Novel druggable space in human KRAS G13D discovered using structural bioinformatics and a P-loop targeting monoclonal antibody.

KRAS belongs to a family of small GTPases that act as binary switches upstream of several signalling cascades, controlling proliferation and survival of cells. Mutations in KRAS drive oncogenesis, especially in pancreatic, lung, and colorectal cancers (CRC). Although historic attempts at targeting mutant KRAS with small molecule inhibitors have proven challenging, there are recent successes with the G12C, and G12D mutations. However, clinically important RAS mutations such as G12V, G13D, Q61L, and A146T, remain elusive drug targets, and insights to their structural landscape is of critical importance to develop novel, and effective therapeutic concepts. We present a fully open, P-loop exposing conformer of KRAS G13D by X-ray crystallography at 1.4-2.4 Å resolution in Mg2+-free phosphate and malonate buffers. The G13D conformer has the switch-I region displaced in an upright position leaving the catalytic core fully exposed. To prove that this state is druggable, we developed a P-loop-targeting monoclonal antibody (mAb). The mAb displayed high-affinity binding to G13D and was shown using high resolution fluorescence microscopy to be spontaneously taken up by G13D-mutated HCT 116 cells (human CRC derived) by macropinocytosis. The mAb inhibited KRAS signalling in phosphoproteomic and genomic studies. Taken together, the data propose novel druggable space of G13D that is reachable in the cellular context. It is our hope that these findings will stimulate attempts to drug this fully open state G13D conformer using mAbs or other modalities.

Blockade of KAT II Facilitates LTP in Kynurenine 3-Monooxygenase Depleted Mice.

Excess of brain kynurenic acid (KYNA), a neuroactive metabolite of the kynurenine pathway, is known to elicit cognitive dysfunction. In the present study, we investigated spatial working memory in mice with elevated levels of KYNA, induced by targeted deletion of kynurenine 3-monooxygenase (KMO), as well as long-term potentiation (LTP) of field excitatory postsynaptic potentials (fEPSPs) in hippocampal brain slices from these mice. The KMO knock-out (KMO-/-) mice performed more poorly in the spatial working memory task as compared to their wild-type (WT) counterparts, as reflected by fewer correct choices in a T-maze. Both fEPSPs, or LTP, did not significantly differ between the 2 mouse strains. However, administration of PF-04859989, a kynurenine aminotransferase (KAT) II inhibitor, limiting the production of KYNA, facilitated fEPSP and enhanced LTP to a greater extent in hippocampal slices from KMO-/- mice compared to WT mice. The results of the present study point to an essential role for KYNA in modulating LTP in the hippocampus of KMO-/- mice which may account for their dysfunctional spatial working memory.

Rational antibody design for undruggable targets using kinetically controlled biomolecular probes.

Several important drug targets, e.g., ion channels and G protein-coupled receptors, are extremely difficult to approach with current antibody technologies. To address these targets classes, we explored kinetically controlled proteases as structural dynamics-sensitive druggability probes in native-state and disease-relevant proteins. By using low-Reynolds number flows, such that a single or a few protease incisions are made, we could identify antibody binding sites (epitopes) that were translated into short-sequence antigens for antibody production. We obtained molecular-level information of the epitope-paratope region and could produce high-affinity antibodies with programmed pharmacological function against difficult-to-drug targets. We demonstrate the first stimulus-selective monoclonal antibodies targeting the transient receptor potential vanilloid 1 (TRPV1) channel, a clinically validated pain target widely considered undruggable with antibodies, and apoptosis-inducing antibodies selectively mediating cytotoxicity in KRAS-mutated cells. It is our hope that this platform will widen the scope of antibody therapeutics for the benefit of patients.

Peripheral and central levels of kynurenic acid in bipolar disorder subjects and healthy controls.

Metabolites of the kynurenine pathway of tryptophan degradation, in particular, the N-Methyl-D-aspartic acid receptor antagonist kynurenic acid (KYNA), are increasingly recognized as primary pathophysiological promoters in several psychiatric diseases. Studies analyzing central KYNA levels from subjects with psychotic disorders have reported increased levels. However, sample sizes are limited and in contrast many larger studies examining this compound in blood from psychotic patients commonly report a decrease. A major question is to what extent peripheral KYNA levels reflect brain KYNA levels under physiological as well as pathophysiological conditions. Here we measured KYNA in plasma from a total of 277 subjects with detailed phenotypic data, including 163 BD subjects and 114 matched healthy controls (HCs), using an HPLC system. Among them, 94 BD subjects and 113 HCs also had CSF KYNA concentrations analyzed. We observe a selective increase of CSF KYNA in BD subjects with previous psychotic episodes although this group did not display altered plasma KYNA levels. In contrast, BD subjects with ongoing depressive symptoms displayed a tendency to decreased plasma KYNA concentrations but unchanged CSF KYNA levels. Sex and age displayed specific effects on KYNA concentrations depending on if measured centrally or in the periphery. These findings implicate brain-specific regulation of KYNA under physiological as well as under pathophysiological conditions and strengthen our previous observation of CSF KYNA as a biomarker in BD. In summary, biomarker and drug discovery studies should include central KYNA measurements for a more reliable estimation of brain KYNA levels.

Electroconvulsive therapy suppresses the neurotoxic branch of the kynurenine pathway in treatment-resistant depressed patients.

BACKGROUND: Neuroinflammation is increasingly recognized as contributing to the pathogenesis of depression. Key inflammatory markers as well as kynurenic acid (KYNA) and quinolinic acid (QUIN), both tryptophan metabolites, have been associated with depressive symptoms and suicidality. The aim of the present study is to investigate the peripheral concentration of cytokines and tryptophan and kynurenine metabolites in patients with unipolar treatment-resistant depression before and after electroconvulsive therapy (ECT), the most effective treatment for depression. METHODS: Cytokines in plasma from patients with major depressive disorder (MDD; n = 19) and healthy volunteers (n = 14) were analyzed with electrochemiluminescence detection. Tryptophan and kynurenine metabolites were detected with high-performance liquid chromatography (HPLC) and LC/MS. KYNA was analyzed in a second healthy control cohort (n = 22). RESULTS: Patients with MDD had increased plasma levels of interleukin (IL)-6 compared to healthy volunteers (P < 0.05). We also found an altered kynurenine metabolism in these patients displayed by decreased plasma levels of KYNA (P < 0.0001) as well as a significantly increased QUIN/KYNA ratio (P < 0.001). Plasma levels of tryptophan, kynurenine, and QUIN did not differ between patients and controls. Treatment with ECT was associated with a significant decrease in the plasma levels of tryptophan (P < 0.05), kynurenine (P < 0.01), and QUIN (P < 0.001), whereas plasma levels of KYNA did not change. The QUIN/KYNA ratio was found to significantly decrease in ECT-treated patients (P < 0.05). There was a significant inverse correlation between symptom severity and kynurenine levels at baseline (r = -0.67, P = 0.002). CONCLUSIONS: This study confirms an imbalanced kynurenine pathway in MDD supporting the hypothesis of a netstimulation of N-methyl-D-aspartic acid (NMDA) receptors in the disorder. Treatment with ECT profoundly decreased QUIN, an NMDA-receptor agonist previously suggested to be implicated in the pathogenesis of depression, an effect that might have bearing for the good clinical outcome of ECT.