Crystal structure of active CDK4-cyclin D and mechanistic basis for abemaciclib efficacy.

Despite the biological and therapeutic relevance of CDK4/6 for the treatment of HR+, HER2- advanced breast cancer, the detailed mode of action of CDK4/6 inhibitors is not completely understood. Of particular interest, phosphorylation of CDK4 at T172 (pT172) is critical for generating the active conformation, yet no such crystal structure has been reported to date. We describe here the x-ray structure of active CDK4-cyclin D3 bound to the CDK4/6 inhibitor abemaciclib and discuss the key aspects of the catalytically-competent complex. Furthermore, the effect of CDK4/6 inhibitors on CDK4 T172 phosphorylation has not been explored, despite its role as a potential biomarker of CDK4/6 inhibitor response. We show mechanistically that CDK4/6i stabilize primed (pT172) CDK4-cyclin D complex and selectively displace p21 in responsive tumor cells. Stabilization of active CDK4-cyclin D1 complex can lead to pathway reactivation following alternate dosing regimen. Consequently, sustained binding of abemaciclib to CDK4 leads to potent cell cycle inhibition in breast cancer cell lines and prevents rebound activation of downstream signaling. Overall, our study provides key insights demonstrating that prolonged treatment with CDK4/6 inhibitors and composition of the CDK4/6-cyclin D complex are both critical determinants of abemaciclib efficacy, with implications for this class of anticancer therapy.

Computational stabilization of T cell receptors allows pairing with antibodies to form bispecifics.

Recombinant T cell receptors (TCRs) can be used to redirect naïve T cells to eliminate virally infected or cancerous cells; however, they are plagued by low stability and uneven expression. Here, we use molecular modeling to identify mutations in the TCR constant domains (Cα/Cß) that increase the unfolding temperature of Cα/Cß by 20 °C, improve the expression of four separate α/ß TCRs by 3- to 10-fold, and improve the assembly and stability of TCRs with poor intrinsic stability. The stabilizing mutations rescue the expression of TCRs destabilized through variable domain mutation. The improved stability and folding of the TCRs reduces glycosylation, perhaps through conformational stabilization that restricts access to N-linked glycosylation enzymes. The Cα/Cß mutations enables antibody-like expression and assembly of well-behaved bispecific molecules that combine an anti-CD3 antibody with the stabilized TCR. These TCR/CD3 bispecifics can redirect T cells to kill tumor cells with target HLA/peptide on their surfaces in vitro.

Comparison of the essential cellular functions of the two murA genes of Bacillus anthracis.

Targeted antisense and gene replacement mutagenesis experiments demonstrate that only the murA1 gene and not the murA2 gene is required for the normal cellular growth of Bacillus anthracis. Antisense-based modulation of murA1 gene expression hypersensitizes cells to the MurA-specific antibiotic fosfomycin despite the normally high resistance of B. anthracis to this drug.

Structure-based design of new dihydrofolate reductase antibacterial agents: 7-(benzimidazol-1-yl)-2,4-diaminoquinazolines.

A new series of dihydrofolate reductase (DHFR) inhibitors, the 7-(benzimidazol-1-yl)-2,4-diaminoquinazolines, were designed and optimized for antibacterial potency and enzyme selectivity. The most potent inhibitors in this series contained a five-membered heterocycle at the 2-position of the benzimidazole, leading to highly potent and selective compounds that exploit the differences in the size of a binding pocket adjacent to the NADPH cofactor between the bacterial and human DHFR enzymes. Typical of these compounds is 7-((2-thiazol-2-yl)benzimidazol-1-yl)-2,4 diaminoquinazoline, which is a potent inhibitor of S. aureus DHFR (Ki = 0.002 nM) with 46700-fold selectivity over human DHFR. This compound also has high antibacterial potency on Gram-positive bacteria with an MIC versus wild type S. aureus of 0.0125 µg/mL and a MIC versus trimethoprim-resistant S. aureus of 0.25 µg/mL. In vivo efficacy versus a S. aureus septicemia was demonstrated, highlighting the potential of this new series.

Identification of bacteria-selective threonyl-tRNA synthetase substrate inhibitors by structure-based design.

A series of potent and bacteria-selective threonyl-tRNA synthetase (ThrRS) inhibitors have been identified using structure-based drug design. These compounds occupied the substrate binding site of ThrRS and showed excellent binding affinities for all of the bacterial orthologues tested. Some of the compounds displayed greatly improved bacterial selectivity. Key residues responsible for potency and bacteria/human ThrRS selectivity have been identified. Antimicrobial activity has been achieved against wild-type Haemophilus influenzae and efflux-deficient mutants of Escherichia coli and Burkholderia thailandensis.

Evaluation of the metS and murB loci for antibiotic discovery using targeted antisense RNA expression analysis in Bacillus anthracis.

The biowarfare-relevant bacterial pathogen Bacillus anthracis contains two paralogs each of the metS and murB genes, which encode the important antibiotic target functions methionyl-tRNA synthetase and UDP-N-acetylenolpyruvoylglucosamine reductase, respectively. Empirical screens were conducted to detect and characterize gene fragments of each of these four genes that could cause growth reduction of B. anthracis when inducibly expressed from a plasmid-borne promoter. Numerous such gene fragments that were overwhelmingly in the antisense orientation were identified for the metS1 and murB2 alleles, while no such orientation bias was seen for the metS2 and murB1 alleles. Gene replacement mutagenesis was used to confirm the essentiality of the metS1 and murB2 alleles, and the nonessentiality of the metS2 and murB1 alleles, for vegetative growth. Induced transcription of RNA from metS1 and murB2 antisense-oriented gene fragments resulted in specific reduction of mRNA of their cognate genes. Attenuation of MetS1 enzyme expression hypersensitized B. anthracis cells to a MetS-specific antimicrobial compound but not to other antibiotics that affect cell wall assembly, fatty acid biosynthesis, protein translation, or DNA replication. Antisense-dependent reduction of MurB2 enzyme expression caused hypersensitivity to beta-lactam antibiotics, a synergistic response that has also been noted for the MurA-specific antibiotic fosfomycin. These experiments form the basis of mode-of-action detection assays that can be used in the discovery of novel MetS- or MurB-specific antibiotic drugs that are effective against B. anthracis or other gram-positive bacterial pathogens.

Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase.

B(12)-dependent methionine synthase (MetH) is a large modular enzyme that utilizes the cobalamin cofactor as a methyl donor or acceptor in three separate reactions. Each methyl transfer occurs at a different substrate-binding domain and requires a different arrangement of modules. In the catalytic cycle, the cobalamin-binding domain carries methylcobalamin to the homocysteine (Hcy) domain to form methionine and returns cob(I)alamin to the folate (Fol) domain for remethylation by methyltetrahydrofolate (CH(3)-H(4)folate). Here, we describe crystal structures of a fragment of MetH from Thermotoga maritima comprising the domains that bind Hcy and CH(3)-H(4)folate. These substrate-binding domains are (beta alpha)(8) barrels packed tightly against one another with their barrel axes perpendicular. The properties of the domain interface suggest that the two barrels remain associated during catalysis. The Hcy and CH(3)-H(4)folate substrates are bound at the C termini of their respective barrels in orientations that position them for reaction with cobalamin, but the two active sites are separated by approximately 50 A. To complete the catalytic cycle, the cobalamin-binding domain must travel back and forth between these distant active sites.

Crystal structures of active fully assembled substrate- and product-bound complexes of UDP-N-acetylmuramic acid:L-alanine ligase (MurC) from Haemophilus influenzae.

UDP-N-acetylmuramic acid:L-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg(2+) and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-L-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn(2+) have been determined to 1.85- and 1.7-A resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the gamma-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates.