Synthesis of [1,2,4]Triazolo[4,3- a]piperazin-6-ones: An Approach to the Triazole-Fused Ketopiperazine Scaffold.

A stereoconservative synthesis to access the triazole-fused ketopiperazine (TKP) scaffold is presented. This underexplored platform offers a wide range of structural modulations with several points of diversity and chiral centers. A series of [1,2,4]triazolo[4,3- a]piperazin-6-ones was synthesized from optically pure dipeptides. The methodology was then successfully applied to access the pyrrolo[1,2- a]triazolo[3,4- c]piperazin-6-one tricycle. Importantly, the crystal structures of representative TKPs confirmed that the configuration of the chiral centers was controlled during the synthetic route and facilitated description of the orientation of the substituents depending on their nature and position on the TKP scaffold.

Discovery of Novel Small-Molecule Inhibitors of LIM Domain Kinase for Inhibiting HIV-1.

A dynamic actin cytoskeleton is necessary for viral entry, intracellular migration, and virion release. For HIV-1 infection, during entry, the virus triggers early actin activity by hijacking chemokine coreceptor signaling, which activates a host dependency factor, cofilin, and its kinase, the LIM domain kinase (LIMK). Although knockdown of human LIM domain kinase 1 (LIMK1) with short hairpin RNA (shRNA) inhibits HIV infection, no specific small-molecule inhibitor of LIMK has been available. Here, we describe the design and discovery of novel classes of small-molecule inhibitors of LIMK for inhibiting HIV infection. We identified R10015 as a lead compound that blocks LIMK activity by binding to the ATP-binding pocket. R10015 specifically blocks viral DNA synthesis, nuclear migration, and virion release. In addition, R10015 inhibits multiple viruses, including Zaire ebolavirus (EBOV), Rift Valley fever virus (RVFV), Venezuelan equine encephalitis virus (VEEV), and herpes simplex virus 1 (HSV-1), suggesting that LIMK inhibitors could be developed as a new class of broad-spectrum antiviral drugs.IMPORTANCE The actin cytoskeleton is a structure that gives the cell shape and the ability to migrate. Viruses frequently rely on actin dynamics for entry and intracellular migration. In cells, actin dynamics are regulated by kinases, such as the LIM domain kinase (LIMK), which regulates actin activity through phosphorylation of cofilin, an actin-depolymerizing factor. Recent studies have found that LIMK/cofilin are targeted by viruses such as HIV-1 for propelling viral intracellular migration. Although inhibiting LIMK1 expression blocks HIV-1 infection, no highly specific LIMK inhibitor is available. This study describes the design, medicinal synthesis, and discovery of small-molecule LIMK inhibitors for blocking HIV-1 and several other viruses and emphasizes the feasibility of developing LIMK inhibitors as broad-spectrum antiviral drugs.

Mixed α/ß-Peptides as a Class of Short Amphipathic Peptide Hydrogelators with Enhanced Proteolytic Stability.

Peptide hydrogels are a highly promising class of materials for biomedical application, albeit facing many challenges with regard to stability and tunability. Here, we report a new class of amphipathic peptide hydrogelators, namely mixed α/ß-peptide hydrogelators. These mixed α/ß-gelators possess good rheological properties (high storage moduli) and form transparent self-supporting gels with shear-thinning behavior. Infrared spectroscopy indicates the presence of ß-sheets as the underlying secondary structure. Interestingly, self-assembled nanofibers of the mixed α/ß-peptides display unique structural morphologies with alteration of the C-terminus (acid vs amide) playing a key role in the fiber formation and gelation properties of the resulting hydrogels. The incorporation of ß3-homoamino acid residues within the mixed α/ß-peptide gelators led to an increase in proteolytic stability of the peptides under nongelating conditions (in solution) as well as gelating conditions (as hydrogel). Under diluted conditions, degradation of mixed α/ß-peptides in the presence of elastase was slowed down 120-fold compared to that of an α-peptide, thereby demonstrating beneficial enzymatic resistance for hydrogel applications in vivo. In addition, increased half-life values were obtained for the mixed α/ß-peptides in human blood plasma, as compared to corresponding α-peptides. It was also found that the mixed α/ß-peptides were amenable to injection via needles used for subcutaneous administrations. The preformed peptide gels could be sheared upon injection and were found to quickly reform to a state close to that of the original hydrogel. The shown properties of enhanced proteolytic stability and injectability hold great promise for the use of these novel mixed α/ß-peptide hydrogels for applications in the areas of tissue engineering and drug delivery.

Therapeutic targeting of casein kinase 1δ in breast cancer.

Identification of specific drivers of human cancer is required to instruct the development of targeted therapeutics. We demonstrate that CSNK1D is amplified and/or overexpressed in human breast tumors and that casein kinase 1δ (CK1δ) is a vulnerability of human breast cancer subtypes overexpressing this kinase. Specifically, selective knockdown of CK1δ, or treatment with a highly selective and potent CK1δ inhibitor, triggers apoptosis of CK1δ-expressing breast tumor cells ex vivo, tumor regression in orthotopic models of triple-negative breast cancer, including patient-derived xenografts, and tumor growth inhibition in human epidermal growth factor receptor 2-positive (HER2(+)) breast cancer models. We also show that Wnt/ß-catenin signaling is a hallmark of human tumors overexpressing CK1δ, that disabling CK1δ blocks nuclear accumulation of ß-catenin and T cell factor transcriptional activity, and that constitutively active ß-catenin overrides the effects of inhibition or silencing of CK1δ. Thus, CK1δ inhibition represents a promising strategy for targeted treatment in human breast cancer with Wnt/ß-catenin involvement.

Casein kinase 1δ is an APC/C(Cdh1) substrate that regulates cerebellar granule cell neurogenesis.

Although casein kinase 1δ (CK1δ) is at the center of multiple signaling pathways, its role in the expansion of CNS progenitor cells is unknown. Using mouse cerebellar granule cell progenitors (GCPs) as a model for brain neurogenesis, we demonstrate that the loss of CK1δ or treatment of GCPs with a highly selective small molecule inhibits GCP expansion. In contrast, CK1δ overexpression increases GCP proliferation. Thus, CK1δ appears to regulate GCP neurogenesis. CK1δ is targeted for proteolysis via the anaphase-promoting complex/cyclosome (APC/C(Cdh1)) ubiquitin ligase, and conditional deletion of the APC/C(Cdh1) activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/C(Cdh1) also downregulates CK1δ during cell-cycle exit. Therefore, we conclude that APC/C(Cdh1) controls CK1δ levels to balance proliferation and cell-cycle exit in the developing CNS. Similar studies in medulloblastoma cells showed that CK1δ holds promise as a therapeutic target.

Bis-aryl urea derivatives as potent and selective LIM kinase (Limk) inhibitors.

The discovery/optimization of bis-aryl ureas as Limk inhibitors to obtain high potency and selectivity and appropriate pharmacokinetic properties through systematic SAR studies is reported. Docking studies supported the observed SAR. Optimized Limk inhibitors had high biochemical potency (IC50 < 25 nM), excellent selectivity against ROCK and JNK kinases (>400-fold), potent inhibition of cofilin phosphorylation in A7r5, PC-3, and CEM-SS T cells (IC50 < 1 µM), and good in vitro and in vivo pharmacokinetic properties. In the profiling against a panel of 61 kinases, compound 18b at 1 µM inhibited only Limk1 and STK16 with ≥80% inhibition. Compounds 18b and 18f were highly efficient in inhibiting cell-invasion/migration in PC-3 cells. In addition, compound 18w was demonstrated to be effective on reducing intraocular pressure (IOP) on rat eyes. Taken together, these data demonstrated that we had developed a novel class of bis-aryl urea derived potent and selective Limk inhibitors.

Rational design of a hexapeptide hydrogelator for controlled-release drug delivery.

The amphiphilic peptide sequence H-Phe-Glu-Phe-Gln-Phe-Lys-OH (MBG-1) is developed as a novel hydrogelator for use in controlled-drug release administration, which is the smallest tunable ionic self-complementary hydrogelating peptide reported to date making it attractive for larger scale preparation. Hydrogelation is demonstrated to result from self-assembly of the peptide into beta-sheet nanofibers that are physically cross-linked by intertwining as well as larger bundle formation. Finally, the release of two small molecule cargos, fluorescein sodium and ciprofloxacin hydrochloride, is demonstrated revealing a two-stage zero-order sustained release profile up to 80% cumulative release over eight days.

Development of highly selective casein kinase 1δ/1ε (CK1δ/ε) inhibitors with potent antiproliferative properties.

The development of a series of potent and highly selective casein kinase 1δ/ε (CK1δ/ε) inhibitors is described. Starting from a purine scaffold inhibitor (SR-653234) identified by high throughput screening, we developed a series of potent and highly kinase selective inhibitors, including SR-2890 and SR-3029, which have IC50 ≤ 50 nM versus CK1δ. The two lead compounds have ≤100 nM EC50 values in MTT assays against the human A375 melanoma cell line and have physical, in vitro and in vivo PK properties suitable for use in proof of principle animal xenograft studies against human cancer cell lines.