Discovery of Ligands for TRIM58, a Novel Tissue-Selective E3 Ligase.

Redirecting E3 ligases to neo-substrates, leading to their proteasomal disassembly, known as targeted protein degradation (TPD), has emerged as a promising alternative to traditional, occupancy-driven pharmacology. Although the field has expanded tremendously over the past years, the choice of E3 ligases remains limited, with an almost exclusive focus on CRBN and VHL. Here, we report the discovery of novel ligands to the PRY-SPRY domain of TRIM58, a RING ligase that is specifically expressed in erythroid precursor cells. A DSF screen, followed by validation using additional biophysical methods, led to the identification of TRIM58 ligand TRIM-473. A basic SAR around the chemotype was established by utilizing a competitive binding assay employing a short FP peptide probe derived from an endogenous TRIM58 substrate. The X-ray co-crystal structure of TRIM58 in complex with TRIM-473 gave insights into the binding mode and potential exit vectors for bifunctional degrader design.

Discovery of a novel, highly potent and orally bioavailable pyrrolidinone indole series of irreversible Myeloperoxidase (MPO) inhibitors.

Myeloperoxidase (MPO) is a heme-containing peroxidase from phagocytic cells, which plays an important role in the innate immune response. The primary anti-microbial function of MPO is achieved by catalyzing the oxidation of halides by hydrogen peroxide (H2O2). Upon activation of phagocytes, MPO activity is detectable in both phagosomes and extracellularly, where it can remain or transcytose into interstitial compartments. Activated MPO leads to oxidative stress and tissue damage in many inflammatory states, including cardiovascular disease. Starting from a low molecular weight (LMW) high throughput screening (HTS) hit, here we report the discovery of a novel pyrrolidinone indole (IN-4) as a highly potent MPO inhibitor. This compound displays similar in vitro potency across peroxidation, plasma and NETosis assays. In a dilution/dialysis study, <5% of the original MPO activity was detected post-incubation of MPO with IN-4, suggesting irreversible enzyme inhibition. A fast MPO inactivation rate (kinact/Ki) and low partition ratio (k3/k4) make IN-4 kinetic properties attractive for an MPO inhibitor. This compound also displays significant selectivity over the closely related thyroid peroxidase (TPO), and is selective for extracellular MPO over intracellular (neutrophil) MPO. Moreover, IN-4 shows good exposure, low clearance and high oral bioavailability in mice, rats and dogs. The high in vitro MPO activity and high oral exposure observed with IN-4 result in a dose-dependent inhibition of MPO activity in three mouse models of inflammation. In conclusion, IN-4 is a novel, potent, mechanism-based and selective MPO inhibitor, which may be used as superior therapeutic agent to treat multiple inflammatory conditions, including cardiovascular disease.

Discovery of a novel indole pharmacophore for the irreversible inhibition of myeloperoxidase (MPO).

Myeloperoxidase (MPO) activity and subsequent generation of hypochlorous acid has been associated with the killing of host-invading microorganisms (e.g. bacteria, viruses, and fungi). However, during oxidative stress, high MPO activity can damage host tissue and is linked to several chronic inflammatory conditions. Herein, we describe the development of a novel biaryl, indole-pyrazole series of irreversible mechanism-based inhibitors of MPO. Derived from an indole-containing high-throughput screen hit, optimization efforts resulted in potent and selective 6-substituted indoles with good oral bioavailability and in vivo activity.

Discovery of 1-((6-Aminopyridin-3-yl)Methyl)-3-(4-Bromophenyl)Urea as a Potent, Irreversible Myeloperoxidase Inhibitor.

Myeloperoxidase (MPO) is a leukocyte-derived redox enzyme that has been linked to oxidative stress and damage in many inflammatory states, including cardiovascular disease. We have discovered aminopyridines that are potent mechanism-based inhibitors of MPO, with significant selectivity over the closely related thyroid peroxidase. 1-((6-Aminopyridin-3-yl)methyl)-3-(4-bromophenyl)urea (Aminopyridine 2) inhibited MPO in human plasma and blocked MPO-dependent vasomotor dysfunction ex vivo in rat aortic rings. Aminopyridine 2 also showed high oral bioavailability and inhibited MPO activity in vivo in a mouse model of peritonitis. Aminopyridine 2 could effectively be administered as a food admixture, making it an important tool for assessing the relative importance of MPO in preclinical models of chronic inflammatory disease.

Non-Directed Allylic C-H Acetoxylation in the Presence of Lewis Basic Heterocycles.

We outline a strategy to enable non-directed Pd(II)-catalyzed C-H functionalization in the presence of Lewis basic heterocycles. In a high-throughput screen of two Pd-catalyzed C-H acetoxylation reactions, addition of a variety of N-containing heterocycles is found to cause low product conversion. A pyridine-containing test substrate is selected as representative of heterocyclic scaffolds that are hypothesized to cause catalyst arrest. We pursue two approaches in parallel that allow product conversion in this representative system: Lewis acids are found to be effective in situ blocking groups for the Lewis basic site, and a pre-formed pyridine N-oxide is shown to enable high yield of allylic C-H acetoxylation. Computational studies with density functional theory (M06) of binding affinities of selected heterocycles to Pd(OAc)2 provide an inverse correlation of the computed heterocycle-Pd(OAc)2 binding affinities with the experimental conversions to products. Additionally, 1H NMR binding studies provide experimental support for theoretical calculations.

Expedient synthesis of N-methyl tubulysin analogues with high cytotoxicity.

An optimized and highly efficient synthesis of potent, bioactive N-methyl tubulysin analogues 2 and 4 has been achieved with > 40% overall yields. This synthesis represents a significant improvement over previously reported syntheses of these and related tubulysin analogues. The stereoselective synthesis of the unnatural amino acid tubuvaline is accomplished using tert-butanesulfinamide chemistry. N-Alkylation to form N-methyl tubuvaline is performed without protection of the tubuvaline alcohol by implementing a unique N-methylation strategy via formation and reduction of a 1,3-tetrahydrooxazine heterocycle. Acylation of the hindered N-methyl tubuvaline amine utilizes a novel sequence of O-acylation followed by an O- to N-acyl transfer to form the hindered amide bond between N-methyl tubuvaline and isoleucine. This high-yielding synthesis should enable the production of large quantities of material for biological studies.

Design, synthesis, and biological properties of highly potent tubulysin D analogues.

Ten analogues of tubulysin D were synthesized and assayed against established mammalian cell lines, including cancer cells measuring inhibition of cell growth by an MTT assay. These experiments establish for the first time the essential features for the potent cytotoxicity of tubulysin D. The activities of analogues 2 to 5 demonstrate that numerous modifications may be introduced at the C-terminus of the natural product with only modest loss in activity, while the activities of analogues 6 to 8 suggest that a basic amine must be present at the N-terminus to maintain activity. Most surprisingly, analogue 10 establishes that replacement of the chemically labile O-acyl N,O-acetal with the stable N-methyl group results in almost no loss in activity. In aggregate, these structure-activity relationships enable the design of analogues such as 11 that are smaller and considerably more stable than tubulysin D but that maintain most of its potent cell-growth inhibitory activity.

Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery.

Substrate activity screening (SAS) is a fragment-based method for the rapid development of novel substrates and their conversion into non-peptidic inhibitors of Cys and Ser proteases. The method consists of three steps: (i) a library of N-acyl aminocoumarins with diverse, low-molecular-weight N-acyl groups is screened to identify protease substrates using a simple fluorescence-based assay; (ii) the identified N-acyl aminocoumarin substrates are optimized by rapid analog synthesis and evaluation; and (iii) the optimized substrates are converted into inhibitors by direct replacement of the aminocoumarin with known mechanism-based pharmacophores. This protocol describes a general procedure for the solid-phase synthesis of a library of N-acyl aminocoumarin substrates and the screening procedure to identify weak binding substrates.

The total synthesis of tubulysin D.

The first total synthesis of tubulysin D is reported. The development and application of new tert-butanesulfinamide methods allowed for rapid syntheses of the tubuvaline and tubuphenylalanine fragments. Most significantly, a route was devised and implemented to introduce and carry forward the highly labile N,O-acetal functionality. Tubulysin D is the most active member of the tubulysin family, and the efficient synthetic route described herein will allow for the rapid syntheses of analogues to probe the biological activity of this important class of natural products.

Identification of selective, nonpeptidic nitrile inhibitors of cathepsin s using the substrate activity screening method.

The substrate activity screening method, a substrate-based fragment identification and optimization method for the development of enzyme inhibitors, was previously applied to cathepsin S to obtain low nanomolar 1,4-disubstituted-1,2,3-triazole-based aldehyde inhibitors (Wood, W. J. L.; Patterson, A. W.; Tsuruoka, H.; Jain, R. K.; Ellman, J. A. J. Am. Chem. Soc. 2005, 127, 15521-15527). Replacement of the metabolically labile aldehyde pharmacophore with the nitrile pharmacophore provided inhibitors with moderate potency for cathepsin S. The inhibitors showed good selectivity over cathepsins B and L but no selectivity over cathepsin K. X-ray structures of two crystal forms (1.5 and 1.9 A) of a complex between cathepsin S and a triazole inhibitor incorporating a chloromethyl ketone pharmacophore guided the design of triazole substrates with increased cleavage efficiency and selectivity for cathepsin S over cathepsins B, L, and K. Conversion of select substrates to nitrile inhibitors yielded a low molecular weight (414 Da) and potent (15 nM) cathepsin S inhibitor that showed >1000-fold selectivity over cathepsins B, L, and K.

Asymmetric synthesis of alpha,alpha-dibranched propargylamines by acetylide additions to N-tert-butanesulfinyl ketimines.

Addition of lithium acetylides prepared from 1-pentyne, phenylacetylene, and trimethylsilylacetylene to diverse N-tert-butanesulfinyl ketimines affords a range of alpha,alpha-dibranched propargyl sulfinamides in generally good yields (up to 87%) and with high diastereoselectivities (up to >99:1). Acidic cleavage of the tert-butanesulfinyl group provides the free alpha,alpha-dibranched propargylamines.

Substrate activity screening: a fragment-based method for the rapid identification of nonpeptidic protease inhibitors.

A new fragment-based method for the rapid development of novel and distinct classes of nonpeptidic protease inhibitors, Substrate Activity Screening (SAS), is described. This method consists of three steps: (1) a library of N-acyl aminocoumarins with diverse, low molecular weight N-acyl groups is screened to identify protease substrates using a simple fluorescence-based assay, (2) the identified N-acyl aminocoumarin substrates are optimized by rapid analogue synthesis and evaluation, and (3) the optimized substrates are converted to inhibitors by direct replacement of the aminocoumarin with known mechanism-based pharmacophores. The SAS method was successfully applied to the cysteine protease cathepsin S, which is implicated in autoimmune diseases. Multiple distinct classes of nonpeptidic substrates were identified upon screening an N-acyl aminocoumarin library. Two of the nonpeptidic substrate classes were optimized to substrates with >8000-fold improvements in cleavage efficiency for each class. Select nonpeptidic substrates were then directly converted to low molecular weight, novel aldehyde inhibitors with nanomolar affinity to cathepsin S. This study demonstrates the unique characteristics and merits of this first substrate-based method for the rapid identification and optimization of weak fragments and provides the framework for the development of completely nonpeptidic inhibitors to many different proteases.