Design and Biochemical Characterization of Peptidic Inhibitors of the Myb/p300 Interaction.

The Myb transcription factor is involved in the proliferation of hematopoietic cells, and deregulation of its expression can lead to cancers such as leukemia. Myb interacts with various proteins, including the histone acetyltransferases p300 and CBP. Myb binds to a small domain of p300, the KIX domain (p300KIX), and inhibiting this interaction is a potential new drug discovery strategy in oncology. The available structures show that Myb binds to a very shallow pocket of the KIX domain, indicating that it might be challenging to identify inhibitors of this interaction. Here, we report the design of Myb-derived peptides which interact with p300KIX. We show that by mutating only two Myb residues that bind in or near a hotspot at the surface of p300KIX, it is possible to obtain single-digit nanomolar peptidic inhibitors of the Myb/p300KIX interaction that bind 400-fold tighter to p300KIX than wildtype Myb. These findings suggest that it might also be possible to design potent low molecular-weight compounds to disrupt the Myb/p300KIX interaction.

JDQ443, a Structurally Novel, Pyrazole-Based, Covalent Inhibitor of KRASG12C for the Treatment of Solid Tumors.

Rapid emergence of tumor resistance via RAS pathway reactivation has been reported from clinical studies of covalent KRASG12C inhibitors. Thus, inhibitors with broad potential for combination treatment and distinct binding modes to overcome resistance mutations may prove beneficial. JDQ443 is an investigational covalent KRASG12C inhibitor derived from structure-based drug design followed by extensive optimization of two dissimilar prototypes. JDQ443 is a stable atropisomer containing a unique 5-methylpyrazole core and a spiro-azetidine linker designed to position the electrophilic acrylamide for optimal engagement with KRASG12C C12. A substituted indazole at pyrazole position 3 results in novel interactions with the binding pocket that do not involve residue H95. JDQ443 showed PK/PD activity in vivo and dose-dependent antitumor activity in mouse xenograft models. JDQ443 is now in clinical development, with encouraging early phase data reported from an ongoing Phase Ib/II clinical trial (NCT04699188).

Discovery, Preclinical Characterization, and Early Clinical Activity of JDQ443, a Structurally Novel, Potent, and Selective Covalent Oral Inhibitor of KRASG12C.

Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors. SIGNIFICANCE: JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155. This article is highlighted in the In This Issue feature, p. 1397.

Redox-Active Heteroatom-Functionalized Polyacetylenes.

The discovery of metallic conductivity in polyacetylene [-HC=CH-]n upon doping represents a landmark achievement. However, the insolubility of polyacetylene and a dearth of methods for its chemical modification have limited its widespread use. Here, we employ a ring-opening metathesis polymerization (ROMP) protocol to prepare functionalized polyacetylenes (fPAs) bearing: (1) electron-deficient boryl (-BR2 ) and phosphoryl (-P(O)R2 ) side chains; (2) electron-donating amino (-NR2 ) groups, and (3) ring-fused 1,2,3-triazolium units via strain-promoted Click chemistry. These functional groups render most of the fPAs soluble and can lead to intense light absorption across the visible to near-IR region. Also, the presence of redox-active boryl and amino groups leads to opposing near-IR optical responses upon (electro)chemical reduction or oxidation. Some of the resulting fPAs show greatly enhanced air stability when compared to known polyacetylenes. Lastly, these fPAs can be cross-linked to yield network materials with the full retention of optical properties.

Discovery of Roblitinib (FGF401) as a Reversible-Covalent Inhibitor of the Kinase Activity of Fibroblast Growth Factor Receptor 4.

FGF19 signaling through the FGFR4/ß-klotho receptor complex has been shown to be a key driver of growth and survival in a subset of hepatocellular carcinomas, making selective FGFR4 inhibition an attractive treatment opportunity. A kinome-wide sequence alignment highlighted a poorly conserved cysteine residue within the FGFR4 ATP-binding site at position 552, two positions beyond the gate-keeper residue. Several strategies for targeting this cysteine to identify FGFR4 selective inhibitor starting points are summarized which made use of both rational and unbiased screening approaches. The optimization of a 2-formylquinoline amide hit series is described in which the aldehyde makes a hemithioacetal reversible-covalent interaction with cysteine 552. Key challenges addressed during the optimization are improving the FGFR4 potency, metabolic stability, and solubility leading ultimately to the highly selective first-in-class clinical candidate roblitinib.

Discovery and Design of First Benzylamine-Based Ligands Binding to an Unlocked Conformation of the Complement Factor D.

Complement Factor D, a serine protease of the S1 family and key component of the alternative pathway amplification loop, represents a promising target for the treatment of several prevalent and rare diseases linked to the innate immune system. Previously reported FD inhibitors have been shown to bind to the FD active site in its self-inhibited conformation characterized by the presence of a salt bridge at the bottom of the S1 pocket between Asp189 and Arg218. We report herein a new set of small-molecule FD ligands that harbor a basic S1 binding moiety directly binding to the carboxylate of Asp189, thereby displacing the Asp189-Arg218 ionic interaction and significantly changing the conformation of the self-inhibitory loop.

Discovery of Highly Potent and Selective Small-Molecule Reversible Factor D Inhibitors Demonstrating Alternative Complement Pathway Inhibition in Vivo.

The highly specific S1 serine protease factor D (FD) plays a central role in the amplification of the complement alternative pathway (AP) of the innate immune system. Genetic associations in humans have implicated AP activation in age-related macular degeneration (AMD), and AP dysfunction predisposes individuals to disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). The combination of structure-based hit identification and subsequent optimization of the center (S)-proline-based lead 7 has led to the discovery of noncovalent reversible and selective human factor D (FD) inhibitors with drug-like properties. The orally bioavailable compound 2 exerted excellent potency in 50% human whole blood in vitro and blocked AP activity ex vivo after oral administration to monkeys as demonstrated by inhibition of membrane attack complex (MAC) formation. Inhibitor 2 demonstrated sustained oral and ocular efficacy in a model of lipopolysaccharide (LPS)-induced systemic AP activation in mice expressing human FD.

Structure-Based Library Design and Fragment Screening for the Identification of Reversible Complement Factor D Protease Inhibitors.

Chronic dysregulation of alternative complement pathway activation has been associated with diverse clinical disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinurea. Factor D is a trypsin-like serine protease with a narrow specificity for arginine in the P1 position, which catalyzes the first enzymatic reaction of the amplification loop of the alternative pathway. In this article, we describe two hit finding approaches leading to the discovery of new chemical matter for this pivotal protease of the complement system: in silico active site mapping for hot spot identification to guide rational structure-based design and NMR screening of focused and diverse fragment libraries. The wealth of information gathered by these complementary approaches enabled the identification of ligands binding to different subpockets of the latent Factor D conformation and was instrumental for understanding the binding requirements for the generation of the first known potent noncovalent reversible Factor D inhibitors.

Small-molecule factor D inhibitors targeting the alternative complement pathway.

Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.

Gift from Nature: Cyclomarin†A Kills Mycobacteria and Malaria Parasites by Distinct Modes of Action.

Malaria continues to be one of the most devastating human diseases despite many efforts to limit its spread by prevention of infection or by pharmaceutical treatment of patients. We have conducted a screen for antiplasmodial compounds by using a natural product library. Here we report on cyclomarin A as a potent growth inhibitor of Plasmodium falciparum and the identification of its molecular target, diadenosine triphosphate hydrolase (PfAp3Aase), by chemical proteomics. Using a biochemical assay, we could show that cyclomarin A is a specific inhibitor of the plasmodial enzyme but not of the closest human homologue hFHIT. Co-crystallisation experiments demonstrate a unique binding mode of the inhibitor. One molecule of cyclomarin A binds a dimeric PfAp3Aase and prevents the formation of the enzyme⋅substrate complex. These results validate PfAp3Aase as a new drug target for the treatment of malaria. We have previously elucidated the structurally unrelated regulatory subunit ClpC1 of the ClpP protease as the molecular target of cyclomarin A in Mycobacterium tuberculosis. Thus, cyclomarin A is a rare example of a natural product with two distinct and specific modes of action.

trans-3,4-Disubstituted pyrrolidines as inhibitors of the human aspartyl protease renin. Part II: prime site exploration using an oxygen linker.

Inhibition of the aspartyl protease renin is considered as an efficient approach for treating hypertension. Lately, we described the discovery of a novel class of direct renin inhibitors which comprised a pyrrolidine scaffold (e.g., 2). Based on the X-ray structure of the lead compound 2 bound to renin we predicted that optimization of binding interactions to the prime site could offer an opportunity to further expand the scope of this chemotype. Pyrrolidine-based inhibitors were synthesized in which the prime site moieties are linked to the pyrrolidine core through an oxygen atom, resulting in an ether or a carbamate linker subseries. Especially the carbamate derivatives showed a pronounced increase in in vitro potency compared to 2. Here we report the structure-activity relationship of both subclasses and demonstrate blood pressure lowering effects for an advanced prototype in a hypertensive double-transgenic rat model after oral dosing.

trans-(3S,4S)-Disubstituted pyrrolidines as inhibitors of the human aspartyl protease renin. Part I: prime site exploration using an amino linker.

Recently, we reported on the discovery of (3S,4S)-disubstituted pyrrolidines (e.g., 2) as inhibitors of the human aspartyl protease renin. In our effort to further expand the scope of this novel class of direct renin inhibitors, a new sub-series was designed in which the prime site substituents are linked to the pyrrolidine core by a (3S)-amino functional group. In particular, analogs bearing the corresponding sulfonamide spacer (50, 51 and 54a) demonstrated a pronounced increase in in vitro potency compared to compound 2.

Structure-based design of substituted piperidines as a new class of highly efficacious oral direct Renin inhibitors.

A cis-configured 3,5-disubstituted piperidine direct renin inhibitor, (syn,rac)-1, was discovered as a high-throughput screening hit from a target-family tailored library. Optimization of both the prime and the nonprime site residues flanking the central piperidine transition-state surrogate resulted in analogues with improved potency and pharmacokinetic (PK) properties, culminating in the identification of the 4-hydroxy-3,5-substituted piperidine 31. This compound showed high in vitro potency toward human renin with excellent off-target selectivity, 60% oral bioavailability in rat, and dose-dependent blood pressure lowering effects in the double-transgenic rat model.

Exploring T cell reactivity to gliadin in young children with newly diagnosed celiac disease.

Class II major histocompatibility molecules confer disease risk in Celiac disease (CD) by presenting gliadin peptides to CD4 T cells in the small intestine. Deamidation of gliadin peptides by tissue transglutaminase creates immunogenic peptides presented by HLA-DQ2 and DQ8 molecules to activate proinflammatory CD4 T cells. Detecting gliadin specific T cell responses from the peripheral blood has been challenging due to low circulating frequencies and heterogeneity in response to gliadin epitopes. We investigated the peripheral T cell responses to alpha and gamma gliadin epitopes in young children with newly diagnosed and untreated CD. Using peptide/MHC recombinant protein constructs, we are able to robustly stimulate CD4 T cell clones previously derived from intestinal biopsies of CD patients. These recombinant proteins and a panel of α- and γ-gliadin peptides were used to assess T cell responses from the peripheral blood. Proliferation assays using peripheral blood mononuclear cells revealed more CD4 T cell responses to α-gliadin than γ-gliadin peptides with a single deamidated α-gliadin peptide able to identify 60% of CD children. We conclude that it is possible to detect T cell responses without a gluten challenge or in vitro stimulus other than antigen, when measuring proliferative responses.

Discovery of C-(1-aryl-cyclohexyl)-methylamines as selective, orally available inhibitors of dipeptidyl peptidase IV.

The successful launches of dipeptidyl peptidase IV (DPP IV) inhibitors as oral anti-diabetics warrant and spur the further quest for additional chemical entities in this promising class of therapeutics. Numerous pharmaceutical companies have pursued their proprietary candidates towards the clinic, resulting in a large body of published chemical structures associated with DPP IV. Herein, we report the discovery of a novel chemotype for DPP IV inhibition based on the C-(1-aryl-cyclohexyl)-methylamine scaffold and its optimization to compounds which selectively inhibit DPP IV at low-nM potency and exhibit an excellent oral pharmacokinetic profile in the rat.

The discovery of novel potent trans-3,4-disubstituted pyrrolidine inhibitors of the human aspartic protease renin from in silico three-dimensional (3D) pharmacophore searches.

The small-molecule trans-3,4-disubstituted pyrrolidine 6 was identified from in silico three-dimensional (3D) pharmacophore searches based on known X-ray structures of renin-inhibitor complexes and demonstrated to be a weakly active inhibitor of the human enzyme. The unexpected binding mode of the more potent enantiomer (3S,4S)-6a in an extended conformation spanning the nonprime and S1' pockets of the recombinant human (rh)-renin active site was elucidated by X-ray crystallography. Initial structure-activity relationship work focused on modifications of the hydrophobic diphenylamine portion positioned in S1 and extending toward the S2 pocket. Replacement with an optimized P3-P1 pharmacophore interacting to the nonsubstrate S3(sp) cavity eventually resulted in significantly improved in vitro potency and selectivity. The prototype analogue (3S,4S)-12a of this new class of direct renin inhibitors exerted blood pressure lowering effects in a hypertensive double-transgenic rat model after oral administration.

A novel class of oral direct renin inhibitors: highly potent 3,5-disubstituted piperidines bearing a tricyclic p3-p1 pharmacophore.

A small library of fragments comprising putative recognition motifs for the catalytic dyad of aspartic proteases was generated by in silico similarity searches within the corporate compound deck based on rh-renin active site docking and scoring filters. Subsequent screening by NMR identified the low-affinity hits 3 and 4 as competitive active site binders, which could be shown by X-ray crystallography to bind to the hydrophobic S3-S1 pocket of rh-renin. As part of a parallel multiple hit-finding approach, the 3,5-disubstituted piperidine (rac)-5 was discovered by HTS using a enzymatic assay. X-ray crystallography demonstrated the eutomer (3S,5R)-5 to be a peptidomimetic inhibitor binding to a nonsubstrate topography of the rh-renin prime site. The design of the potent and selective (3S,5R)-12 bearing a P3(sp)-tethered tricyclic P3-P1 pharmacophore derived from 3 is described. (3S,5R)-12 showed oral bioavailability in rats and demonstrated blood pressure lowering activity in the double-transgenic rat model.

Novel heterocyclic DPP-4 inhibitors for the treatment of type 2 diabetes.

Novel deazaxanthine-based DPP-4 inhibitors have been identified that are potent (IC(50) <10nM) and highly selective versus other dipeptidyl peptidases. Their synthesis and SAR are reported, along with initial efforts to improve the PK profile through decoration of the deazaxanthine core. Optimisation of compound 3a resulted in the identification of compound (S)-4i, which displayed an improved in vitro and ADME profile. Further enhancements to the PK profile were possible by changing from the deazahypoxanthine to the deazaxanthine template, culminating in compound 12g, which displayed good ex vivo DPP-4 inhibition and a superior PK profile in rat, suggestive of once daily dosing in man.

Crystal structure of human BACE2 in complex with a hydroxyethylamine transition-state inhibitor.

BACE2 is a membrane-bound aspartic protease of the A1 family with a high level of sequence homology to BACE1. While BACE1 is involved in the generation of amyloid plaques in Alzheimer's disease by cleaving Abeta-peptides from the amyloid precursor protein, the physiological function of BACE2 is not well understood. BACE2 appears to be associated with the early onset of dementia in patients with Down's syndrome, and it has been shown to be highly expressed in breast cancers. Further, it may participate in the function of normal and abnormal processes of human muscle biology. Similar to other aspartic proteases, BACE2 is expressed as an inactive zymogen requiring the cleavage of its pro-sequence during the maturation process. We have produced mature BACE2 by expression of pro-BACE2 in Escherichia coli as inclusion bodies, followed by refolding and autocatalytic activation at pH 3.4. Using a C and N-terminally truncated BACE2 variant, we were able to crystallize and determine the crystal structure of mature BACE2 in complex with a hydroxyethylamine transition-state mimetic inhibitor at 3.1 angstroms resolution. The structure of BACE2 follows the general fold of A1 aspartic proteases. However, similar to BACE1, its C-terminal domain is significantly larger than that of the other family members. Furthermore, the structure of BACE2 reveals differences in the S3, S2, S1' and S2' active site substrate pockets as compared to BACE1, and allows, therefore, for a deeper understanding of the structural features that may facilitate the design of selective BACE1 or BACE2 inhibitors.

Crystal structure of an activation intermediate of cathepsin E.

Cathepsin E is an intracellular, non-lysosomal aspartic protease expressed in a variety of cells and tissues. The protease has proposed physiological roles in antigen presentation by the MHC class II system, in the biogenesis of the vasoconstrictor peptide endothelin, and in neurodegeneration associated with brain ischemia and aging. Cathepsin E is the only A1 aspartic protease that exists as a homodimer with a disulfide bridge linking the two monomers. Like many other aspartic proteases, it is synthesized as a zymogen which is catalytically inactive towards its natural substrates at neutral pH and which auto-activates in an acidic environment. Here we report the crystal structure of an activation intermediate of human cathepsin E at 2.35A resolution. The overall structure follows the general fold of aspartic proteases of the A1 family, and the intermediate shares many features with the intermediate 2 on the proposed activation pathway of aspartic proteases like pepsin C and cathepsin D. The pro-sequence is cleaved from the protease and remains stably associated with the mature enzyme by forming the outermost sixth strand of the interdomain beta-sheet. However, different from these other aspartic proteases the pro-sequence of cathepsin E remains intact after cleavage from the mature enzyme. In addition, the active site of cathepsin E in the crystal is occupied by N-terminal amino acid residues of the mature protease in the non-primed binding site and by an artificial N-terminal extension of the pro-sequence from a neighboring molecule in the primed site. The crystal structure of the cathepsin E/pro-sequence complex, therefore, provides further insight into the activation mechanism of aspartic proteases.