NCS-1 protein regulates TRPA1 channel through the PI3K pathway in breast cancer and neuronal cells.

The physical and functional interaction between transient receptor potential channel ankyrin 1 (TRPA1) and neuronal calcium sensor 1 (NCS-1) was assessed. NCS-1 is a calcium (Ca2+) sensor found in many tissues, primarily neurons, and TRPA1 is a Ca2+ channel involved not only in thermal and pain sensation but also in conditions such as cancer and chemotherapy-induced peripheral neuropathy, in which NCS-1 is also a regulatory component.We explored the interactions between these two proteins by employing western blot, qRT-PCR, co-immunoprecipitation, Ca2+ transient monitoring with Fura-2 spectrophotometry, and electrophysiology assays in breast cancer cells (MDA-MB-231) with different levels of NCS-1 expression and neuroblastoma cells (SH-SY5Y).Our findings showed that the expression of TRPA1 was directly correlated with NCS-1 levels at both the protein and mRNA levels. Additionally, we found a physical and functional association between these two proteins. Physically, the NCS-1 and TRPA1 co-immunoprecipitate. Functionally, NCS-1 enhanced TRPA1-dependent Ca2+ influx, current density, open probability, and conductance, where the functional effects depended on PI3K. Conclusion: NCS-1 appears to act not only as a Ca2+ sensor but also modulates TRPA1 protein expression and channel function in a direct fashion through the PI3K pathway. These results contribute to understanding how Ca2+ homeostasis is regulated and provides a mechanism underlying conditions where Ca2+ dynamics are compromised, including breast cancer. With a cellular pathway identified, targeted treatments can be developed for breast cancer and neuropathy, among other related diseases.

Multi-objective evolutionary design of adenosine receptor ligands.

A novel multiobjective evolutionary algorithm (MOEA) for de novo design was developed and applied to the discovery of new adenosine receptor antagonists. This method consists of several iterative cycles of structure generation, evaluation, and selection. We applied an evolutionary algorithm (the so-called Molecule Commander) to generate candidate A1 adenosine receptor antagonists, which were evaluated against multiple criteria and objectives consisting of high (predicted) affinity and selectivity for the receptor, together with good ADMET properties. A pharmacophore model for the human A1 adenosine receptor (hA1AR) was created to serve as an objective function for evolution. In addition, three support vector machine models based on molecular fingerprints were developed for the other adenosine receptor subtypes (hA2A, hA2B, and hA3) and applied as negative objective functions, to aim for selectivity. Structures with a higher evolutionary fitness with respect to ADMET and pharmacophore matching scores were selected as input for the next generation and thus developed toward overall fitter ("better") compounds. We finally obtained a collection of 3946 unique compounds from which we derived chemical scaffolds. As a proof-of-principle, six of these templates were selected for actual synthesis and subsequently tested for activity toward all adenosine receptors subtypes. Interestingly, scaffolds 2 and 3 displayed low micromolar affinity for many of the adenosine receptor subtypes. To further investigate our evolutionary design method, we performed systematic modifications on scaffold 3. These modifications were guided by the substitution patterns as observed in the set of generated compounds that contained scaffold 3. We found that an increased affinity with appreciable selectivity for hA1AR over the other adenosine receptor subtypes was achieved through substitution of the scaffold; compound 3a had a Ki value of 280 nM with approximately 10-fold selectivity with respect to hA2AR, while 3g had a 1.6 µM affinity for hA1AR with negligible affinity for the hA2A, hA2B, and hA3 receptor subtypes.