Identification and Development of Cyclic Peptide Inhibitors of Hypoxia Inducible Factors 1 and 2 That Disrupt Hypoxia-Response Signaling in Cancer Cells.

Hypoxia inducible factor (HIF) is a heterodimeric transcription factor composed of an oxygen-regulated α subunit and a constitutively expressed ß subunit that serves as the master regulator of the cellular response to low oxygen concentrations. The HIF transcription factor senses and responds to hypoxia by significantly altering transcription and reprogramming cells to enable adaptation to a hypoxic microenvironment. Given the central role played by HIF in the survival and growth of tumors in hypoxia, inhibition of this transcription factor serves as a potential therapeutic approach for treating a variety of cancers. Here, we report the identification, optimization, and characterization of a series of cyclic peptides that disrupt the function of HIF-1 and HIF-2 transcription factors by inhibiting the interaction of both HIF-1α and HIF-2α with HIF-1ß. These compounds are shown to bind to HIF-α and disrupt the protein-protein interaction between the α and ß subunits of the transcription factor, resulting in disruption of hypoxia-response signaling by our lead molecule in several cancer cell lines.

Micelle-Promoted Reductive Amination of DNA-Conjugated Amines for DNA-Encoded Library Synthesis.

DNA-encoded libraries (DELs) have become a leading technology for hit identification in drug discovery projects as large, diverse libraries can be generated. DELs are commonly synthesised via split-and-pool methodology; thus, chemical transformations utilised must be highly efficient, proceeding with high conversions. Reactions performed in DEL synthesis also require a broad substrate scope to produce diverse, drug-like libraries. Many pharmaceutical compounds incorporate multiple C-N bonds, over a quarter of which are synthesised via reductive aminations. However, few on-DNA reductive amination procedures have been developed. Herein is reported the application of the micelle-forming surfactant, TPGS-750-M, to the on-DNA reductive amination of DNA-conjugated amines, yielding highly efficient conversions with a broad range of aldehydes, including medicinally relevant heterocyclic and aliphatic substrates. The procedure is compatible with DNA amplification and sequencing, demonstrating its applicability to DEL synthesis.

Scope of on-DNA nucleophilic aromatic substitution on weakly-activated heterocyclic substrates for the synthesis of DNA-encoded libraries.

DNA-Encoded Libraries (DEL) represent a promising hit finding strategy for drug discovery. Nonetheless, the available DNA-compatible chemistry remains of limited scope. Nucleophilic aromatic substitution (SNAr) has been extensively used in DEL synthesis but has generally been restricted to highly activated (hetero)arenes. Herein, we report an optimised procedure for SNAr reactions through the use of factorial experimental design (FED) on-DNA using 15% THF as a co-solvent. This method gave conversions of >95% for pyridine and pyrazine scaffolds for 36 secondary cyclic amines. This analysis provides a new DNA-compatible SNAr reaction to produce high yielding libraries. The scope of this reaction on other amines is described. This work identifies challenges for the further development for DNA-compatible SNAr reactions. 2009 Elsevier Ltd. All rights reserved.

Functional Group Tolerance of a Micellar on-DNA Suzuki-Miyaura Cross-Coupling Reaction for DNA-Encoded Library Design.

DNA-encoded libraries (DELs) offer great promise for the discovery of new ligands for proteins. Many current reactions used for DEL synthesis do not proceed efficiently over a wide range of substrates. Combining a diverse array of multicomponent reactions with micellar-promoted Suzuki-Miyaura cross-coupling provides a strategy for synthesizing highly diverse DELs with exceptionally high fidelity. These results demonstrate that the micellar Suzuki-Miyaura reaction has exceptional functional group tolerance and broad applicability.

High Fidelity Suzuki-Miyaura Coupling for the Synthesis of DNA Encoded Libraries Enabled by Micelle Forming Surfactants.

DNA encoded chemical libraries provide a highly efficient means of screening vast numbers of small molecules against an immobilized protein target. Their potential is currently restricted by the constraints of carrying out library synthesis in the presence of attached DNA tags, for which a limited number of reactions and substrates can be used. Even established reactions, such as Suzuki-Miyaura couplings, do not give efficient coupling reactions across a wide range of substrates and can lead to significant DNA degradation. We developed an efficient protocol for carrying out Suzuki-Miyaura couplings on DNA tagged substrates that proceeds with unprecedented efficiency to the desired biaryl products (>98% on average with no detectable DNA degradation) across a wide range of drug-like substrates using a micellar promoted process with commercial TPGS-750-M surfactant. We have demonstrated the applicability of this method in DEL synthesis by preparing a prototypical two-dimensional 36-member library employing the Suzuki-Miyaura coupling methodology as the final library synthesis step. This work shows, for the first time, that standard micellar surfactants can promote reactions for encoded library synthesis, leading to libraries of exceptional fidelity, and demonstrates the potential to expand the range of accessible DNA compatible chemistry.

A genetically selected cyclic peptide inhibitor of BCL6 homodimerization.

We report an inhibitor of the homodimeric protein-protein interaction of the BCL6 oncoprotein, identified from a genetically encoded SICLOPPS library of 3.2 million cyclic hexapeptides in combination with a bacterial reverse two-hybrid system. This cyclic peptide is shown to bind the BTB domain of BCL6, disrupts its homodimerization, and subsequent binding of the SMRT2 corepressor peptide.

Potent reversible inhibition of myeloperoxidase by aromatic hydroxamates.

The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation.

Collaborative Virtual Screening Identifies a 2-Aryl-4-aminoquinazoline Series with Efficacy in an In Vivo Model of Trypanosoma cruzi Infection.

Probing multiple proprietary pharmaceutical libraries in parallel via virtual screening allowed rapid expansion of the structure-activity relationship (SAR) around hit compounds with moderate efficacy against Trypanosoma cruzi, the causative agent of Chagas Disease. A potency-improving scaffold hop, followed by elaboration of the SAR via design guided by the output of the phenotypic virtual screening efforts, identified two promising hit compounds 54 and 85, which were profiled further in pharmacokinetic studies and in an in vivo model of T. cruzi infection. Compound 85 demonstrated clear reduction of parasitemia in the in vivo setting, confirming the interest in this series of 2-(pyridin-2-yl)quinazolines as potential anti-trypanosome treatments.

From libraries to candidate: the discovery of new ultra long-acting dibasic ß2-adrenoceptor agonists.

Libraries of dibasic compounds designed around the molecular scaffold of the DA(2)/ß(2) dual agonist sibenadet (Viozan™) have yielded a number of promising starting points that have been further optimised into novel potent and selective target molecules with required pharmacokinetic properties. From a shortlist, 31 was discovered as a novel, high potency, and highly efficacious ß(2)-agonist with high selectivity and a duration of action commensurable with once daily dosing.

Design-driven LO: the discovery of new ultra long acting dibasic ß2-adrenoceptor agonists.

Starting with the molecular scaffold of the DA(2)/ß(2) dual agonist sibenadet (Viozan™), a number of molecular changes were incorporated, which were designed to increase the potency and selectivity of the target molecule, and improve its pharmacokinetics. Through this process a novel, high potency, full ß(2)-agonist with high selectivity and long duration capable of being dosed once daily has been discovered.

Design driven HtL: The discovery and synthesis of new high efficacy ß2-agonists.

The design and synthesis of a new series of high efficacy ß(2)-agonists devoid of the key benzylic alcohol present in previously described highly efficacious ß(2)-agonists is reported. A hypothesis for the unprecedented level of efficacy is proposed based on considerations of ß(2)-adrenoceptor crystal structure, other biophysical data and modeling studies.

Highly efficient on-DNA amide couplings promoted by micelle forming surfactants for the synthesis of DNA encoded libraries.

DNA encoded libraries (DELs) represent powerful new technology for finding small molecule ligands for proteins and are increasingly being applied to hit finding in medicinal chemistry. Crucial to the synthesis of high quality DELs is the identification of chemical reactions for their assembly that proceed with very high conversion across a range of different substrates, under conditions compatible with DNA-tagged substrates. Many current chemistries used in DEL synthesis do not meet this requirement, resulting in libraries of low fidelity. Amide couplings are the most commonly used reaction in synthesis of screening libraries and also in DELs. The ability to carry out highly efficient, widely applicable amide couplings in DEL synthesis would therefore be highly desirable. We report a method for amide coupling using micelle forming surfactants, promoted by a modified linker, that is broadly applicable across a wide range of substrates. Most significantly, this works exceptionally well for coupling of DNA-conjugated carboxylic acids (N-to-C) with amines in solution, a procedure that is currently very inefficient. The optimisation of separate procedures for coupling of DNA-conjugated acids and amines by reagent screening and statistically driven optimisation is described. The generality of the method is illustrated by the application to a wide range of examples with unprecedented levels of conversion. The utility of the (N-to-C) coupling of DNA-conjugated acids in DEL synthesis is illustrated by the three cycle synthesis of a fully DNA-encoded compound by two cycles of coupling of an aminoester, with intermediate ester hydrolysis, followed by capping with an amine. This methodology will be of great utility in the synthesis of high fidelity DELs.

Collaborative virtual screening to elaborate an imidazo[1,2-a]pyridine hit series for visceral leishmaniasis.

An innovative pre-competitive virtual screening collaboration was engaged to validate and subsequently explore an imidazo[1,2-a]pyridine screening hit for visceral leishmaniasis. In silico probing of five proprietary pharmaceutical company libraries enabled rapid expansion of the hit chemotype, alleviating initial concerns about the core chemical structure while simultaneously improving antiparasitic activity and selectivity index relative to the background cell line. Subsequent hit optimization informed by the structure-activity relationship enabled by this virtual screening allowed thorough investigation of the pharmacophore, opening avenues for further improvement and optimization of the chemical series.

The discovery of new spirocyclic muscarinic M3 antagonists.

The optimisation of a new series of high potency muscarinic M3 antagonists, derived from high throughput screening library hit is described.

Identification of Small-Molecule Positive Modulators of Calcitonin-like Receptor-Based Receptors.

Class B G protein-coupled receptors are highly therapeutically relevant but challenges remain in identifying suitable small-molecule drugs. The calcitonin-like receptor (CLR) in particular is linked to conditions such as migraine, cardiovascular disease, and inflammatory bowel disease. The CLR cannot act as a cell-surface receptor alone but rather must couple to one of three receptor activity-modifying proteins (RAMPs), forming heterodimeric receptors for the peptides adrenomedullin and calcitonin gene-related peptide. These peptides have extended binding sites across their receptors. This is one reason why there are few small-molecule ligands that can modulate these receptors. Here we describe small molecules that are able to positively modulate the signaling of the CLR with all three RAMPs but are not active at the related calcitonin receptor. These compounds were selected from a ß-arrestin recruitment screen, coupled with rounds of medicinal chemistry to improve their activity. Translational potential is shown as the compounds can positively modulate cAMP signaling in a vascular cell line model. Binding experiments do not support an extracellular domain binding site; however, molecular modeling reveals potential allosteric binding sites in multiple receptor regions. These are the first small-molecule positive modulators described for the CLR:RAMP complexes.

Discovery of small molecule human C5a receptor antagonists.

A novel series of small molecule C5a antagonists is reported. In particular, in vitro metabolic studies and solution based combinatorial synthesis are demonstrated as useful tools for the rapid identification of antagonists with low in vitro clearance. Members of this series specifically inhibited the binding of (125)I-labeled C5a to human recombinant C5a receptor (C5aR). In functional cell assays these compounds displayed surmountable antagonism against C5a and did not demonstrate any detectable agonist activity.

Inhibition of low-density lipoprotein receptor degradation with a cyclic peptide that disrupts the homodimerization of IDOL E3 ubiquitin ligase.

Cellular uptake of circulating cholesterol occurs via the low density lipoprotein receptor (LDLR). The E3 ubiquitin ligase IDOL is a mediator of LDLR degradation, with IDOL homodimerization thought to be required for its activity. To probe the possibility of modulating LDLR levels with an inhibitor of IDOL homodimerization, we screened a SICLOPPS library of 3.2 million cyclic peptides for compounds that disrupt this protein-protein interaction. We identified cyclo-CFFLYT as the lead inhibitor, and improved its activity through the incorporation of non-natural amino acids. The activity of the optimized cyclic peptide was assessed in hepatic cells, with a dose-dependent increase in LDLR levels observed in the presence of our IDOL homodimerization inhibitor.

Towards a hit for every target.

Technological advances coupled with novel collaborative strategies for compound sourcing and management are poised to transform the utility of high-throughput screening.

An analysis of the attrition of drug candidates from four major pharmaceutical companies.

The pharmaceutical industry remains under huge pressure to address the high attrition rates in drug development. Attempts to reduce the number of efficacy- and safety-related failures by analysing possible links to the physicochemical properties of small-molecule drug candidates have been inconclusive because of the limited size of data sets from individual companies. Here, we describe the compilation and analysis of combined data on the attrition of drug candidates from AstraZeneca, Eli Lilly and Company, GlaxoSmithKline and Pfizer. The analysis reaffirms that control of physicochemical properties during compound optimization is beneficial in identifying compounds of candidate drug quality and indicates for the first time a link between the physicochemical properties of compounds and clinical failure due to safety issues. The results also suggest that further control of physicochemical properties is unlikely to have a significant effect on attrition rates and that additional work is required to address safety-related failures. Further cross-company collaborations will be crucial to future progress in this area.