Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse ...monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl
mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8
T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.
The PI3K/AKT/mTOR pathway has been shown to play an important role in cancer. Starting with compounds 1 and 2 (GDC-0941) as templates, (thienopyrimidin-2-yl)aminopyrimidines were discovered as potent ...inhibitors of PI3K or both PI3K and mTOR. Structural information derived from PI3Kγ−ligand cocrystal structures of 1 and 2 were used to design inhibitors that maintained potency for PI3K yet improved metabolic stability and oral bioavailability relative to 1. The addition of a single methyl group to the optimized 5 resulted in 21, which had significantly reduced potency for mTOR. The lead compounds 5 (GNE-493) and 21 (GNE-490) have good pharmacokinetic (PK) parameters, are highly selective, demonstrate knock down of pathway markers in vivo, and are efficacious in xenograft models where the PI3K pathway is deregulated. Both compounds were compared in a PI3Kα mutated MCF7.1 xenograft model and were found to have equivalent efficacy when normalized for exposure.
Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is a serine/threonine kinase implicated in the regulation of many biological processes. A fragment-based lead discovery approach was ...used to generate potent and selective MAP4K4 inhibitors. The fragment hit pursued in this article had excellent ligand efficiency (LE), an important attribute for subsequent successful optimization into drug-like lead compounds. The optimization efforts eventually led us to focus on the pyridopyrimidine series, from which 6-(2-fluoropyridin-4-yl)pyrido3,2-dpyrimidin-4-amine (29) was identified. This compound had low nanomolar potency, excellent kinase selectivity, and good in vivo exposure, and demonstrated in vivo pharmacodynamic effects in a human tumor xenograft model.
Polo-like kinase 1 (Plk1) is an attractive target for the development of anticancer agents due to its importance in regulating cell-cycle progression. Overexpression of Plk1 has been detected in a ...variety of cancers, and expression levels often correlate with poor prognosis. Despite high interest in Plk1-targeted therapeutics, there is currently no structure publicly available to guide structure-based drug design of specific inhibitors. We determined the crystal structures of the T210V mutant of the kinase domain of human Plk1 complexed with the nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMPPNP) or the pyrrolo-pyrazole inhibitor PHA-680626 at 2.4 and 2.1 Å resolution, respectively. Plk1 adopts the typical kinase domain fold and crystallized in a conformation resembling the active state of other kinases. Comparison of the kinetic parameters determined for the (unphosphorylated) wild-type enzyme, as well as the T210V and T210D mutants, shows that the mutations primarily affect the k cat of the reaction, with little change in the apparent K m for the protein or nucleotide substrates (k cat = 0.0094, 0.0376, and 0.0049 s-1 and K m(ATP) = 3.2, 4.0, and 3.0 μM for WT, T210D, and T210V, respectively). The structure highlights features of the active site that can be exploited to obtain Plk1-specific inhibitors with selectivity over other kinases and Plk isoforms. These include the presence of a phenylalanine at the bottom of the ATP pocket, combined with a cysteine (as opposed to the more commonly found leucine) in the roof of the binding site, a pocket created by Leu132 in the hinge region, and a cluster of positively charged residues in the solvent-exposed area outside of the adenine pocket adjacent to the hinge region.
Efforts to identify potent small molecule inhibitors of PI3 kinase and mTOR led to the evaluation of tetrasubstituted thienopyrimidines. These molecules generally have reduced in vivo clearance ...relative to the trisubstituted thienopyrimidines culminating in the identification of
GNE-477 (
8).
Efforts to identify potent small molecule inhibitors of PI3 kinase and mTOR led to the discovery of the exceptionally potent 6-aryl morpholino thienopyrimidine
6. In an effort to reduce the melting point in analogs of
6, the thienopyrimidine was modified by the addition of a methyl group to disrupt planarity. This modification resulted in a general improvement in in vivo clearance. This discovery led to the identification of
GNE-477 (
8), a potent and efficacious dual PI3K/mTOR inhibitor.
Interest in covalent drug discovery has surged in recent years, following the high-profile FDA approvals of covalent inhibitors that target BTK and KRAS G12C. High-throughput screening by intact ...protein mass spectrometry is a popular method for identifying lead matter from covalent fragment libraries. While the technique is proven in its capacity to confirm covalent binding, it does not provide binding site information on its own. Follow-up assays to identify binding sites can be time- and resource-intensive, potentially extending the hit confirmation timeline by weeks or months. Here, we describe the development of CoMPAS, a novel, targeted mass spectrometry-based covalent screening method that provides binding site information in the initial screen. The high sensitivity of targeted detection confers additional advantages over the intact protein method, including the ability to characterize more potent binders and reduced protein reagent requirements. Interpretation of the structure–activity relationship is simplified by enabling the use of binding site-specific EC50 values. To investigate higher-throughput screening beyond what is possible with standard liquid chromatography, we acquired data in parallel on an Agilent RapidFire system and compared the screening results by statistical analysis. To demonstrate the multiplexing capabilities of CoMPAS, we determined the target selectivity of screening hits against a pool of off-target kinases.
PROteolysis TArgeting Chimeras (PROTACs) promote the degradation, rather than inhibition, of a drug target as a mechanism for therapeutic treatment. Bifunctional PROTAC molecules allow simultaneous ...binding of both the target protein and an E3‐Ubiquitin ligase, bringing the two proteins into close spatial proximity to allow ubiquitinylation and degradation of the target protein via the cell's endogenous protein degradation pathway. We utilized native mass spectrometry (MS) to study the ternary complexes promoted by the previously reported PROTAC GNE‐987 between Brd4 bromodomains 1 and 2, and Von Hippel Lindeau E3‐Ubiquitin Ligase. Native MS at high resolution allowed us to measure ternary complex formation as a function of PROTAC concentration to provide a measure of complex affinity and stability, whilst simultaneously measuring other intermediate protein species. Native MS provides a high‐throughput, low sample consumption, direct screening method to measure ternary complexes for PROTAC development.
MS for PROTACS! Native mass spectrometry (MS) can measure ternary complexes between PROTACs, target proteins and E3‐ubiqutin ligases, together with other protein species present at equilibrium, with accurate mass. Stable ternary complexes are a requirement for PROTAC‐mediated target protein degradation. Native MS can be used as a high‐throughput screening technique to directly detect PROTAC ternary complexes that will result in efficient target degradation.
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