Liposomal Packaging Generates Wnt Protein with In Vivo Biological Activity

Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context

Discovery of Selective LRRK2 Inhibitors Guided by Computational Analysis and Molecular Modeling

Mutations in the genetic sequence of leucine-rich repeat kinase 2 (LRRK2) have been linked to increased LRRK2 activity and risk for the development of Parkinson’s disease (PD). Potent and selective small molecules capable of inhibiting the kinase activity of LRRK2 will be important tools for establishing a link between the kinase activity of LRRK2 and PD. In the absence of LRRK2 kinase domain crystal structures, a LRRK2 homology model was developed that provided robust guidance in the hit-to-lead optimization of small molecule LRRK2 inhibitors. Through a combination of molecular modeling, sequence analysis, and matched molecular pair (MMP) activity cliff analysis, a potent and selective lead inhibitor was discovered. The selectivity of this compound could be understood using the LRRK2 homology model, and application of this learning to a series of 2,4-diaminopyrimidine inhibitors in a scaffold hopping exercise led to the identification of highly potent and selective LRRK2 inhibitors that were also brain penetrable

Discovery of a potent, selective, and orally available class I phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) kinase inhibitor (GDC-0980) for the treatment of cancer

The discovery of 2 (GDC-0980), a class I PI3K and mTOR kinase inhibitor for oncology indications, is described. mTOR inhibition was added to the class I PI3K inhibitor 1 (GDC-0941) scaffold primarily through the substitution of the indazole in 1 for a 2-aminopyrimidine. This substitution also increased the microsomal stability and the free fraction of compounds as evidenced through a pairwise comparison of molecules that were otherwise identical. Highlighted in detail are analogues of an advanced compound 4 that were designed to improve solubility, resulting in 2. This compound, is potent across PI3K class I isoforms with IC(50)s of 5, 27, 7, and 14 nM for PI3Kα, β, δ, and γ, respectively, inhibits mTOR with a K(i) of 17 nM yet is highly selective versus a large panel of kinases including others in the PIKK family. On the basis of the cell potency, low clearance in mouse, and high free fraction, 2 demonstrated significant efficacy in mouse xenografts when dosed as low as 1 mg/kg orally and is currently in phase I clinical trials for cancer

Structure-Based Identification of Ureas as Novel Nicotinamide Phosphoribosyltransferase (Nampt) Inhibitors

Nicotinamide phosphoribosyltransferase (Nampt) is a promising anticancer target. Virtual screening identified a thiourea analogue, compound <b>5</b>, as a novel highly potent Nampt inhibitor. Guided by the cocrystal structure of <b>5</b>, SAR exploration revealed that the corresponding urea compound <b>7</b> exhibited similar potency with an improved solubility profile. These studies also indicated that a 3-pyridyl group was the preferred substituent at one inhibitor terminus and also identified a urea moiety as the optimal linker to the remainder of the inhibitor structure. Further SAR optimization of the other inhibitor terminus ultimately yielded compound <b>50</b> as a urea-containing Nampt inhibitor which exhibited excellent biochemical and cellular potency (enzyme IC₅₀ = 0.007 μM; A2780 IC₅₀ = 0.032 μM). Compound <b>50</b> also showed excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model (TGI of 97% was observed on day 17)

Pyrrolobenzodiazepine Dimer Antibody–Drug Conjugates: Synthesis and Evaluation of Noncleavable Drug-Linkers

Three rationally designed pyrrolobenzodiazepine (PBD) drug-linkers have been synthesized via intermediate <b>19</b> for use in antibody–drug conjugates (ADCs). They lack a cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a hydrophilic spacer, and either an alkyne (<b>6</b>), triazole (<b>7</b>), or piperazine (<b>8</b>) link to the PBD. In vitro IC₅₀ values were 11–48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (<b>7</b> inactive) for the anti-HER2 ADCs (HER2 0 MCF7, all inactive) and 0.10–1.73 μg/mL (<b>7</b> inactive) in CD22 3+ BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5–1 mg/kg, 1 mg/kg, and 3–6 mg/kg for <b>6</b>, <b>8</b>, and <b>7</b>, respectively. Tumor stasis at 2 mg/kg was observed for anti-CD22 <b>6</b> in WSU-DLCL2. In summary, noncleavable PBD-ADCs exhibit potent activity, particularly in HER2 models

Structure-Based Discovery of Novel Amide-Containing Nicotinamide Phosphoribosyltransferase (Nampt) Inhibitors

Crystal structures of several urea- and thiourea-derived compounds in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein were utilized to design a potent amide-containing inhibitor bearing an aza-indole moiety (<b>7</b>, Nampt BC IC₅₀ = 9.0 nM, A2780 cell proliferation IC₅₀ = 10 nM). The Nampt–<b>7</b> cocrystal structure was subsequently obtained and enabled the design of additional amide-containing inhibitors which incorporated various other fused 6,5-heterocyclic moieties and biaryl sulfone or sulfonamide motifs. Additional modifications of these molecules afforded many potent biaryl sulfone-containing Nampt inhibitors which also exhibited favorable in vitro ADME properties (microsomal and hepatocyte stability, MDCK permeability, plasma protein binding). An optimized compound (<b>58</b>) was a potent inhibitor of multiple cancer cell lines (IC₅₀ <10 nM vs U251, HT1080, PC3, MiaPaCa2, and HCT116 lines), displayed acceptable mouse PK properties (F = 41%, CL = 52.4 mL/min/kg), and exhibited robust efficacy in a U251 mouse xenograft model

Fragment-Based Identification of Amides Derived from <i>trans</i>-2-(Pyridin-3-yl)cyclopropanecarboxylic Acid as Potent Inhibitors of Human Nicotinamide Phosphoribosyltransferase (NAMPT)

Potent, <i>trans</i>-2-(pyridin-3-yl)­cyclopropane­carboxamide-containing inhibitors of the human nicotinamide phosphoribosyl­transferase (NAMPT) enzyme were identified using fragment-based screening and structure-based design techniques. Multiple crystal structures were obtained of initial fragment leads, and this structural information was utilized to improve the biochemical and cell-based potency of the associated molecules. Many of the optimized compounds exhibited nanomolar antiproliferative activities against human tumor lines in in vitro cell culture experiments. In a key example, a fragment lead (<b>13</b>, <i>K</i>_D = 51 μM) was elaborated into a potent NAMPT inhibitor (<b>39</b>, NAMPT IC₅₀ = 0.0051 μM, A2780 cell culture IC₅₀ = 0.000 49 μM) which demonstrated encouraging in vivo efficacy in an HT-1080 mouse xenograft tumor model

Discovery of Peptidomimetic Antibody–Drug Conjugate Linkers with Enhanced Protease Specificity

Antibody–drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine–citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs