-pyrrolo[3,2- b]pyridin-1-yl)-2-[(2 R,5 R)-5-methyl-2-([(3R)-3-methylmorpholin-4-yl]methyl)piperazin-1-yl]ethan-1-one (ASTX660).

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).

Application of fragment screening by X-ray crystallography to beta-secretase.

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.

Diverse, high-quality test set for the validation of protein-ligand docking performance.

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.

Application of fragment screening by X-ray crystallography to the discovery of aminopyridines as inhibitors of beta-secretase.

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).

Application of fragment-based lead generation to the discovery of novel, cyclic amidine beta-secretase inhibitors with nanomolar potency, cellular activity, and high ligand efficiency.

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.

Discovery of a Potent Nonpeptidomimetic, Small-Molecule Antagonist of Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) and X-Linked Inhibitor of Apoptosis Protein (XIAP).

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.

Application of fragment screening and fragment linking to the discovery of novel thrombin inhibitors.

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.

Sequence-specific minor groove binding by bis-benzimidazoles: water molecules in ligand recognition.

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.

PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy.

PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy.