A new tissue-agnostic microfluidic device to model physiology and disease: the lattice platform.

To accurately phenocopy human biology in vitro, researchers have been reducing their dependence on standard, static two-dimensional (2D) cultures and instead are moving towards three-dimensional (3D) and/or multicellular culture techniques. While these culture innovations are becoming more commonplace, there is a growing body of research that illustrates the benefits and even necessity of recapitulating the dynamic flow of nutrients, gas, waste exchange and tissue interactions that occur in vivo. However, cost and engineering complexity are two main factors that hinder the adoption of these technologies and incorporation into standard laboratory workflows. We developed LATTICE, a plug-and-play microfluidic platform able to house up to eight large tissue or organ models that can be cultured individually or in an interconnected fashion. The functionality of the platform to model both healthy and diseased tissue states was demonstrated using 3D cultures of reproductive tissues including murine ovarian tissues and human fallopian tube explants (hFTE). When exogenously exposed to pathological doses of gonadotropins and androgens to mimic the endocrinology of polycystic ovarian syndrome (PCOS), subsequent ovarian follicle development, hormone production and ovulation copied key features of this endocrinopathy. Further, hFTE cilia beating decreased significantly only when experiencing continuous media exchanges. We were then able to endogenously recreate this phenotype on the platform by dynamically co-culturing the PCOS ovary and hFTE. LATTICE was designed to be customizable with flexibility in 3D culture formats and can serve as a powerful automated tool to enable the study of tissue and cellular dynamics in health and disease in all fields of research.

Identifying Anti-prion Chemical Compounds Using a Newly Established Yeast High-Throughput Screening System.

Prion-like protein aggregation underlies the pathology of a group of fatal neurodegenerative diseases in humans, including Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis, and transmissible spongiform encephalopathy. At present, few high-throughput screening (HTS) systems are available for anti-prion small-molecule identification. Here we describe an innovative phenotypic HTS system in yeast that allows for efficient identification of chemical compounds that eliminate the yeast prion [SWI+]. We show that some identified anti-[SWI+] compounds can destabilize other non-[SWI+] prions, and their antagonizing effects can be prion- and/or variant specific. Intriguingly, among the identified hits are several previously identified anti-PrPSc compounds and a couple of US Food and Drug Administration-approved drugs for AD treatment, validating the efficacy of this HTS system. Moreover, a few hits can reduce proteotoxicity induced by expression of several pathogenic mammalian proteins. Thus, we have established a useful HTS system for identifying compounds that can potentially antagonize prionization and human proteinopathies.

Antagonism of L-type Ca2+ channels CaV1.3 and CaV1.2 by 1,4-dihydropyrimidines and 4H-pyrans as dihydropyridine mimics.

The L-type calcium channel (LTCC) CaV1.3 is regarded as a new potential therapeutic target for Parkinson's disease. Calcium influx through CaV1.3 LTCC during autonomous pacemaking in adult dopaminergic neurons of the substantia nigra pars compacta is related to the generation of mitochondrial oxidative stress in animal models. Development of a CaV1.3 antagonist selective over CaV1.2 is essential because CaV1.2 pore-forming subunits are the predominant form of LTCCs and are abundant in the central nervous and cardiovascular systems. We have explored 1,4-dihydropyrimidines and 4H-pyrans to identify potent and selective antagonists of CaV1.3 relative to CaV1.2 LTCCs. A library of 36 dihydropyridine (DHP)-mimic 1,4-dihydropyrimidines and 4H-pyrans was synthesized, and promising chiral compounds were resolved. The antagonism studies of CaV1.3 and CaV1.2 LTCCs using DHP mimic compounds showed that dihydropyrimidines and 4H-pyrans are effective antagonists of DHPs for CaV1.3 LTCCs. Some 1,4-dihydropyrimidines are more selective than isradipine for CaV1.3 over CaV1.2, shown here by both calcium flux and patch-clamp electrophysiology experiments, where the ratio of antagonism is around 2-3. These results support the hypothesis that the modified hydrogen bonding donor/acceptors in DHP-mimic dihydropyrimidines and 4H-pyrans can interact differently with DHP binding sites, but, in addition, the data suggest that the binding sites of DHP in CaV1.3 and CaV1.2 LTCCs are very similar.

Structure-activity relationship of N,N'-disubstituted pyrimidinetriones as Ca(V)1.3 calcium channel-selective antagonists for Parkinson's disease.

CaV1.3 L-type calcium channels (LTCCs) have been a potential target for Parkinson's disease since calcium ion influx through the channel was implicated in the generation of mitochondrial oxidative stress, causing cell death in the dopaminergic neurons. Selective inhibition of CaV1.3 over other LTCC isoforms, especially CaV1.2, is critical to minimize potential side effects. We recently identified pyrimidinetriones (PYTs) as a CaV1.3-selective scaffold; here we report the structure-activity relationship of PYTs with both CaV1.3 and CaV1.2 LTCCs. By variation of the substituents on the cyclopentyl and arylalkyl groups of PYT, SAR studies allowed characterization of the CaV1.3 and CaV1.2 LTCCs binding sites. The SAR also identified four important moieties that either retain selectivity or enhance binding affinity. Our study represents a significant enhancement of the SAR of PYTs at CaV1.3 and CaV1.2 LTCCs and highlights several advances in the lead optimization and diversification of this family of compounds for drug development.

Antagonism of 4-substituted 1,4-dihydropyridine-3,5-dicarboxylates toward voltage-dependent L-type Ca2+ channels Ca V 1.3 and Ca V 1.2.

L-type Ca(2+) channels in mammalian brain neurons have either a Ca(V)1.2 or Ca(V)1.3 pore-forming subunit. Recently, it was shown that Ca(V)1.3 Ca(2+) channels underlie autonomous pacemaking in adult dopaminergic neurons in the substantia nigra pars compacta, and this reliance renders them sensitive to toxins used to create animal models of Parkinson's disease. Antagonism of these channels with the dihydropyridine antihypertensive drug isradipine diminishes the reliance on Ca(2+) and the sensitivity of these neurons to toxins, pointing to a potential neuroprotective strategy. However, for neuroprotection without an antihypertensive side effect, selective Ca(V)1.3 channel antagonists are required. In an attempt to identify potent and selective antagonists of Ca(V)1.3 channels, 124 dihydropyridines (4-substituted-1,4-dihydropyridine-3,5-dicarboxylic diesters) were synthesized. The antagonism of heterologously expressed Ca(V)1.2 and Ca(V)1.3 channels was then tested using electrophysiological approaches and the FLIPR Calcium 4 assay. Despite the large diversity in substitution on the dihydropyridine scaffold, the most Ca(V)1.3 selectivity was only about twofold. These results support a highly similar dihydropyridine binding site at both Ca(V)1.2 and Ca(V)1.3 channels and suggests that other classes of compounds need to be identified for Ca(V)1.3 selectivity.