Vladimir Riazanski

(R)-Roscovitine, a pharmacological inhibitor of kinases, is currently in phase II clinical trial as a drug candidate for the treatment of cancers, Cushing's disease and rheumatoid arthritis. We here review the data that support the investigation of (R)-roscovitine as a potential therapeutic agent for the treatment of cystic fibrosis (CF). (R)-Roscovitine displays four independent properties that may favorably combine against CF: (1) it partially protects F508del-CFTR from proteolytic degradation and favors its trafficking to the plasma membrane; (2) by increasing membrane targeting of the TRPC6 ion channel, it rescues acidification in phagolysosomes of CF alveolar macrophages (which show abnormally high pH) and consequently restores their bactericidal activity; (3) its effects on neutrophils (induction of apoptosis), eosinophils (inhibition of degranulation/induction of apoptosis) and lymphocytes (modification of the Th17/Treg balance in favor of the differentiation of anti-inflammatory lymphocytes and reduced production of various interleukins, notably IL-17A) contribute to the resolution of inflammation and restoration of innate immunity, and (4) roscovitine displays analgesic properties in animal pain models. The fact that (R)-roscovitine has undergone extensive preclinical safety/pharmacology studies, and phase I and II clinical trials in cancer patients, encourages its repurposing as a CF drug candidate.

Bone differs from other connective tissues; it is isolated by a layer of osteoblasts that are connected by tight and gap junctions. This allows bone to create dense lamellar type I collagen, control pH, mineral deposition, and regulate water content forming a compact and strong structure. New woven bone formed after degradation of mineralized cartilage is rapidly degraded and resynthesized to impart structural order for local bone strength. Ossification is regulated by thickness of bone units and by patterning via bone morphogenetic receptors including activin, other bone morphogenetic protein receptors, transforming growth factor-β receptors, all part of a receptor superfamily. This superfamily interacts with receptors for additional signals in bone differentiation. Important features of the osteoblast environment were established using recent tools including osteoblast differentiation in vitro. Osteoblasts deposit matrix protein, over 90% type I collagen, in lamellae with orientation alternating parallel or orthogonal to the main stress axis of the bone. Into this organic matrix, mineral is deposited as hydroxyapatite. Mineral matrix matures from amorphous to crystalline hydroxyapatite. This process includes at least two-phase changes of the calcium-phosphate mineral as well as intermediates involving tropocollagen fibrils to form the bone composite. Beginning with initiation of mineral deposition, there is uncertainty regarding cardinal processes, but the driving force is not merely exceeding the calcium-phosphate solubility product. It occurs behind a epithelial-like layer of osteoblasts, which generate phosphate and remove protons liberated during calcium-phosphate salt deposition. The forming bone matrix is discontinuous from the general extracellular fluid. Required adjustment of ionic concentrations and water removal from bone matrix are important details remaining to be addressed.

1. Fundamental to the understanding of CNS function is the question of how individual neurons integrate multiple synaptic inputs into an output consisting of a sequence of action potentials carrying information coded as spike frequency. The availability for activation of neuronal Na(+) channels is critical for this process and is regulated both by fast and slow inactivation processes. Here, we have investigated slow inactivation processes in detail in hippocampal neurons. 2. Slow inactivation was induced by prolonged (10-300 s) step depolarisations to -10 mV at room temperature. In isolated hippocampal dentate granule cells (DGCs), recovery from this inactivation was biexponential, with time constants for the two phases of slow inactivation tau(slow,1) and tau(slow,2) ranging from 1 to 10 s and 20 to 50 s, respectively. Both (slow,1) and tau(slow,2) were related to the duration of prior depolarisation by a power law function of the form tau(t) = a (t/a)b, where t is the duration of the depolarisation, a is a constant kinetic setpoint and b is a scaling power. This analysis yielded values of a = 0.034 s and b = 0.62 for tau(slow,1) and a = 24 s and b = 0.30 for tau(slow,2) in the rat. 3. When a train of action potential-like depolarisations of different frequencies (50, 100, 200 Hz) was used to induce inactivation, a similar relationship was found between the frequency of depolarisation and both tau(slow,1) and tau(slow,2) (a = 0.58 s, b = 0.39 for tau(slow,1) and a = 3.77 s and b = 0.42 for tau(slow,2)). 4. Using nucleated patches from rat hippocampal slices, we have addressed possible cell specific differences in slow inactivation. In fast-spiking basket cells a similar scaling relationship can be found (a = 3.54 s and b = 0.39) as in nucleated patches from DGCs (a = 2.3 s and b = 0.48) and non-fast-spiking hilar neurons (a = 2.57 s and b = 0.49). 5. Likewise, comparison of human and rat granule cells showed that properties of ultra-slow recovery from inactivation are conserved across species. In both species ultra-slow recovery was biexponential with both tau(slow,1) and tau(slow,2) being related to the duration of depolarisation t, with a = 0.63 s and b = 0.44 for tau(slow,1) and a = 25 s and b = 0.37 for tau(slow,2) for the human subject. 6. In summary, we describe in detail how the biophysical properties of Na(+) channels result in a complex interrelationship between availability of sodium channels and membrane potential or action potential frequency that may contribute to temporal integration on a time scale of seconds to minutes in different types of hippocampal neurons.

Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H(+) into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired.