Global signal peptide profiling reveals principles of selective Sec61 inhibition.

Cotransins target the Sec61 translocon and inhibit the biogenesis of an undefined subset of secretory and membrane proteins. Remarkably, cotransin inhibition depends on the unique signal peptide (SP) of each Sec61 client, which is required for cotranslational translocation into the endoplasmic reticulum. It remains unknown how an SP's amino acid sequence and biophysical properties confer sensitivity to structurally distinct cotransins. Here we describe a fluorescence-based, pooled-cell screening platform to interrogate nearly all human SPs in parallel. We profiled two cotransins with distinct effects on cancer cells and discovered a small subset of SPs, including the oncoprotein human epidermal growth factor receptor 3 (HER3), with increased sensitivity to the more selective cotransin, KZR-9873. By comparing divergent mouse and human orthologs, we unveiled a position-dependent effect of arginine on SP sensitivity. Our multiplexed profiling platform reveals how cotransins can exploit subtle sequence differences to achieve SP discrimination.

Zetomipzomib (KZR-616) attenuates lupus in mice via modulation of innate and adaptive immune responses.

Zetomipzomib (KZR-616) is a selective inhibitor of the immunoproteasome currently undergoing clinical investigation in autoimmune disorders. Here, we characterized KZR-616 in vitro and in vivo using multiplexed cytokine analysis, lymphocyte activation and differentiation, and differential gene expression analysis. KZR-616 blocked production of >30 pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs), polarization of T helper (Th) cells, and formation of plasmablasts. In the NZB/W F1 mouse model of lupus nephritis (LN), KZR-616 treatment resulted in complete resolution of proteinuria that was maintained at least 8 weeks after the cessation of dosing and was mediated in part by alterations in T and B cell activation, including reduced numbers of short and long-lived plasma cells. Gene expression analysis of human PBMCs and tissues from diseased mice revealed a consistent and broad response focused on inhibition of T, B, and plasma cell function and the Type I interferon pathway and promotion of hematopoietic cell lineages and tissue remodeling. In healthy volunteers, KZR-616 administration resulted in selective inhibition of the immunoproteasome and blockade of cytokine production following ex vivo stimulation. These data support the ongoing development of KZR-616 in autoimmune disorders such as systemic lupus erythematosus (SLE)/LN.

Required Immunoproteasome Subunit Inhibition Profile for Anti-Inflammatory Efficacy and Clinical Candidate KZR-616 ((2 S,3 R)- N-(( S)-3-(Cyclopent-1-en-1-yl)-1-(( R)-2-methyloxiran-2-yl)-1-oxopropan-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-(( S)-2-(2-morpholinoacetamido)propanamido)propenamide).

Selective immunoproteasome inhibition is a promising approach for treating autoimmune disorders, but optimal proteolytic active site subunit inhibition profiles remain unknown. We reveal here our design of peptide epoxyketone-based selective low molecular mass polypeptide-7 (LMP7) and multicatalytic endopeptidase complex subunit-1 (MECL-1) subunit inhibitors. Utilizing these and our previously disclosed low molecular mass polypeptide-2 (LMP2) inhibitor, we demonstrate a requirement of dual LMP7/LMP2 or LMP7/MECL-1 subunit inhibition profiles for potent cytokine expression inhibition and in vivo efficacy in an inflammatory disease model. These and additional findings toward optimized solubility led the design and selection of KZR-616 disclosed here and presently in clinical trials for treatment of rheumatic disease.

Synthesis, in Vitro Covalent Binding Evaluation, and Metabolism of (14)C-Labeled Inhibitors of 11ß-HSD1.

In this letter, we reported the design and synthesis of three potent, selective, and orally bioavailable 11ß-HSD1 inhibitors labeled with (14)C: AMG 456 (1), AM-6949 (2), and AM-7715 (3). We evaluated the covalent protein binding of the labeled inhibitors in human liver microsomes in vitro and assessed their potential bioactivation risk in humans. We then studied the in vitro mechanism of 2 in human hepatocytes and the formation of reactive intermediates. Our study results suggest that 1 and 3 have low potential for metabolic bioactivation in humans, while 2 has relatively high risk.

Discovery of AMG 925, a FLT3 and CDK4 dual kinase inhibitor with preferential affinity for the activated state of FLT3.

We describe the structural optimization of a lead compound 1 that exhibits dual inhibitory activities against FLT3 and CDK4. A series of pyrido[4',3':4,5]pyrrolo[2,3-d]pyrimidine derivatives was synthesized, and SAR analysis, using cell-based assays, led to the discovery of 28 (AMG 925), a potent and orally bioavailable dual inhibitor of CDK4 and FLT3, including many FLT3 mutants reported to date. Compound 28 inhibits the proliferation of a panel of human tumor cell lines including Colo205 (Rb(+)) and U937 (FLT3(WT)) and induced cell death in MOLM13 (FLT3(ITD)) and even in MOLM13 (FLT3(ITD, D835Y)), which exhibits resistance to a number of FLT3 inhibitors currently under clinical development. At well-tolerated doses, compound 28 leads to significant growth inhibition of MOLM13 xenografts in nude mice, and the activity correlates with inhibition of STAT5 and Rb phosphorylation.

Selective and potent morpholinone inhibitors of the MDM2-p53 protein-protein interaction.

We previously reported the discovery of AMG 232, a highly potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Our continued search for potent and diverse analogues led to the discovery of novel morpholinone MDM2 inhibitors. This change to a morpholinone core has a significant impact on both potency and metabolic stability compared to the piperidinone series. Within this morpholinone series, AM-8735 emerged as an inhibitor with remarkable biochemical potency (HTRF IC50 = 0.4 nM) and cellular potency (SJSA-1 EdU IC50 = 25 nM), as well as pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 41 mg/kg. Lead optimization toward the discovery of this inhibitor as well as key differences between the morpholinone and the piperidinone series will be described herein.

Discovery and optimization of benzenesulfonanilide derivatives as a novel class of 11ß-HSD1 inhibitors.

A novel series of benzenesulfonanilide derivatives of 11ß-HSD1 inhibitors were identified via modification of the sulfonamide core of the arylsulfonylpiperazine lead structures. The synthesis, in vitro biological evaluation, and structure-activity relationship of these compounds are presented. Optimization of this series rapidly resulted in the discovery of compounds (S)-10 and (S)-23 (11ß-HSD1 SPA IC(50)=1.8 and 1.4 nM, respectively).

Discovery of amide replacements that improve activity and metabolic stability of a bis-amide smoothened antagonist hit.

A bis-amide antagonist of Smoothened, a seven-transmembrane receptor in the Hedgehog signaling pathway, was discovered via high throughput screening. In vitro and in vivo experiments demonstrated that the bis-amide was susceptible to N-acyl transferase mediated amide scission. Several bioisosteric replacements of the labile amide that maintained in vitro potency were identified and shown to be metabolically stable in vitro and in vivo.

Synthesis and optimization of novel 4,4-disubstituted cyclohexylbenzamide derivatives as potent 11ß-HSD1 inhibitors.

The synthesis and SAR of a series of 4,4-disubstituted cyclohexylbenzamide inhibitors of 11ß-HSD1 are described. Optimization rapidly led to potent, highly selective, and orally bioavailable inhibitors demonstrating efficacy in both rat and non-human primate ex vivo pharmacodynamic models.

Addressing PXR liabilities of phthalazine-based hedgehog/smoothened antagonists using novel pyridopyridazines.

Pyridopyridazine antagonists of the hedgehog signaling pathway are described. Designed to optimize our previously described phthalazine smoothened antagonists, a representative compound eliminates a PXR liability while retaining potency and in vitro metabolic stability. Moreover, the compound has improved efficacy in a hedgehog/smoothened signaling mouse pharmacodynamic model.

Design of 1-piperazinyl-4-arylphthalazines as potent Smoothened antagonists.

The Hedgehog (Hh) signaling pathway regulates cell proliferation and differentiation in developing tissues, and abnormal activation of the Hh pathway has been linked to several tumor subsets. As a transducer of Hh signaling, the GPCR-like protein Smoothened (Smo) is a promising target for disruption of unregulated Hh signaling. A series of 1-amino-4-arylphthalazines was developed as potent and orally bioavailable inhibitors of Smo. A representative compound from this class demonstrated significant tumor volume reduction in a mouse medulloblastoma model.

Optimization of novel di-substituted cyclohexylbenzamide derivatives as potent 11 beta-HSD1 inhibitors.

Novel 4,4-disubstituted cyclohexylbenzamide inhibitors of 11beta-HSD1 were optimized to account for liabilities relating to in vitro pharmacokinetics, cytotoxicity and protein-related shifts in potency. A representative compound showing favorable in vivo pharmacokinetics was found to be an efficacious inhibitor of 11beta-HSD1 in a rat pharmacodynamic model (ED(50)=10mg/kg).

Discovery and optimization of piperidyl benzamide derivatives as a novel class of 11beta-HSD1 inhibitors.

Discovery and optimization of a piperidyl benzamide series of 11beta-HSD1 inhibitors is described. This series was derived from a cyclohexyl benzamide lead structures to address PXR selectivity, high non-specific protein binding, poor solubility, limited in vivo exposure, and in vitro cytotoxicity issues observed with the cyclohexyl benzamide structures. These efforts led to the discovery of piperidyl benzamide 15 which features improved properties over the cyclohexyl benzamide derivatives.

An efficient and scalable one-pot double Michael addition-Dieckmann condensation for the synthesis of 4,4-disubstituted cyclohexane beta-keto esters.

A simple, scalable, and efficient one-pot methodology for the synthesis of 4,4-disubstituted cyclohexane beta-keto esters from benzylic nitriles or esters and methyl acrylate promoted by potassium tert-butoxide is described. The process relies on a tandem double Michael addition-Dieckmann condensation reaction, which results in the formation of three discrete carbon-carbon bonds in a single pot, including a quaternary center. The method allows for the convenient and rapid synthesis of a variety of 4-aryl-4-cyano-2-carbomethoxycyclohexanone and 4-aryl-2,4-biscarbomethoxycyclohexanone building blocks for use in natural products synthesis and medicinal chemistry.

Inhibition of human papilloma virus E2 DNA binding protein by covalently linked polyamides.

Polyamides are a class of heterocyclic small molecules with the potential of controlling gene expression by binding to the minor groove of DNA in a sequence-specific manner. To evaluate the feasibility of this class of compounds as antiviral therapeutics, molecules were designed to essential sequence elements occurring numerous times in the HPV genome. This sequence element is bound by a virus-encoded transcription and replication factor E2, which binds to a 12 bp recognition site as a homodimeric protein. Here, we take advantage of polyamide:DNA and E2:DNA co-crystal structural information and advances in polyamide synthetic chemistry to design tandem hairpin polyamides that are capable of displacing the major groove-binding E2 homodimer from its DNA binding site. The binding of tandem hairpin polyamides and the E2 DNA binding protein to the DNA site is mutually exclusive even though the two ligands occupy opposite faces of the DNA double helix. We show with circular permutation studies that the tandem hairpin polyamide prevents the intrinsic bending of the E2 DNA site important for binding of the protein. Taken together, these results illustrate the feasibility of inhibiting the binding of homodimeric, major groove-binding transcription factors by altering the local DNA geometry using minor groove-binding tandem hairpin polyamides.