Phosphorylation of SAMHD1 Thr592 increases C-terminal domain dynamics, tetramer dissociation and ssDNA binding kinetics.

SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) is driven into its activated tetramer form by binding of GTP activator and dNTP activators/substrates. In addition, the inactive monomeric and dimeric forms of the enzyme bind to single-stranded (ss) nucleic acids. During DNA replication SAMHD1 can be phosphorylated by CDK1 and CDK2 at its C-terminal threonine 592 (pSAMHD1), localizing the enzyme to stalled replication forks (RFs) to promote their restart.

Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants.

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (M) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants.

Dissecting the molecular determinants of clinical PARP1 inhibitor selectivity for tankyrase1.

Poly-ADP-ribosyltransferases play a critical role in DNA repair and cell death, and poly(ADP-ribosyl) polymerase 1 (PARP1) is a particularly important therapeutic target for the treatment of breast cancer because of its synthetic lethal relationship with breast cancer susceptibility proteins 1 and 2. Numerous PARP1 inhibitors have been developed, and their efficacy in cancer treatment is attributed to both the inhibition of enzymatic activity and their ability to trap PARP1 on to the damaged DNA, which is cytotoxic. Of the clinical PARP inhibitors, talazoparib is the most effective at trapping PARP1 on damaged DNA.

Reviving B-Factors: Activating ALK Mutations Increase Protein Dynamics of the Unphosphorylated Kinase.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that can become oncogenic by activating mutations or overexpression. Full kinetic characterization of both phosphorylated and nonphosphorylated wildtype and mutant ALK kinase domain was done. Our structure-based drug design programs directed at ALK allowed us to interrogate whether X-ray crystallography data could be used to support the hypothesis that activation of ALK by mutation occurs due to increased protein dynamics.

Conserved RNA-binding specificity of polycomb repressive complex 2 is achieved by dispersed amino acid patches in EZH2.

Polycomb repressive complex 2 (PRC2) is a key chromatin modifier responsible for methylation of lysine 27 in histone H3. PRC2 has been shown to interact with thousands of RNA species in vivo, but understanding the physiological function of RNA binding has been hampered by the lack of separation-of-function mutants. Here, we use comprehensive mutagenesis and hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify critical residues for RNA interaction in PRC2 core complexes from and , for which crystal structures are known.

The Axl kinase domain in complex with a macrocyclic inhibitor offers first structural insights into an active TAM receptor kinase.

The receptor tyrosine kinase family consisting of Tyro3, Axl, and Mer (TAM) is one of the most recently identified receptor tyrosine kinase families. TAM receptors are up-regulated postnatally and maintained at high levels in adults. They all play an important role in immunity, but Axl has also been implicated in cancer and therefore is a target in the discovery and development of novel therapeutics.

Targeting S-adenosylmethionine biosynthesis with a novel allosteric inhibitor of Mat2A.

S-Adenosyl-L-methionine (SAM) is an enzyme cofactor used in methyl transfer reactions and polyamine biosynthesis. The biosynthesis of SAM from ATP and L-methionine is performed by the methionine adenosyltransferase enzyme family (Mat; EC 2.5.

Polycomb repressive complex 2 structure with inhibitor reveals a mechanism of activation and drug resistance.

Polycomb repressive complex 2 (PRC2) mediates gene silencing through chromatin reorganization by methylation of histone H3 lysine 27 (H3K27). Overexpression of the complex and point mutations in the individual subunits of PRC2 have been shown to contribute to tumorigenesis. Several inhibitors of the PRC2 activity have shown efficacy in EZH2-mutated lymphomas and are currently in clinical development, although the molecular basis of inhibitor recognition remains unknown.

Enzyme kinetics and distinct modulation of the protein kinase N family of kinases by lipid activators and small molecule inhibitors.

The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate.

Covalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistance.

Covalent inhibition is a reemerging paradigm in kinase drug design, but the roles of inhibitor binding affinity and chemical reactivity in overall potency are not well-understood. To characterize the underlying molecular processes at a microscopic level and determine the appropriate kinetic constants, specialized experimental design and advanced numerical integration of differential equations are developed. Previously uncharacterized investigational covalent drugs reported here are shown to be extremely effective epidermal growth factor receptor (EGFR) inhibitors (kinact/Ki in the range 10(5)-10(7) M(-1)s(-1)), despite their low specific reactivity (kinact ≤ 2.

Development of a long acting human growth hormone analog suitable for once a week dosing.

Human growth hormone was conjugated to a carrier aldolase antibody, using a novel linker by connecting a disulphide bond in growth hormone to a lysine-94 amine located on the Fab arm of the antibody. The resulting CovX body showed reduced affinity towards human growth hormone receptor, reduced cell-based activity, but improved pharmacodynamic properties. We have demonstrated that this CovX-body, given once a week, showed comparable activity as growth hormone given daily in an in vivo hypophysectomized rat model.

Development of a potential high-throughput workflow to characterize sites of bioconjugation by immuno-affinity capture coupled to MALDI-TOF mass spectrometry.

The characterization of conjugation sites in bioconjugates is critical in the early discovery phase because site-specific conjugation improves in vivo stability and drug efficacy. We previously developed an engineered monoclonal antibody (mAb) scaffold which enables site-specific conjugation toward a reactive lysine (Lys) residue on each heavy chain (HC) by using an azetidinone (AZD) linker. In order to explore conjugations in other location which avoids potential interference with target binding, other chemical linkers have been studied and the investigation of N-hydroxysuccinimade (NHS) linker is reported here.

Structural and biophysical characterization of XIAP BIR3 G306E mutant: insights in protein dynamics and application for fragment-based drug design.

Previous reports describe modulators of X-linked inhibitor of apoptosis (XIAP)-caspase interaction designed from the AVPI N-terminal peptide sequence of second mitochondria-derived activator of caspase. A fragment-based drug design strategy was initiated to identify therapeutic non-peptidomimetic antagonists of X-linked inhibitor of apoptosis protein-protein interactions. Fragments that bind to the AVPI binding site of BIR3 (bacculoviral inhibitory repeat) were identified, and to further localize the fragment binding within the AVPI binding site, a point mutation was designed which alters the dynamics of flexible loops and blocks PI region of the binding cleft, thus enabling definition of weakly bound small molecules in the AV portion of the binding cleft.

Supercritical fluid chromatography reduction of hydrogen/deuterium back exchange in solution-phase hydrogen/deuterium exchange with mass spectrometric analysis.

The single biggest problem with solution-phase H/D exchange as a mass spectrometric probe of surface exposure in a protein (or protein complex) is back-exchange of H for D after the initial H/D exchange has been quenched. Back-exchange results in loss of pertinent data and also greatly hampers data analysis. Previously, very fast, cold (0-4 degrees C) HPLC was performed to help reduce back-exchange, but calculated back-exchange still averages approximately 30%.

Fourier transform ion cyclotron resonance mass spectrometry using atmospheric pressure photoionization for high-resolution analyses of corticosteroids.

Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) was coupled with atmospheric pressure photoionization (APPI) for the first time and used for the analysis of several corticosteroids.1 The analytes showed excellent response using APPI when compared with both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). APPI has the advantage of requiring less heat for desolvation, resulting in less thermal degradation of the analytes and higher signal-to-noise than APCI.

Preserving the chromatographic integrity of high-speed supercritical fluid chromatography separations using time-of-flight mass spectrometry.

The advantage of high-speed time-of-flight-mass spectrometry (TOF-MS) detection for ultrafast qualitative supercritical fluid chromatography/mass spectrometry (SFC/MS) applications allows the superior resolving power of SFC to be exploited in high-throughput analysis. A chromatographic comparison of quadrupole MS and TOF-MS shows high-speed TOF total ion current data point sampling to be more indicative of fast SFC separations and corresponding short (1-2 s) baseline peak widths. Results shown for analysis of a six-compound mixture with two peaks eluting at 0.