Christian Raetz: scientist and friend extraordinaire.

Chris Raetz passed away on August 16, 2011, still at the height of his productive years. His seminal contributions to biomedical research were in the genetics, biochemistry, and structural biology of phospholipid and lipid A biosynthesis in Escherichia coli and other gram-negative bacteria. He defined the catalytic properties and structures of many of the enzymes responsible for the "Raetz pathway for lipid A biosynthesis." His deep understanding of chemistry, coupled with knowledge of medicine, biochemistry, genetics, and structural biology, formed the underpinnings for his contributions to the lipid field. He displayed an intense passion for science and a broad interest that came from a strong commitment to curiosity-driven research, a commitment he imparted to his mentees and colleagues. What follows is a testament to both Chris's science and humanity from his friends and colleagues.

The biochemistry of disease: desperately seeking syzygy.

The elucidation of the precise molecular structure and dynamics of biological processes is the great work of biochemistry. From this, insights into the changes leading to process dysfunction or disease are derived, as well as the possible approaches to restore healthy function. Translating this information into effective and safe treatments for disease requires a coordinated interdisciplinary effort, a fusion of creativity and practicality, and a healthy dose of luck. Using several reviews in this volume as springboards, I discuss the broader issues of drug development, highlighting some recent successes and future directions. Such occurrences inspire awe but remain too rare for comfort.

Welcome to Biochemistry Volume 1: No Hyphen Required.

The founding of the journal Biochemistry by the American Chemical Society 60 years ago was a highlight of the Society's growing commitment to chemically driven biochemistry. It was a commitment that was nearly an additional 60 years in the making. In that time, biological chemistry was becoming more molecularly focused. Its relationship to the traditional chemical disciplines became apparent to a generation of young chemists, who grappled with defining the field's core chemical principles and creating new areas of research for a new journal. The path to Biochemistry was exciting, but it was also complex and difficult. Even its naming was arguable.

Chemoproteomics Using Nucleotide Acyl Phosphates Reveals an ATP Binding Site at the Dimer Interface of Procaspase-6.

Acyl phosphates of ATP (ATPAc) and related nucleotides have proven to be useful for the interrogation of known nucleotide binding sites via specific acylation of conserved lysines (K). In addition, occasional K acylations are identified in proteins without such known sites. Here we present a robust and specific acylation of procaspase-6 by ATPAc at K133 in Jurkat cell lysates. The K133 acylation is dependent on π-π stacking interactions between the adenine moiety of ATPAc and a conserved Y198-Y198 site formed at the homodimeric interface of procaspase-6. Significantly, the Y198A mutation in procaspase-6 abolishes K133 acylation but has no effect on the proteolytic activity of the mature, active caspase-6 Y198A variant. Additional in vitro studies show that ATP can inhibit the autoproteolytic activation of procaspase-6. These observations suggest that ATP, and possibly other nucleotides, may serve as the endogenous ligands for the allosteric site at the procaspase-6 dimer interface, a site that has persisted in its "orphan" status for more than a decade.

ERK5 kinase activity is dispensable for cellular immune response and proliferation.

Unlike other members of the MAPK family, ERK5 contains a large C-terminal domain with transcriptional activation capability in addition to an N-terminal canonical kinase domain. Genetic deletion of ERK5 is embryonic lethal, and tissue-restricted deletions have profound effects on erythroid development, cardiac function, and neurogenesis. In addition, depletion of ERK5 is antiinflammatory and antitumorigenic. Small molecule inhibition of ERK5 has been shown to have promising activity in cell and animal models of inflammation and oncology. Here we report the synthesis and biological characterization of potent, selective ERK5 inhibitors. In contrast to both genetic depletion/deletion of ERK5 and inhibition with previously reported compounds, inhibition of the kinase with the most selective of the new inhibitors had no antiinflammatory or antiproliferative activity. The source of efficacy in previously reported ERK5 inhibitors is shown to be off-target activity on bromodomains, conserved protein modules involved in recognition of acetyl-lysine residues during transcriptional processes. It is likely that phenotypes reported from genetic deletion or depletion of ERK5 arise from removal of a noncatalytic function of ERK5. The newly reported inhibitors should be useful in determining which of the many reported phenotypes are due to kinase activity and delineate which can be pharmacologically targeted.

Chemoproteomic Evaluation of Target Engagement by the Cyclin-Dependent Kinase 4 and 6 Inhibitor Palbociclib Correlates with Cancer Cell Response.

Palbociclib is a cyclin-dependent kinase (CDK) 4/CDK6 inhibitor approved for breast cancer that is estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative. We profiled palbociclib in cells either sensitive or resistant to the drug using an ATP/ADP probe-based chemoproteomics platform. Palbociclib only engaged CDK4 or CDK6 in sensitive cells. In resistant cells, no inhibition of CDK4 or CDK6 was observed, although the off-target profiles were similar in both cell types. Prolonged incubation of sensitive cells with the compound (24 h) resulted in the downregulation of additional kinases, including kinases critical for cell cycle progression. This downregulation is consistent with cell cycle arrest caused by palbociclib treatment. Both the direct and indirect targets were also observed in a human tumor xenograft study using the COLO-205 cell line in which phosphorylation of the retinoblastoma protein was tracked as the pharmacodyanamic marker. Together, these results suggest that this probe-based approach could be an important strategy toward predicting patient responsiveness to palbociclib.

ATP Acyl Phosphate Reactivity Reveals Native Conformations of Hsp90 Paralogs and Inhibitor Target Engagement.

Hsp90 is an ATP-dependent chaperone of widespread interest as a drug target. Here, using an LC-MS/MS chemoproteomics platform based on a lysine-reactive ATP acyl phosphate probe, several Hsp90 inhibitors were profiled in native cell lysates. Inhibitor specificities for all four human paralogs of Hsp90 were simultaneously monitored at their endogenous relative abundances. Equipotent inhibition of probe labeling in each paralog occurred at sites both proximal to and distal from bound ATP observed in Hsp90 cocrystal structures, suggesting that the ATP probe is assaying a native conformation not predicted by available structures. Inhibitor profiling against a comprehensive panel of protein kinases and other ATP-binding proteins detected in native cell lysates identified PMS2, a member of the GHKL ATPase superfamily as an off-target of NVP-AUY922 and radicicol. Because of the endogenously high levels of Hsp90 paralogs in typical cell lysates, the measured potency of inhibitors was weaker than published IC50 values. Significant inhibition of Hsp90 required inhibitor concentrations above a threshold where off-target activity was detectable. Direct on- and off-target engagement was measured by profiling lysates derived from cells treated with Hsp90 inhibitors. These studies also assessed the downstream cellular pathway effects of Hsp90 inhibition, including the down regulation of several known Hsp90 client proteins and some previously unknown client proteins. Overall, the ATP probe-based assay methodology enabled a broad characterization of Hsp90 inhibitor activity and specificity in native cell lysates.

Monitoring native p38α:MK2/3 complexes via trans delivery of an ATP acyl phosphate probe.

Here we describe a chemical proteomics strategy using ATP acyl phosphates to measure the formation of a protein:protein complex between p38α and mapkap kinases 2 and/or 3. Formation of the protein:protein complex results in a new probe labeling site on p38α that can be used to quantify the extent of interaction in cell lysates and the equilibrium binding constant for the interaction in vitro. We demonstrate through RNA interference that the labeling site is dependent on formation of the protein:protein complex in cells. Further, we identify that active-site-directed, small-molecule inhibitors of MK2/3 selectively inhibit the heterodimer-dependent probe labeling, whereas p38α inhibitors do not. These findings afford a new method to evaluate p38α and MK2/3 inhibitors within native biological systems and a new tool for improved understanding of p38α signaling pathways.

Amides of 4-hydroxy-8-methanesulfonylamino-quinoline-2-carboxylic acid as zinc-dependent inhibitors of Lp-PLA2.

AX10479, the phenyl amide of 4-hydroxy-8-methanesulfonylamino-quinoline-2-carboxylic acid, was identified as a Zn(2+)-dependent, 27nM inhibitor of human plasma Lp-PLA(2). Structure-activity relationship studies focused on the AX10479 2-phenylamide group identified equipotent cycloaliphatic amides, an enantioselective preference for chiral amides, and phenyl substitution patterns (e.g., 2-methyl-3-fluoro) that increased potency.

Hit-to-lead optimization and kinase selectivity of imidazo[1,2-a]quinoxalin-4-amine derived JNK1 inhibitors.

As the result of a rhJNK1 HTS, the imidazo[1,2-a]quinoxaline 1 was identified as a 1.6 µM rhJNK1 inhibitor. Optimization of this compound lead to AX13587 (rhJNK1 IC50=160 nM) which was co-crystallized with JNK1 to identify key molecular interactions. Kinase profiling against 125+ kinases revealed AX13587 was an inhibitor of JNK, MAST3, and MAST4 whereas its methylene homolog AX14373 (native JNK1 IC50=47 nM) was a highly specific JNK inhibitor.

6-Position optimization of tricyclic 4-quinolone-based inhibitors of glycogen synthase kinase-3ß: discovery of nitrile derivatives with picomolar potency.

We previously disclosed tricylic, 6-carboxylic acid-bearing 4-quinolones as GSK-3ß inhibitors. Herein we discuss the optimization of this series to yield a series of more potent 6-nitrile analogs with insignificant anti-microbial activity. Finally, kinase profiling indicated that members of this class were highly specific GSK-3 inhibitors.

Amides of xanthurenic acid as zinc-dependent inhibitors of Lp-PLA(2).

AX10185, the phenyl amide of xanthurenic acid, was found to be a sub-100nM inhibitor of Lp-PLA(2). However, in the presence of EDTA the inhibitory activity of AX10185 was extinguished while the enzymatic activity of Lp-PLA(2) did not change. Subsequent metal screening experiments determined the inhibition to be Zn(2+) dependent. Structure-activity relationship studies indicated the presence of the 4-hydroxy group to be critical and selected substituted phenyl, polycyclic, and cycloaliphatic amides of xanthurenic acid to be well tolerated.

Synthesis and structure-activity relationship of (1-halo-2-naphthyl) carbamate-based inhibitors of KIAA1363 (NCEH1/AADACL1).

KIAA1363 is a serine hydrolase whose activity has been shown to be positively associated with tumor cell invasiveness. Thus, inhibitors of KIAA1363 represent a novel targeted therapy approach towards cancer. AX11890 ((1-bromo-2-naphthyl) N,N-dimethylcarbamate) was identified as a KIAA1363 inhibitor with an IC(50) value of 1.2 µM and was shown using ESI-MS to carbamylate the catalytic residue Ser(191). SAR studies explored both substitution of the 1-bromo group and derivatization of the 6-position. Activity-based protein profiling demonstrated AX13057 inhibited tumor-localized KIAA1363 in SK-OV-3 xenograft-bearing mice.

Synthesis and structure-activity relationship of 4-quinolone-3-carboxylic acid based inhibitors of glycogen synthase kinase-3ß.

The synthesis, GSK-3ß inhibitory activity, and anti-microbial activity of bicyclic and tricyclic derivatives of the 5,7-diamino-6-fluoro-4-quinolone-3-carboxylic acid scaffold were studied. Kinase selectivity profiling indicated that members of this class were potent and highly selective GSK-3 inhibitors.

S28 peptidases: lessons from a seemingly 'dysfunctional' family of two.

A recent paper in BMC Structural Biology reports the crystal structure of human prolylcarboxypeptidase (PRCP), one of the two members of the S28 peptidase family. Comparison of the substrate-binding site of PRCP with that of its family partner, dipeptidyl dipeptidase 7 (DPP7), helps to explain the different enzymatic activities of these structurally similar proteins, and also reveals a novel apparent charge-relay system in PRCP involving the active-site catalytic histidine. See research article: http://www.biomedcentral.com/1472-6807/10/16/

Synthesis and optimization of 2-pyridin-3-yl-benzo[d][1,3]oxazin-4-one based inhibitors of human neutrophil elastase.

The hit-to-lead optimization of the HNE inhibitor 5-methyl-2-(2-phenoxy-pyridin-3-yl)-benzo[d][1,3]oxazin-4-one is described. A structure-activity relationship study that focused on the 5 and 7 benzoxazinone positions yielded the optimized 5-ethyl-7-methoxy-benzo[d][1,3]oxazin-4-one core structure. 2-[2-(4-Methyl-piperazin-1-yl)-pyridin-3-yl] derivatives of this core were shown to yield HNE inhibitors of similar potency with significantly different stabilities in rat plasma.

High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity.

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 µM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.

Erratum: Author Correction: High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity.

[This corrects the article DOI: 10.1038/s42003-019-0587-z.].

New LSD therapies unfolding.

NIH researchers report results of a qHTS hunt for new chemical chaperones for the treatment of Gaucher disease.

Design and synthesis of AX4697, a bisindolylmaleimide exo-affinity probe that labels protein kinase C alpha and beta.

The synthesis and biochemical characterization of AX4697, a fluorescent, bisindolylmaleimide-derived probe for PKCalpha and beta, is described. AX4697 was able to quantify changes in PKC expression in drug-treated Jurkat cells and was shown to covalently label PKCalpha on C619, a residue that sits just outside the active site.