Long-term assessment of haematological recovery following somatic genetic rescue in a MYSM1-deficient patient: Implications for in vivo gene therapy.

MYSM1 deficiency causes inherited bone marrow failure syndrome (IBMFS). We have previously identified an IBMFS patient with a homozygous pathogenic variant in MYSM1 who recovered from cytopenia due to spontaneous correction of one MYSM1 variant in the haematopoietic compartment, an event called somatic genetic rescue (SGR). The study of the genetic and biological aspects of the patient's haematopoietic/lymphopoietic system over a decade after SGR shows that one genetically corrected haematopoietic stem cell (HSC) can restore a healthy and stable haematopoietic system.

Spontaneous sensations reveal distorted body perception in complex regional pain syndrome (CRPS).

Distortions of body representation have been reported in Complex Regional Pain Syndrome (CRPS). The perception of sensations arising without external triggers (spontaneous sensations or SPS) was assessed here as a means of investigating distortions of body representation and awareness in CRPS. To avoid confounds between CRPS symptoms and SPS, lower-limb CRPS patients were included, whereas SPS were tested on the hands.

Discovery and Optimization of wt-RET/KDR-Selective Inhibitors of RET Kinase.

A combination of focused library and virtual screening, hit expansion, and rational design has resulted in the development of a series of inhibitors of RET kinase, the anticipated drug-resistant mutant of RET kinase. These agents do not inhibit the wild type (wt) isoforms of RET or KDR and therefore offer a potential adjunct to RET inhibitors currently undergoing clinical evaluation.

The SGC beyond structural genomics: redefining the role of 3D structures by coupling genomic stratification with fragment-based discovery.

The ongoing explosion in genomics data has long since outpaced the capacity of conventional biochemical methodology to verify the large number of hypotheses that emerge from the analysis of such data. In contrast, it is still a gold-standard for early phenotypic validation towards small-molecule drug discovery to use probe molecules (or tool compounds), notwithstanding the difficulty and cost of generating them. Rational structure-based approaches to ligand discovery have long promised the efficiencies needed to close this divergence; in practice, however, this promise remains largely unfulfilled, for a host of well-rehearsed reasons and despite the huge technical advances spearheaded by the structural genomics initiatives of the noughties.

Explicit treatment of active-site waters enhances quantum mechanical/implicit solvent scoring: Inhibition of CDK2 by new pyrazolo[1,5-a]pyrimidines.

We present comprehensive testing of solvent representation in quantum mechanics (QM)-based scoring of protein-ligand affinities. To this aim, we prepared 21 new inhibitors of cyclin-dependent kinase 2 (CDK2) with the pyrazolo[1,5-a]pyrimidine core, whose activities spanned three orders of magnitude. The crystal structure of a potent inhibitor bound to the active CDK2/cyclin A complex revealed that the biphenyl substituent at position 5 of the pyrazolo[1,5-a]pyrimidine scaffold was located in a previously unexplored pocket and that six water molecules resided in the active site.

Structural and biochemical characterization of the Cop9 signalosome CSN5/CSN6 heterodimer.

The Cop9 signalosome complex (CSN) regulates the functional cycle of the major E3 ubiquitin ligase family, the cullin RING E3 ubiquitin ligases (CRLs). Activated CRLs are covalently modified by the ubiquitin-like protein Nedd8 (neural precursor cell expressed developmentally down-regulated protein 8). CSN serves an essential role in myriad cellular processes by reversing this modification through the isopeptidase activity of its CSN5 subunit.

An inhibitor's-eye view of the ATP-binding site of CDKs in different regulatory states.

We have used a chemically diverse panel of kinase inhibitors to assess the chemical similarity of the ATP-binding sites of cyclin-dependent kinase (CDK) subfamily members in a range of activation states. Using this approach, we find that different activation states of a particular CDK may differ from each other as much as different CDKs in the same activation state. We also find that inhibitors discriminate more effectively among CDK family members in their monomeric state than in their cyclin-bound state, providing direct evidence for the belief that selective binding to inactive kinase states might be more readily achieved than selective binding to active states.

Structure and function of MPN (Mpr1/Pad1 N-terminal) domain-containing proteins.

MPN (Mpr1/Pad1 N-terminal) domain-containing proteins are present throughout all domains of life. In eukaryotes, MPN domain-containing proteins are commonly found in association with other molecules in large protein complexes, where examples comprise; the 26S proteasome and the COP9 (Constitutive photomorphogenesis 9) signalosome complexes, including the MPN subunits, POH1 and Mov34, CSN5 and CSN6, respectively. Examples of MPN domaincontaining proteins that are not incorporated in a large multi-protein complex have also been reported and include AMSH (for associated molecule with the SH3 domain of STAM) and the AMSH-Like Protein (AMSH-LP).

8-Substituted O(6)-cyclohexylmethylguanine CDK2 inhibitors: using structure-based inhibitor design to optimize an alternative binding mode.

Evaluation of the effects of purine C-8 substitution within a series of CDK1/2-selective O(6)-cyclohexylmethylguanine derivatives revealed that potency decreases initially with increasing size of the alkyl substituent. Structural analysis showed that C-8 substitution is poorly tolerated, and to avoid unacceptable steric interactions, these compounds adopt novel binding modes. Thus, 2-amino-6-cyclohexylmethoxy-8-isopropyl-9H-purine adopts a "reverse" binding mode where the purine backbone has flipped 180°.

Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1.

The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN), a large multiprotein complex that resembles the 19S lid of the 26S proteasome, plays a central role in the regulation of the E3-cullin RING ubiquitin ligases (CRLs). The catalytic activity of the CSN complex, carried by subunit 5 (CSN5/Jab1), resides in the deneddylation of the CRLs that is the hydrolysis of the cullin-neural precursor cell expressed developmentally downregulated gene 8 (Nedd8)isopeptide bond. Whereas CSN-dependent CSN5 displays isopeptidase activity, it is intrinsically inactive in other physiologically relevant forms.

An integrated chemical biology approach provides insight into Cdk2 functional redundancy and inhibitor sensitivity.

Cdk2 promotes DNA replication and is a promising cancer therapeutic target, but its functions appear redundant with Cdk1, an essential Cdk affected by most Cdk2 inhibitors. Here, we present an integrated multidisciplinary approach to address Cdk redundancy. Mathematical modeling of enzymology data predicted conditions allowing selective chemical Cdk2 inhibition.

Recent developments in cyclin-dependent kinase biochemical and structural studies.

The cyclin-dependent kinases (CDKs) have been intensely studied because of their involvement in regulating essential cellular activities that include proliferation and transcription. A series of CDK2-containing structures have informed a general model for the molecular details of CDK activation and regulation. Recent structural studies of other members of the CDK family have lead to a re-appraisal of this model.

The structure of CDK4/cyclin D3 has implications for models of CDK activation.

Cyclin-dependent kinase 4 (CDK4)/cyclin D complexes are expressed early in the G(1) phase of the cell cycle and stimulate the expression of genes required for G(1) progression by phosphorylation of the product of the retinoblastoma gene, pRb. To elaborate the molecular pathway of CDK4 activation and substrate selection we have determined the structure of nonphosphorylated CDK4/cyclin D3. This structure of an authentic CDK/cyclin complex shows that cyclin binding may not be sufficient to drive the CDK active site toward an active conformation.

N-&-N, a new class of cell death-inducing kinase inhibitors derived from the purine roscovitine.

Cyclin-dependent kinases (CDKs) and their regulators show frequent abnormalities in tumors. Ten low molecular weight pharmacologic inhibitors of CDKs are currently in clinical trials against various cancers, including the 2,6,9-trisubstituted purine (R)-roscovitine (CYC202/Seliciclib). We here report the characterization of N-&-N1, a bioisoster of roscovitine displaying improved antitumoral properties.

CR8, a potent and selective, roscovitine-derived inhibitor of cyclin-dependent kinases.

Among the ten pharmacological inhibitors of cyclin-dependent kinases (CDKs) currently in clinical trials, the purine roscovitine (CYC202, Seliciclib) is undergoing phase 2 trials against non-small-cell lung and nasopharyngeal cancers. An extensive medicinal chemistry study, designed to generate more potent analogues of roscovitine, led to the identification of an optimal substitution at the N6 position (compound CR8). An extensive selectivity study (108 kinases) highlights the exquisite selectivity of CR8 for CDK1/2/3/5/7/9.

Structures of P. falciparum protein kinase 7 identify an activation motif and leads for inhibitor design.

Malaria is a major threat to world health. The identification of parasite targets for drug development is a priority and parasitic protein kinases suggest themselves as suitable targets as many display profound structural and functional divergences from their host counterparts. In this paper, we describe the structure of the orphan protein kinase, Plasmodium falciparum protein kinase 7 (PFPK7).

Meriolins (3-(pyrimidin-4-yl)-7-azaindoles): synthesis, kinase inhibitory activity, cellular effects, and structure of a CDK2/cyclin A/meriolin complex.

We report the synthesis and biological characterization of 3-(pyrimidin-4-yl)-7-azaindoles (meriolins), a chemical hybrid between the natural products meridianins and variolins, derived from marine organisms. Meriolins display potent inhibitory activities toward cyclin-dependent kinases (CDKs) and, to a lesser extent, other kinases (GSK-3, DYRK1A). The crystal structures of 1e (meriolin 5) and variolin B (Bettayeb, K.

Redox-linked structural changes associated with the formation of a catalytically competent form of the diheme cytochrome c peroxidase from Pseudomonas aeruginosa.

A recombinant form of the prototypic diheme bacterial cytochrome c peroxidase (BCCP) from Pseudomonas aeruginosa (PsaCCP) has been expressed in Escherichia coli and purified to homogeneity. This material was used to carry out the first integrated biochemical, spectroscopic and structural investigation of the factors leading to reductive activation of this class of enzymes. A single, tightly bound, Ca2+ ion (K = 3 x 10(10) M-1) found at the domain interface of both the fully oxidized and mixed-valence forms of the enzyme is absolutely required for catalytic activity.

Searching for cyclin-dependent kinase inhibitors using a new variant of the cope elimination.

beta-Piperidinoethylsulfides are oxidized by m-chloroperbenzoic acid to intermediates containing both N-oxide and sulfone functions. These undergo a Cope-type elimination to a vinylsulfone that can be captured by amines to afford beta-aminoethylsulfones. When a beta-aminoethylsulfone group is linked to the 4-position of a phenyl group attached at N-2 of O6-cyclohexylmethylguanine, the resulting derivatives are inhibitors of the cyclin-dependent kinase CDK2.

Structure and mechanism in the bacterial dihaem cytochrome c peroxidases.

The bacterial cytochrome c peroxidases contain an electron-transferring haem c (E) and a peroxidatic haem c (P). Many are isolated in an inactive oxidised state. Reduction of the E haem promotes Ca(2+)-dependent spin state and coordination changes at the P haem rendering it accessible to ligand.

Activation and catalysis of the di-heme cytochrome c peroxidase from Paracoccus pantotrophus.

Bacterial cytochrome c peroxidases contain an electron transferring (E) heme domain and a peroxidatic (P) heme domain. All but one of these enzymes are isolated in an inactive oxidized state and require reduction of the E heme by a small redox donor protein in order to activate the P heme. Here we present the structures of the inactive oxidized and active mixed valence enzyme from Paracoccus pantotrophus.

Crystal structure and solution NMR dynamics of a D (type II) peroxiredoxin glutaredoxin and thioredoxin dependent: a new insight into the peroxiredoxin oligomerism.

Peroxiredoxins (Prxs) constitute a family of thiol peroxidases that reduce hydrogen peroxide, peroxinitrite, and hydroperoxides using a strictly conserved cysteine. Very abundant in all organisms, Prxs are produced as diverse isoforms characterized by different catalytic mechanisms and various thiol-containing reducing agents. The oligomeric state of Prxs and the link with their functionality is a subject of intensive research.

Assessing crystallization droplets using birefringence.

In this paper, the detection of crystalline elements in protein crystallization droplets containing precipitate is illustrated using the rotating-polarizer microscope technique. The sensitivity of this automated birefringence technique enables the detection of microcrystals in a precipitate that appears to be amorphous using traditional methods of inspection. The technique is illustrated with lysozyme and glucose isomerase.