2,4-Diaminopyrimidine MK2 inhibitors. Part II: Structure-based inhibitor optimization

We describe structure-based optimization of a series of novel 2,4-diaminopyrimidine MK2 inhibitors. Co-crystal structures (see accompanying Letter) demonstrated a unique inhibitor binding mode. Resulting inhibitors had IC₅₀ values as low as 19 nM and moderate selectivity against a kinase panel. Compounds 15, 31a, and 31b inhibit TNFα production in peripheral human monocytes.     

Sic1 as a timer of Clb cyclin waves in the yeast cell cycle - design principle of not just an inhibitor

Cellular systems biology aims to uncover design principles that describe the properties of biological networks through interaction of their components in space and time. The cell cycle is a complex system regulated by molecules that are integrated into functional modules to ensure genome integrity and faithful cell division. In budding yeast, cyclin-dependent kinases (Cdk1/Clb) drive cell cycle progression, being activated and inactivated in a precise temporal sequence. In this module, which we refer to as the 'Clb module', different Cdk1/Clb complexes are regulated to generate waves of Clb activity, a functional property of cell cycle control. The inhibitor Sic1 plays a critical role in the Clb module by binding to and blocking Cdk1/Clb activity, ultimately setting the timing of DNA replication and mitosis. Fifteen years of research subsequent to the identification of Sic1 have lead to the development of an integrative approach that addresses its role in regulating the Clb module. Sic1 is an intrinsically disordered protein and achieves its inhibitory function by cooperative binding, where different structural regions stretch on the Cdk1/Clb surface. Moreover, Sic1 promotes S?phase entry, facilitating Cdk1/Clb5 nuclear transport, and therefore revealing a double function of inhibitor/activator that rationalizes a mechanism to prevent precocious DNA replication. Interestingly, the investigation of Clb temporal dynamics by mathematical modelling and experimental validation provides evidence that Sic1 acts as a timer to coordinate oscillations of Clb cyclin waves. Here we review these findings, focusing on the design principle underlying the Clb module, which highlights the role of Sic1 in regulating phase-specific Cdk1/Clb activities.     

Discovery of thieno[2,3-c]pyridines as potent COT inhibitors

Evaluation of hit chemotypes from high throughput screening identified a novel series of 2,4-disubstituted thieno[2,3-c]pyridines as COT kinase inhibitors. Structural modifications exploring SAR at the 2- and 4-positions resulting in inhibitors with improved enzyme potency and cellular activity are disclosed.     

Polyamines and Anaerobic Elongation of Rice Coleoptile

The role of polyamines in the anaerobic elongation of rice (Oryzasativa L.) coleoptiles was studied. The reduced growth of ricecoleoptiles under anoxic conditions was accompanied by a massiveaccumulation of free putrescine. Putrescine was synthesizedfrom arginine in a reaction catalyzed by arginine decarboxylase(ADC). The anoxic titer of putrescine was closely correlatedwith elongation of coleoptiles. In experiments in which putrescineand inhibitors [-difluoromethylarginine (DFMA) and -difluoromethylornithine(DFMO)] of the synthesis of polyamines were exogenously supplied,we demonstrated an absolute requirement for putrescine, synthesizedby ADC, for anaerobic elongation of coleoptiles. The presenceof exogenous putrescine (alone or in combination with DFMA)increased the rate of anaerobic elongation of coleoptile by30–40%. (Received December 1, 1988; Accepted June 19, 1989)     

Integrin function and regulation in development

Integrins are a large family of membrane receptors, consisting of alpha and beta subunits, that play a pivotal role in the interaction of cells with the extracellular matrix. Such interaction regulates the organization of cells in organs and tissues during development as well as cell differentiation and proliferation. We have shown that unfertilized oocytes express integrins that might be important during fertilization. We also analyzed nervous system and muscle tissue development showing that integrin expression is precisely regulated during organization of these tissues. The results indicate that two distinct integrin alpha subunits mediate the outgrowth of processes in nerve and glial cells. Alpha1 integrin, a laminin receptor, is up-regulated by nerve growth factor and other differentiation stimuli and is involved in neurite extension by nerve cells. In contrast, process extension by glial cells is likely to involve the alphaV integrin. Moreover, the latter integrin subunit is also transiently expressed in muscle of the embryo body where it localizes predominantly at developing myotendinous junctions. After birth this integrin disappears and is substituted by the alpha7 subunit. At the same time, important changes also occur in the expression of the associated beta subunit. In fact, the beta1A isoform which is expressed in fetal muscles, is substituted by beta1D. These isoforms are generated by alternative splicing and differ in only a few amino acid residues at the COOH terminus of the protein. This region of the molecule is exposed at the cytoplasmic face of the plasma membrane and is connected to the actin filaments. Our results show that beta1D, which is expressed only in striated muscle tissues, binds to both cytoskeletal and extracellular matrix proteins with an affinity higher than beta1A. Thus, beta1D provides a stronger link between the cytoskeleton and extracellular matrix necessary to support mechanical tension during muscle contraction. These results indicate that cells can regulate their interactions with the extracellular matrix by changing their expression of alpha integrin subunits and thus ligand specificity, or by more subtle changes involving alternative usage of different cytoplasmic domains. The important role of both alpha and beta integrin subunit cytoplasmic domains during development is further illustrated by the analysis of targeted mutations which we have generated by homologous recombination in mice.     

Adenosine Transport into Guinea-pig Synaptosomes

Abstract: Kinetics for transport of adenosine into guinea-pig neocortex synaptosomes were studied by incubating them with [¹⁴C]adenosine for up to 30 s. The apparent K _m value of the high-affinity transport system for adenosine was 21.1 μM and the V _max value was 257.3 pmol/min/mg protein. The transport system was inhibited by both compounds structurally related (compounds 554 and 555) and unrelated (dipyridamole) to adenosine. Because electrically stimulated synaptosomes release up to 1.5% of the adenosine derivative content per min, the physiological significance of adenosine uptake is discussed as a possible mechanism to compensate for the loss of adenine nucleotides from synaptosomes preparations.     

Virulence genes are carried by a megaplasmid of the plant pathogen Pseudomonas solanacearum

Summary A class of avirulent mutants of the plant pathogenic bacterium Pseudomonas solanacearum, strain GMI1000, resistant to acridine orange (Acr^r), harbour a deletion of over 85 kb in their genome. This deletion affects, a1,000 kb megaplasmid which has previously been shown to be present in most of the strains of this species. In addition at least 11 out of 13 independent Tn5 insertions, leading to loss of virulence, are located on the megaplasmid. Nine of them are present in the region which is deleted from the Acr^r mutants. These results suggest that the majority of virulence genes identified so far are plasmid borne.