Synthesis and biological evaluation of solubilized sulfonamide analogues of the phosphatidylinositol 3-kinase inhibitor ZSTK474.

Replacing one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 with a variety of sulfonamide-linked solubilizing substituents produced a new class of active and potent PI3Kα inhibitors, with several derivatives demonstrating high PI3Kα enzyme potency and good cellular potency in two human derived cell lines. The overall results suggest a preference for linear and somewhat flexible solubilizing functions. From this series, compound 16, also known as SN32976, was selected for advanced preclinical evaluation.

Combining properties of different classes of PI3Kα inhibitors to understand the molecular features that confer selectivity.

Phosphoinositide 3-kinases (PI3Ks) are major regulators of many cellular functions, and hyperactivation of PI3K cell signalling pathways is a major target for anticancer drug discovery. PI3Kα is the isoform most implicated in cancer, and our aim is to selectively inhibit this isoform, which may be more beneficial than concurrent inhibition of all Class I PI3Ks. We have used structure-guided design to merge high-selectivity and high-affinity characteristics found in existing compounds. Molecular docking, including the prediction of water-mediated interactions, was used to model interactions between the ligands and the PI3Kα affinity pocket. Inhibition was tested using lipid kinase assays, and active compounds were tested for effects on PI3K cell signalling. The first-generation compounds synthesized had IC50 (half maximal inhibitory concentration) values >4 µM for PI3Kα yet were selective for PI3Kα over the other Class I isoforms (ß, δ and γ). The second-generation compounds explored were predicted to better engage the affinity pocket through direct and water-mediated interactions with the enzyme, and the IC50 values decreased by ∼30-fold. Cell signalling analysis showed that some of the new PI3Kα inhibitors were more active in the H1047R mutant bearing cell lines SK-OV-3 and T47D, compared with the E545K mutant harbouring MCF-7 cell line. In conclusion, we have used a structure-based design approach to combine features from two different compound classes to create new PI3Kα-selective inhibitors. This provides new insights into the contribution of different chemical units and interactions with different parts of the active site to the selectivity and potency of PI3Kα inhibitors.

Synthesis and biological evaluation of sulfonamide analogues of the phosphatidylinositol 3-kinase inhibitor ZSTK474.

Replacement of one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 (1) with sulfonamide containing substituents produced a new class of active and potent PI3Kα inhibitors. Solubility issues prevented all but the 6-amino derivative 17 from being evaluated in vivo, but the clear activity of this compound demonstrated that this class of PI3K inhibitor shows great promise.

Preptin Deficiency Does Not Protect against High-Fat Diet-Induced Metabolic Dysfunction or Bone Loss in Mice.

Preptin is derived from the cleavage of the E-peptide of pro-insulin-like growth factor (IGF)-II and is an insulin secretagogue. Observational studies have linked elevated circulating preptin to metabolic dysfunction in humans; however, a causal role for preptin in metabolic dysfunction has not been established. Additionally, preptin can promote osteoblast proliferation and differentiation, suggesting a link with skeletal health. We previously described a global preptin knockout (KO) model. In this study, we sought to uncover the impact of preptin KO in mice on the response to a moderately high-fat diet (HFD) and low-fat diet (LFD). HFD groups had higher weight and fat mass gain, lower trabecular and cortical bone volume and fracture load, and higher liver triglycerides. In males, preptin deficiency led to lower blood glucose than wild-type (WT) mice under LFD conditions. This was accompanied by differences in bone microarchitecture, including lower trabecular bone volume fraction, trabecular number, and lower cortical thickness. These differences were absent in female mice, although KO females had a HFD-driven increase in fat mass and liver triglycerides that was absent in WT mice. Female WT mice had increased glucose-stimulated insulin secretion under HFD conditions that was absent in female KO mice. Overall, preptin may have a detrimental impact on metabolism and a positive impact on bone health in male mice and may protect against liver fat storage in females while enabling islet compensation under HFD conditions. When we consider that serum preptin levels are elevated in humans of both sexes in pathological states in which insulin levels are elevated, the impact of preptin on comorbidity risk needs to be better understood. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

DMXAA (Vadimezan, ASA404) is a multi-kinase inhibitor targeting VEGFR2 in particular.

The flavone acetic acid derivative DMXAA [5,6-dimethylXAA (xanthenone-4-acetic acid), Vadimezan, ASA404] is a drug that displayed vascular-disrupting activity and induced haemorrhagic necrosis and tumour regression in pre-clinical animal models. Both immune-mediated and non-immune-mediated effects contributed to the tumour regression. The vascular disruption was less in human tumours, with immune-mediated effects being less prominent, but nonetheless DMXAA showed promising effects in Phase II clinical trials in non-small-cell lung cancer. However, these effects were not replicated in Phase III clinical trials. It has been difficult to understand the differences between the pre-clinical findings and the later clinical trials as the molecular targets for the agent have never been clearly established. To investigate the mechanism of action, we sought to determine whether DMXAA might target protein kinases. We found that, at concentrations achieved in blood during clinical trials, DMXAA has inhibitory effects against several kinases, with most potent effects being on members of the VEGFR (vascular endothelial growth factor receptor) tyrosine kinase family. Some analogues of DMXAA were even more effective inhibitors of these kinases, in particular 2-MeXAA (2-methylXAA) and 6-MeXAA (6-methylXAA). The inhibitory effects were greatest against VEGFR2 and, consistent with this, we found that DMXAA, 2-MeXAA and 6-MeXAA were able to block angiogenesis in zebrafish embryos and also inhibit VEGFR2 signalling in HUVECs (human umbilical vein endothelial cells). Taken together, these results indicate that at least part of the effects of DMXAA are due to it acting as a multi-kinase inhibitor and that the anti-VEGFR activity in particular may contribute to the non-immune-mediated effects of DMXAA on the vasculature.

A brief relaxation intervention reduces stress and improves surgical wound healing response: a randomised trial.

Psychological stress has been shown to impair wound healing, but experimental research in surgical patients is lacking. This study investigated whether a brief psychological intervention could reduce stress and improve wound healing in surgical patients. This randomised controlled trial was conducted at a surgical centre. Inclusion criteria were English-speaking patients over 18 years booked to undergo elective laparoscopic cholecystectomy; exclusion criteria were cancellation of surgery, medical complications, and refusal of consent. Seventy five patients were randomised and 15 patients were excluded; 60 patients completed the study (15 male, 45 female). Participants were randomised to receive standard care or standard care plus a 45-min psychological intervention that included relaxation and guided imagery with take-home relaxation CDs for listening to for 3 days before and 7 days after surgery. In both groups ePTFE tubes were inserted during surgery and removed at 7 days after surgery and analysed for hydroxyproline as a measure of collagen deposition and wound healing. Change in perceived stress from before surgery to 7-day follow-up was assessed using questionnaires. Intervention group patients showed a reduction in perceived stress compared with the control group, controlling for age. Patients in the intervention group had higher hydroxyproline deposition in the wound than did control group patients (difference in means 0.35, 95% CI 0.66-0.03; t(43)=2.23, p=0.03). Changes in perceived stress were not associated with hydroxyproline deposition. A brief relaxation intervention prior to surgery can reduce stress and improve the wound healing response in surgical patients. The intervention may have particular clinical application for those at risk of poor healing following surgery.

Response to BRAF-targeted Therapy Is Enhanced by Cotargeting VEGFRs or WNT/ß-Catenin Signaling in BRAF-mutant Colorectal Cancer Models.

The fact that 10% of colorectal cancer tumors harbor BRAF V600E mutations suggested targeting BRAF as a potential therapy. However, BRAF inhibitors have only limited single-agent efficacy in this context. The potential for combination therapy has been shown by the BEACON trial where targeting the EGF receptor with cetuximab greatly increased efficacy of BRAF inhibitors in BRAF-mutant colorectal cancer. Therefore, we explored whether efficacy of the mutant BRAF inhibitor vemurafenib could be enhanced by cotargeting of either oncogenic WNT/ß-catenin signaling or VEGFR signaling. We find the WNT/ß-catenin inhibitors pyrvinium, ICG-001 and PKF118-310 attenuate growth of colorectal cancer cell lines in vitro with BRAF-mutant lines being relatively more sensitive. Pyrvinium combined with vemurafenib additively or synergistically attenuated growth of colorectal cancer cell lines in vitro. The selective and potent VEGFR inhibitor axitinib was most effective against BRAF-mutant colorectal cancer cell lines in vitro, but the addition of vemurafenib did not significantly increase these effects. When tested in vivo in animal tumor models, both pyrvinium and axitinib were able to significantly increase the ability of vemurafenib to attenuate tumor growth in xenografts of BRAF-mutant colorectal cancer cells. The magnitude of these effects was comparable with that induced by a combination of vemurafenib and cetuximab. This was associated with additive effects on release from tumor cells and tumor microenvironment cell types of substances that would normally aid tumor progression. Taken together, these preclinical data indicate that the efficacy of BRAF inhibitor therapy in colorectal cancer could be increased by cotargeting either WNT/ß-catenin or VEGFRs with small-molecule inhibitors.

The chaperone proteins HSP70, HSP40/DnaJ and GRP78/BiP suppress misfolding and formation of ß-sheet-containing aggregates by human amylin: a potential role for defective chaperone biology in Type 2 diabetes.

Misfolding of the islet ß-cell peptide hA (human amylin) into ß-sheet-containing oligomers is linked to ß-cell apoptosis and the pathogenesis of T2DM (Type 2 diabetes mellitus). In the present study, we have investigated the possible effects on hA misfolding of the chaperones HSP (heat-shock protein) 70, GRP78/BiP (glucose-regulated protein of 78 kDa/immunoglobulin heavy-chain-binding protein) and HSP40/DnaJ. We demonstrate that hA underwent spontaneous time-dependent ß-sheet formation and aggregation by thioflavin-T fluorescence in solution, whereas rA (rat amylin) did not. HSP70, GRP78/BiP and HSP40/DnaJ each independently suppressed hA misfolding. Maximal molar protein/hA ratios at which chaperone activity was detected were 1:200 (HSP70, HSP40/DnaJ and GRP78/BiP). By contrast, none of the chaperones modified the secondary structure of rA. hA, but not rA, was co-precipitated independently with HSP70 and GRP78/BiP by anti-amylin antibodies. As these effects occur at molar ratios consistent with chaperone binding to relatively rare misfolded hA species, we conclude that HSP70 and GRP78/BiP can detect and bind misfolded hA oligomers, thereby effectively protecting hA against bulk misfolding and irreversible aggregation. Defective ß-cell chaperone biology could contribute to hA misfolding and initiation of apoptosis in T2DM.

Evolution of Molecular Targets in Melanoma Treatment.

Melanoma is the deadliest type of skin cancers, accounting for more than 80% of skin cancer mortality. Although melanoma was known very early in the history of medicine, treatment for this disease had remained largely the same until very recently. Previous treatment options, including removal surgery and systemic chemotherapy, offered little benefit in extending the survival of melanoma patients. However, the last decade has seen breakthroughs in melanoma treatment, which all emerged following new insight into the oncogenic signaling of melanoma. This paper reviewed the evolution of drug targets for melanoma treatment based on the emergence of novel findings in the molecular signaling of melanoma. One of the findings that are most influential in melanoma treatment is that more than 50% of melanoma tumors contain BRAF mutations. This is fundamental for the development of BRAF inhibitors, which is the first group of drugs that significantly improves the overall survival of melanoma patients compared to the traditional chemotherapeutic dacarbazine. More recently, findings of the role of immune checkpoint molecules such as CTLA-4 and PD1/PD-L1 in melanoma biology have led to the development of a new therapeutic category: immune checkpoint inhibitors, which, for the first time in the history of cancer treatment, produced a durable response in a subset of melanoma patients. However, as this paper discussed next, there is still an unmet need for melanoma treatment. A significant population of patients did not respond to either BRAF inhibitors or immune checkpoint inhibitors. Of those patients who gained an initial response from those therapies, a remarkable percentage would develop drug resistance even when MEK inhibitors were added to the treatment. Finally, this paper discusses some possible targets for melanoma treatment.

For Better or Worse: The Potential for Dose Limiting the On-Target Toxicity of PI 3-Kinase Inhibitors.

The hyper-activation of the phosphoinositide (PI) 3-kinase signaling pathway is a hallmark of many cancers and overgrowth syndromes, and as a result, there has been intense interest in the development of drugs that target the various isoforms of PI 3-kinase. Given the key role PI 3-kinases play in many normal cell functions, there is significant potential for the disruption of essential cellular functions by PI 3-kinase inhibitors in normal tissues; so-called on-target drug toxicity. It is, therefore, no surprise that progress within the clinical development of PI 3-kinase inhibitors as single-agent anti-cancer therapies has been slowed by the difficulty of identifying a therapeutic window. The aim of this review is to place the cellular, tissue and whole-body effects of PI 3-kinase inhibition in the context of understanding the potential for dose limiting on-target toxicities and to introduce possible strategies to overcome these.

Biological characterization of SN32976, a selective inhibitor of PI3K and mTOR with preferential activity to PI3Kα, in comparison to established pan PI3K inhibitors.

Multiple therapeutic agents have been developed to target the phosphatidylinositol 3-kinase (PI3K) signaling pathway, which is frequently dysregulated in cancer promoting tumor growth and survival. These include pan PI3K inhibitors, which target class Ia PI3K isoforms and have largely shown limited single agent activity with narrow therapeutic windows in clinical trials. Here, we characterize SN32976, a novel pan PI3K inhibitor, for its biochemical potency against PI3K isoforms and mTOR, kinase selectivity, cellular activity, pharmacokinetics, pharmacodynamics and antitumor efficacy relative to five clinically-evaluated pan PI3K inhibitors: buparlisib, dactolisib, pictilisib, omipalisib and ZSTK474. SN32976 potently inhibited PI3K isoforms and mTOR, displaying preferential activity for PI3Kα and sparing of PI3Kδ relative to the other inhibitors, while showing less off-target activity than the clinical inhibitors in a panel of 442 kinases. The major metabolites of SN32976 were also potent PI3K inhibitors with similar selectivity for PI3Kα as the parent compound. SN32976 compared favorably with the clinically-evaluated PI3K inhibitors in cellular assays, inhibiting pAKT expression and cell proliferation at nM concentrations, and in animal models, inducing a greater extent and duration of pAKT inhibition in tumors than pictilisib, dactolisib and omipalisib at similarly tolerated dose levels and inhibiting tumor growth to a greater extent than dactolisib and ZSTK474 and with similar efficacy to pictilisib and omipalisib. These results suggest that SN32976 is a promising clinical candidate for cancer therapy with enhanced kinase selectivity and preferential inhibition of PI3Kα compared to first generation pan PI3K inhibitors, while retaining comparable anticancer activity.

Preptin analogues: chemical synthesis, secondary structure and biological studies.

Peptide hormones that modulate insulin secretion have been recognized to have therapeutic potential, with peptides such as amylin (pramlintide acetate, Symlin) and exendin-4 (exenatide, Byetta) now commercially available. Preptin is a peptide that has been shown to increase insulin secretion in vitro and in vivo. Here, we describe the first chemical synthesis and analysis of a short series of preptin analogues based on the rat preptin sequence. Phe 21 in the preptin sequence was substituted with the non-protein amino acids D-Phe, D-Hphe, 3-aminobenzoic acid and 1-aminocyclooctane-1-carboxylic acid, which rendered the preptin analogues resistant to chymotryptic protease hydrolysis at this position. Substitution of Phe 21 with these non-protein amino acids did not abrogate the insulin secretory effect of preptin, with analogues showing a similar dose-dependent effect on insulin secretion from ßTC6-F7 mouse ß-cells in both the presence and absence of glucose as unmodified rat preptin. Further studies on the stability of the preptin analogues and their effect on insulin secretion are in progress.

Enzyme activity effects of N-terminal His-tag attached to catalytic sub-unit of phosphoinositide-3-kinase.

NTT (N-terminal tags) on the catalytic (p110) sub-unit of PI 3-K (phosphoinositol 3-kinase) have previously been shown to increase cell signalling and oncogenic transformation. Here we test the impact of an NT (N-terminal) His-tag on in vitro lipid and protein kinase activity of all class-1 PI 3-K isoforms and two representative oncogenic mutant forms (E545K and H1047R), in order to elucidate the mechanisms behind this elevated signalling and transformation observed in vivo. Our results show that an NT His-tag has no impact on lipid kinase activity as measured by enzyme titration, kinetics and inhibitor susceptibility. Conversely, the NT His-tag did result in a differential effect on protein kinase activity, further potentiating the elevated protein kinase activity of both the helical domain and catalytic domain oncogenic mutants with relation to p110 phosphorylation. All other isoforms also showed elevated p110 phosphorylation (although not statistically significant). We conclude that the previously reported increase in cell signalling and oncogenic-like transformation in response to p110 NTT is not mediated via an increase in the lipid kinase activity of PI 3-K, but may be mediated by increased p110 autophosphorylation and/or other, as yet unidentified, intracellular protein/protein interactions. We further observe that tagged recombinant protein is suitable for use in in vitro lipid kinase screens to identify PI 3-K inhibitors; however, we recommend that in vivo (including intracellular) experiments and investigations into the protein kinase activity of PI 3-K should be conducted with untagged constructs.

Oncogenic mutations of p110α isoform of PI 3-kinase upregulate its protein kinase activity.

In addition to lipid kinase activity, the class-I PI 3-kinases also function as protein kinases targeting regulatory autophosphorylation sites and exogenous substrates. The latter include a recently identified regulatory phosphorylation of the GM-CSF/IL-3 ßc receptor contributing to survival of acute myeloid leukaemia cells. Previous studies suggested differences in the protein kinase activity of the 4 isoforms of class-I PI 3-kinase so we compared the ability of all class-I PI 3-kinases and 2 common oncogenic mutants to autophosphorylate, and to phosphorylate an intracellular fragment of the GM-CSF/IL-3 ßc receptor (ßic). We find p110α, p110ß and p110γ all phosphorylate ßic but p110δ is much less effective. The two most common oncogenic mutants of p110α, H1047R and E545K have stronger protein kinase activity than wildtype p110α, both in terms of autophosphorylation and towards ßic. Importantly, the lipid kinase activity of the oncogenic mutants is still inhibited by autophosphorylation to a similar extent as wildtype p110α. Previous evidence indicates the protein kinase activity of p110α is Mn(2+) dependent, casting doubt over its role in vivo. However, we show that the oncogenic mutants of p110α plus p110ß and p110γ all display significant activity in the presence of Mg(2+). Furthermore we demonstrate that some small molecule inhibitors of p110α lipid kinase activity (PIK-75 and A66) are equally effective against the protein kinase activity, but other inhibitors (e.g. wortmannin and TGX221) show different patterns of inhibition against the lipid and protein kinases activities. These findings have implications for the function of PI 3-kinase, especially in tumours carrying p110α mutations.

Synthesis and biological evaluation of novel phosphatidylinositol 3-kinase inhibitors: Solubilized 4-substituted benzimidazole analogs of 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474).

A range of 4-substituted derivatives of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di-(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) were prepared in a search for more soluble analogs. 4-Aminoalkoxy substituents provided the most potent derivatives, with the 4-O(CH2)3NMe2 analog (compound 14) being identified as displaying the best overall activity in combination with good aqueous solubility (25 mg/mL for the hydrochloride salt). This compound was tested in a U87MG xenograft model, but displayed less potency than ZSTK474 as a result of an unfavorable pharmacokinetic profile.

Synthesis and biological evaluation of novel analogues of the pan class I phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474).

A structure-activity relationship (SAR) study of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) identified substitution at the 4 and 6 positions of the benzimidazole ring as having significant effects on the potency of substituted derivatives. The 6-amino-4-methoxy analogue displayed a greater than 1000-fold potency enhancement over the corresponding 6-aza-4-methoxy analogue against all three class Ia PI 3-kinase enzymes (p110α, p110ß, and p110δ) and also displayed significant potency against two mutant forms of the p110α isoform (H1047R and E545K). This compound was also evaluated in vivo against a U87MG human glioblastoma tumor xenograft model in Rag1(-/-) mice, and at a dose of 50 mg/kg given by ip injection at a qd × 10 dosing schedule it dramatically reduced cancer growth by 81% compared to untreated controls.

A novel two-chain IGF-II-derived peptide from purified ß-cell granules.

OBJECTIVE: Insulin-like growth factor II (IGF-II) is a potent mitogen that regulates prenatal growth and development in both humans and rodents. Its role in post-natal life is less clear although immunohistochemical studies have observed IGF-II-like immunoreactivity (IGF-II-LI) associated with insulin-producing pancreatic ß-cells. Here we isolated secretory granules from a ß-cell line, ßTC6-F7, and characterized the nature of the IGF-II-LI located therein. DESIGN: Secretory granules were isolated from cultured mouse ßTC6-F7 cells by ultracentrifugation. Granule protein content was separated by reversed-phase HPLC, and assayed for IGF-II (radioimmunoassay) prior to identification by gas-phase NH(2)-terminal sequencing and MALDI-TOF MS. Effects of glucose incorporation into muscle glycogen were determined by incubating with isolated rat soleus muscle strips. RESULTS: ßTC6-F7 cells contained 60 ± 8 pmol of IGF-II-LI per 106 cells compared to 340 ± 44 pmol insulin-LI per 106 cells. IGF-II immunoreactive fractions were found to contain an IGF-II-like molecule with a molecular mass of 6847.6 Da. The protein was found to be a two-chain insulin-like product of Igf2 that corresponds to mouse des(37-40)IGF-II, which we termed 'vesiculin'. This molecule was also detectable in ßTC6-F7 cells by intact-cell mass spectrometry. Mouse vesiculin evoked concentration-dependent stimulation of muscle glycogen synthesis ex vivo with an EC(50) value of 131 nM ± 1.35. CONCLUSIONS: Vesiculin, des(37-40)IGF-II, is a novel two-chain insulin-like hormone and the major "IGF-II-like" peptide found in purified mouse ßTC6-F7 secretory granules. It stimulated ex vivo muscle glycogen synthesis with an efficacy greater than or equal to the intrinsic potency of IGF-II when compared to insulin derived from the same species.

Proteins associated with immunopurified granules from a model pancreatic islet beta-cell system: proteomic snapshot of an endocrine secretory granule.

beta-Cell granules contain proteins involved in fuel regulation, which when altered, contribute to metabolic disorders including diabetes mellitus. We analyzed proteins present in purified granules from the INS-1E beta-cell model. Fifty-one component proteins were identified by LC-MS/MS including hormones, granins, protein processing components, cellular trafficking components, enzymes implicated in cellular metabolism and chaperone proteins. These findings may increase understanding of granule secretion and the processes leading to protein aggregation and beta-cell death in type-2 diabetes.

The proteome of rodent mesenteric lymph.

Mesenteric lymph contributes to normal homeostasis and has an emerging role in the pathogenesis of multiple organ dysfunction syndrome. The aim of this study was to define the proteome of normal rodent mesenteric lymph in the fasted and fed states. Eight male Wistar rats fed a standard rodent diet were randomized to two groups. Group 1 (fasted, n = 4) were fasted for 24 h before anesthetized collection of mesenteric lymph. Group 2 (fed, n = 4) were allowed ad libitum access to food before lymph collection. Mesenteric lymph was subjected to proteomic analysis using iTRAQ and liquid chromatography-tandem mass spectrometry (LC-MS/MS). One hundred fifty proteins, including 26 hypothetical proteins, were identified in this study. All proteins were identified in lymph from both the fasted and fed states. The relative distribution profiles of protein functional classes in the mesenteric lymph differed significantly from that reported for plasma. The most abundant classes identified in lymph were protease inhibitors (16%) and proteins related to innate immunity (12%). In conclusion, this study provides the first detailed description of the normal mesenteric lymph proteome in the fed and fasted states using iTRAQ and LC-MS/MS.