USP7 small-molecule inhibitors interfere with ubiquitin binding.

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

Potent LpxC Inhibitors with In Vitro Activity against Multidrug-Resistant Pseudomonas aeruginosa.

New drugs with novel mechanisms of resistance are desperately needed to address both community and nosocomial infections due to Gram-negative bacteria. One such potential target is LpxC, an essential enzyme that catalyzes the first committed step of lipid A biosynthesis. Achaogen conducted an extensive research campaign to discover novel LpxC inhibitors with activity against Pseudomonas aeruginosa We report here the in vitro antibacterial activity and pharmacodynamics of ACHN-975, the only molecule from these efforts and the first ever LpxC inhibitor to be evaluated in phase 1 clinical trials. In addition, we describe the profiles of three additional LpxC inhibitors that were identified as potential lead molecules. These efforts did not produce an additional development candidate with a sufficiently large therapeutic window and the program was subsequently terminated.

Structures of Pseudomonas aeruginosa LpxA Reveal the Basis for Its Substrate Selectivity.

In Gram-negative bacteria, the first step of lipid A biosynthesis is catalyzed by UDP-N-acetylglucosamine acyltransferase (LpxA) through the transfer of a R-3-hydroxyacyl chain from the acyl carrier protein (ACP) to the 3-hydroxyl group of UDP-GlcNAc. Previous studies suggest that LpxA is a critical determinant of the acyl chain length found in lipid A, which varies among species of bacteria. In Escherichia coli and Leptospira interrogans, LpxA prefers to incorporate longer R-3-hydroxyacyl chains (C14 and C12, respectively), whereas in Pseudomonas aeruginosa, the enzyme is selective for R-3-hydroxydecanoyl, a 10-hydrocarbon long acyl chain. We now report three P. aeruginosa LpxA crystal structures: apo protein, substrate complex with UDP-GlcNAc, and product complex with UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc. A comparison between the apo form and complexes identifies key residues that position UDP-GlcNAc appropriately for catalysis and supports the role of catalytic His121 in activating the UDP-GlcNAc 3-hydroxyl group for nucleophilic attack during the reaction. The product-complex structure, for the first time, offers structural insights into how Met169 serves to constrain the length of the acyl chain and thus functions as the so-called hydrocarbon ruler. Furthermore, compared with ortholog LpxA structures, the purported oxyanion hole, formed by the backbone amide group of Gly139, displays a different conformation in P. aeruginosa LpxA, which suggests flexibility of this structural feature important for catalysis and the potential need for substrate-induced conformational change in catalysis. Taken together, the three structures provide valuable insights into P. aeruginosa LpxA catalysis and substrate specificity as well as templates for future inhibitor discovery.

Discovery and Preclinical Pharmacology of NX-2127, an Orally Bioavailable Degrader of Bruton's Tyrosine Kinase with Immunomodulatory Activity for the Treatment of Patients with B Cell Malignancies.

Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, is an essential effector of B-cell receptor (BCR) signaling. Chronic activation of BTK-mediated BCR signaling is a hallmark of many hematological malignancies, which makes it an attractive therapeutic target. Pharmacological inhibition of BTK enzymatic function is now a well-proven strategy for the treatment of patients with these malignancies. We report the discovery and characterization of NX-2127, a BTK degrader with concomitant immunomodulatory activity. By design, NX-2127 mediates the degradation of transcription factors IKZF1 and IKZF3 through molecular glue interactions with the cereblon E3 ubiquitin ligase complex. NX-2127 degrades common BTK resistance mutants, including BTKC481S. NX-2127 is orally bioavailable, exhibits in vivo degradation across species, and demonstrates efficacy in preclinical oncology models. NX-2127 has advanced into first-in-human clinical trials and achieves deep and sustained degradation of BTK following daily oral dosing at 100 mg.

Antagonists of inhibitor of apoptosis proteins based on thiazole amide isosteres.

A series of IAP antagonists based on thiazole or benzothiazole amide isosteres was designed and synthesized. These compounds were tested for binding to the XIAP-BIR3 and ML-IAP BIR using a fluorescence polarization assay. The most potent of these compounds, 19a and 33b, were found to have K(i)'s of 20-30 nM against ML-IAP and 50-60 nM against XIAP-BIR3.

Discovery of dual-activity small-molecule ligands of Pseudomonas aeruginosa LpxA and LpxD using SPR and X-ray crystallography.

The lipid A biosynthesis pathway is essential in Pseudomonas aeruginosa. LpxA and LpxD are the first and third enzymes in this pathway respectively, and are regarded as promising antibiotic targets. The unique structural similarities between these two enzymes make them suitable targets for dual-binding inhibitors, a characteristic that would decrease the likelihood of mutational resistance and increase cell-based activity. We report the discovery of multiple small molecule ligands that bind to P. aeruginosa LpxA and LpxD, including dual-binding ligands. Binding poses were determined for select compounds by X-ray crystallography. The new structures reveal a previously uncharacterized magnesium ion residing at the core of the LpxD trimer. In addition, ligand binding in the LpxD active site resulted in conformational changes in the distal C-terminal helix-bundle, which forms extensive contacts with acyl carrier protein (ACP) during catalysis. These ligand-dependent conformational changes suggest a potential allosteric influence of reaction intermediates on ACP binding, and vice versa. Taken together, the novel small molecule ligands and their crystal structures provide new chemical scaffolds for ligand discovery targeting lipid A biosynthesis, while revealing structural features of interest for future investigation of LpxD function.

Optimization and Mechanistic Characterization of Pyridopyrimidine Inhibitors of Bacterial Biotin Carboxylase.

A major challenge for new antibiotic discovery is predicting the physicochemical properties that enable small molecules to permeate Gram-negative bacterial membranes. We have applied physicochemical lessons from previous work to redesign and improve the antibacterial potency of pyridopyrimidine inhibitors of biotin carboxylase (BC) by up to 64-fold and 16-fold against Escherichia coli and Pseudomonas aeruginosa, respectively. Antibacterial and enzyme potency assessments in the presence of an outer membrane-permeabilizing agent or in efflux-compromised strains indicate that penetration and efflux properties of many redesigned BC inhibitors could be improved to various extents. Spontaneous resistance to the improved pyridopyrimidine inhibitors in P. aeruginosa occurs at very low frequencies between 10-8 and 10-9. However, resistant isolates had alarmingly high minimum inhibitory concentration shifts (16- to >128-fold) compared to the parent strain. Whole-genome sequencing of resistant isolates revealed that either BC target mutations or efflux pump overexpression can lead to the development of high-level resistance.

Optimization of LpxC Inhibitors for Antibacterial Activity and Cardiovascular Safety.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a Zn2+ deacetylase that is essential for the survival of most pathogenic Gram-negative bacteria. ACHN-975 (N-((S)-3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-(((1R,2R)-2-(hydroxymethyl)cyclopropyl)buta-1,3-diyn-1-yl)benzamide) was the first LpxC inhibitor to reach human clinical testing and was discovered to have a dose-limiting cardiovascular toxicity of transient hypotension without compensatory tachycardia. Herein we report the effort beyond ACHN-975 to discover LpxC inhibitors optimized for enzyme potency, antibacterial activity, pharmacokinetics, and cardiovascular safety. Based on its overall profile, compound 26 (LPXC-516, (S)-N-(2-(hydroxyamino)-1-(3-methoxy-1,1-dioxidothietan-3-yl)-2-oxoethyl)-4-(6-hydroxyhexa-1,3-diyn-1-yl)benzamide) was chosen for further development. A phosphate prodrug of 26 was developed that provided a solubility of >30 mg mL-1 for parenteral administration and conversion into the active drug with a t1/2 of approximately two minutes. Unexpectedly, and despite our optimization efforts, the prodrug of 26 still possesses a therapeutic window insufficient to support further clinical development.

Discovery of Small-Molecule Inhibitors of Ubiquitin Specific Protease 7 (USP7) Using Integrated NMR and in Silico Techniques.

USP7 is a deubiquitinase implicated in destabilizing the tumor suppressor p53, and for this reason it has gained increasing attention as a potential oncology target for small molecule inhibitors. Herein we describe the biophysical, biochemical, and computational approaches that led to the identification of 4-(2-aminopyridin-3-yl)phenol compounds described by Kategaya ( Nature 2017 , 550 , 534 - 538 ) as specific inhibitors of USP7. Fragment based lead discovery (FBLD) by NMR combined with virtual screening and re-mining of biochemical high-throughput screening (HTS) hits led to the discovery of a series of ligands that bind in the "palm" region of the catalytic domain of USP7 and inhibit its catalytic activity. These ligands were then optimized by structure-based design to yield cell-active molecules with reasonable physical properties. This discovery process not only involved multiple techniques working in concert but also illustrated a unique way in which hits from orthogonal screening approaches complemented each other for lead identification.

Discovery of dual leucine zipper kinase (DLK, MAP3K12) inhibitors with activity in neurodegeneration models.

Dual leucine zipper kinase (DLK, MAP3K12) was recently identified as an essential regulator of neuronal degeneration in multiple contexts. Here we describe the generation of potent and selective DLK inhibitors starting from a high-throughput screening hit. Using proposed hinge-binding interactions to infer a binding mode and specific design parameters to optimize for CNS druglike molecules, we came to focus on the di(pyridin-2-yl)amines because of their combination of desirable potency and good brain penetration following oral dosing. Our lead inhibitor GNE-3511 (26) displayed concentration-dependent protection of neurons from degeneration in vitro and demonstrated dose-dependent activity in two different animal models of disease. These results suggest that specific pharmacological inhibition of DLK may have therapeutic potential in multiple indications.

Assembling polycyclic bisguanidine motifs resembling batzelladine alkaloids by double tethered Biginelli condensations.

[reaction: see text] Double tethered Biginelli condensations furnish linked polycyclic bisguanidines or bisureas. Alteration of the bis-beta-ketoester component allows bispolycyclic guanidine motifs to be constructed that resemble natural batzelladine alkaloids or have novel linkages.

Tailoring small molecules for an allosteric site on procaspase-6.

Although they represent attractive therapeutic targets, caspases have so far proven recalcitrant to the development of drugs targeting the active site. Allosteric modulation of caspase activity is an alternate strategy that potentially avoids the need for anionic and electrophilic functionality present in most active-site inhibitors. Caspase-6 has been implicated in neurodegenerative disease, including Huntington's and Alzheimer's diseases. Herein we describe a fragment-based lead discovery effort focused on caspase-6 in its active and zymogen forms. Fragments were identified for procaspase-6 using surface plasmon resonance methods and subsequently shown by X-ray crystallography to bind a putative allosteric site at the dimer interface. A fragment-merging strategy was employed to produce nanomolar-affinity ligands that contact residues in the L2 loop at the dimer interface, significantly stabilizing procaspase-6. Because rearrangement of the L2 loop is required for caspase-6 activation, our results suggest a strategy for the allosteric control of caspase activation with drug-like small molecules.

Discovery and Biological Profiling of Potent and Selective mTOR Inhibitor GDC-0349.

Aberrant activation of the PI3K-Akt-mTOR signaling pathway has been observed in human tumors and tumor cell lines, indicating that these protein kinases may be attractive therapeutic targets for treating cancer. Optimization of advanced lead 1 culminated in the discovery of clinical development candidate 8h, GDC-0349, a potent and selective ATP-competitive inhibitor of mTOR. GDC-0349 demonstrates pathway modulation and dose-dependent efficacy in mouse xenograft cancer models.

A whole cell assay to measure caspase-6 activity by detecting cleavage of lamin A/C.

Caspase-6 is a cysteinyl protease implicated in neurodegenerative conditions including Alzheimer's and Huntington's disease making it an attractive target for therapeutic intervention. A greater understanding of the role of caspase-6 in disease has been hampered by a lack of suitable cellular assays capable of specifically detecting caspase-6 activity in an intact cell environment. This is mainly due to the use of commercially available peptide substrates and inhibitors which lack the required specificity to facilitate development of this type of assay. We report here a 384-well whole-cell chemiluminescent ELISA assay that monitors the proteolytic degradation of endogenously expressed lamin A/C during the early stages of caspase-dependent apoptosis. The specificity of lamin A/C proteolysis by caspase-6 was demonstrated against recombinant caspase family members and further confirmed in genetic deletion studies. In the assay, plasma membrane integrity remained intact as assessed by release of lactate dehydrogenase from the intracellular environment and the exclusion of cell impermeable peptide inhibitors, despite the induction of an apoptotic state. The method described here is a robust tool to support drug discovery efforts targeting caspase-6 and is the first reported to specifically monitor endogenous caspase-6 activity in a cellular context.

Mechanistic and structural understanding of uncompetitive inhibitors of caspase-6.

Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound's inhibitory activity is also dependent on the amino acid sequence and P1' character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.

Discovery of a potent small-molecule antagonist of inhibitor of apoptosis (IAP) proteins and clinical candidate for the treatment of cancer (GDC-0152).

A series of compounds were designed and synthesized as antagonists of cIAP1/2, ML-IAP, and XIAP based on the N-terminus, AVPI, of mature Smac. Compound 1 (GDC-0152) has the best profile of these compounds; it binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domains of cIAP1 and cIAP2 with K(i) values of 28, 14, 17, and 43 nM, respectively. These compounds promote degradation of cIAP1, induce activation of caspase-3/7, and lead to decreased viability of breast cancer cells without affecting normal mammary epithelial cells. Compound 1 inhibits tumor growth when dosed orally in the MDA-MB-231 breast cancer xenograft model. Compound 1 was advanced to human clinical trials, and it exhibited linear pharmacokinetics over the dose range (0.049 to 1.48 mg/kg) tested. Mean plasma clearance in humans was 9 ± 3 mL/min/kg, and the volume of distribution was 0.6 ± 0.2 L/kg.

Potent, selective, and orally bioavailable inhibitors of mammalian target of rapamycin (mTOR) kinase based on a quaternary substituted dihydrofuropyrimidine.

A series of inhibitors of mTOR kinase based on a quaternary-substituted dihydrofuropyrimidine was designed and synthesized. The most potent compounds in this series inhibited mTOR kinase with K(i) < 1.0 nM and were highly (>100×) selective for mTOR over the closely related PI3 kinases. Compounds in this series showed inhibition of the pathway and antiproliferative activity in cell-based assays. Furthermore, these compounds had excellent mouse PK, and showed a robust PK-PD relationship in a mouse model of cancer.

Targeting inhibitor of apoptosis proteins for therapeutic intervention.

The inhibitors of apoptosis (IAP) proteins have emerged over the last decade as important targets for therapeutic intervention in human malignancies. Overexpression of IAPs has been implicated in cell survival and resistance against stress-induced apoptosis brought on by radiation and/or chemotherapeutics (currently the standard-of-care in a variety of different cancer diseases). In addition, evasion from death receptor-mediated apoptosis and regulation of NF-κB pathways and cell division have also been associated with IAP proteins. Efforts to target IAP proteins in tumors have focused mainly on designing small molecules that mimic the IAP-binding motif of the endogenous IAP antagonist, second mitochondrial activator of caspases. In addition, several other IAP-targeting strategies, including antisense oligonucleotides and transcriptional repression, have also been initiated, with the hope of providing therapeutic benefit to cancer patients.

Orally bioavailable antagonists of inhibitor of apoptosis proteins based on an azabicyclooctane scaffold.

A series of IAP antagonists based on an azabicyclooctane scaffold was designed and synthesized. The most potent of these compounds, 14b, binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domain of c-IAP1 with K(i) values of 140, 38, and 33 nM, respectively. These compounds promote degradation of c-IAP1, activate caspases, and lead to decreased viability of breast cancer cells without affecting normal mammary epithelial cells. Finally, compound 14b inhibits tumor growth when dosed orally in a breast cancer xenograft model.

Evolution of a strategy for the synthesis of structurally complex batzelladine alkaloids. Enantioselective total synthesis of the proposed structure of batzelladine F and structural revision.

Stereoselective synthesis of octahydro-5,6,6a-triazaacenaphthalenes 29 and 34 having the anti-relationship of the angular hydrogens flanking the pyrrolidine nitrogen confirmed suspicions that the relative configuration of the left-hand tricyclic guanidine fragment of batzelladine F should be revised to have the syn relationship of these hydrogens. Several strategies were examined for coupling tricyclic guanidine fragments to prepare potential structures for batzelladine F. Eventually, a convergent synthesis strategy was devised, whose central step was a fragment-coupling tethered-Biginelli reaction (Scheme 17). Using this approach we synthesized four potential structures of batzelladine F, 35-38. None of these compounds, nor their enantiomers, were identical to natural batzelladine F. Reinvestigation of mass spectra of natural batzelladine F, and fragments 88 and 89 obtained upon saponification of batzelladine F, demonstrated that the originally proposed connectivity of this alkaloid was also incorrect. The revised connectivity, 90, of natural batzelladine F depicted in Scheme 21 is proposed.