MALDI-TOF Mass Spectrometry-Based Assay for Measuring Covalent Target Engagement of KRAS G12C Inhibitors.

MALDI-TOF mass spectrometry enables high-throughput screening of covalent fragment libraries and SAR compound progressions of selective KRAS G12C inhibitors. Using the MALDI-TOF platform instead of the more traditional ESI-MS TOF/orbitrap instrumentation can radically shorten sample acquisition time, allowing up to 384 samples to be screened in 30 min. The typical throughput for a covalent library screen is 1152 samples per 8 h, including processing, calculation, and reporting steps. The throughput can be doubled without any significant assay modification.

High-Throughput Cell-Based Screening of Small Molecule KRAS Signaling Inhibitors Using a Homogeneous Time-Resolved Fluorescence (HTRF) Assay.

With recent advances proving that effective inhibition of KRAS is possible, there have been significant efforts made to develop inhibitors of specific mutant alleles. Here we describe a detailed protocol that employs homogeneous time-resolved fluorescence (HTRF) to identify compounds acting on KRAS signaling in malignant cell lines. This method allows for high-throughput, cell-based screens of large compound libraries for the development of RAS-targeted therapeutics.

FLAG-KRAS4B as a Model System for KRAS4B Proteoform and PTM Evaluation by Mass Spectrometry.

Prior analysis of intact and modified protein forms (proteoforms) of KRAS4B isolated from cell lines and tumor samples by top-down mass spectrometry revealed the presence of novel posttranslational modifications (PTMs) and potential evidence of context-specific KRAS4B modifications. However, low endogenous proteoform signal resulted in ineffective characterization, making it difficult to visualize less abundant PTMs or perform follow-up PTM validation using standard proteomic workflows. The NCI RAS Initiative has developed a model system, whereby KRAS4B bearing an N-terminal FLAG tag can be stably expressed within a panel of cancer cell lines. Herein, we present a method for combining immunoprecipitation with complementary proteomic methods to directly analyze N-terminally FLAG-tagged KRAS4B proteoforms and PTMs. We provide detailed protocols for FLAG-KRAS4B purification, proteoform analysis by targeted top-down LC-MS/MS, and validation of abundant PTMs by bottom-up LC-MS/MS with example results.

A Top-Down Proteomic Assay to Evaluate KRAS4B-Compound Engagement.

Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4BG12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site.

Revealing the mechanism of action of a first-in-class covalent inhibitor of KRASG12C (ON) and other functional properties of oncogenic KRAS by 31P NMR.

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-[(ß,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1-state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1-RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.

NMR 1H, 13C, 15N backbone resonance assignments of the T35S and oncogenic T35S/Q61L mutants of human KRAS4b in the active, GppNHp-bound conformation.

RAS proteins cycling between the active-form (GTP-bound) and inactive-form (GDP-bound) play a key role in cell signaling pathways that control cell survival, proliferation, and differentiation. Mutations at codon 12, 13, and 61 in RAS are known to attenuate its GTPase activity favoring the RAS active state and constitutively active downstream signaling. This hyperactivation accounts for various malignancies including pancreatic, lung, and colorectal cancers. Active KRAS is found to exist in equilibrium between two rapidly interconverting conformational states (State1-State2) in solution. Due to this dynamic feature of the protein, the 1H-15N correlation cross-peak signals of several amino acid (AA) residues of KRAS belonging to the flexible loop regions are absent from its 2D 1H-15N HSQC spectrum within and near physiological solution pH. A threonine to serine mutation at position 35 (T35S) shifts the interconverting equilibrium to State1 conformation and enables the emergence of such residues in the 2D 1H-15N HSQC spectrum due to gained conformational rigidity. We report here the 1HN, 15N, and 13C backbone resonance assignments for the 19.2 kDa (AA 1-169) protein constructs of KRAS-GppNHp harboring T35S mutation (KRAST35S/C118S-GppNHp) and of its oncogenic counterpart harboring the Q61L mutation (KRAST35S/Q61L/C118S-GppNHp) using heteronuclear, multidimensional NMR spectroscopy at 298 K. High resolution NMR data allowed the unambiguous assignments of 1H-15N correlation cross-peaks for all the residues except for Met1. Furthermore, 2D 1H-15N HSQC overlay of two proteins assisted in determination of Q61L mutation-induced chemical shift perturbations for select residues in the regions of P-loop, Switch-II, and helix α3.

PABA/NO lead optimization: Improved targeting of cytotoxicity to glutathione S-transferase P1-overexpressing cancer cells.

PABA/NO [O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino) diazen-1-ium-1,2-diolate] is a nitric oxide (NO)-releasing arylating agent designed to be selectively activated by reaction with glutathione (GSH) on catalysis by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancer cells. PABA/NO has proven active in several cancer models in vitro and in vivo, but its tendency to be metabolized via a variety of pathways, some that generate inactive metabolites and hydrolysis products, limits its potential as a drug. Here we show that a simple replacement of cyano for nitro at the 4 position to give compound 4b ('p-cyano-PABA/NO') has the dual effect of slowing the undesired side reactions while enhancing the proportion of NO release and arylating activity on catalysis by GSTP1. Compound 4b showed increased resistance to hydrolysis and uncatalyzed reaction with GSH, along with a more favorable product distribution in the presence of GSTP1. It also showed significant proapoptotic activity. The data suggest p-cyano-PABA/NO to be a more promising prodrug than PABA/NO, with better selectivity toward cancer cells.

Nitric oxide (NO) releasing poly ADP-ribose polymerase 1 (PARP-1) inhibitors targeted to glutathione S-transferase P1-overexpressing cancer cells.

We report the antitumor effects of nitric oxide (NO) releasing derivatives of the PARP-1 inhibitor olaparib (1). Compound 5b was prepared by coupling the carboxyl group of 3b and the free amino group of arylated diazeniumdiolated piperazine 4. Analogue 5a has the same structure except that the F is replaced by H. Compound 13 is the same as 5b except that a Me2N-N(O)═NO- group was added para and ortho to the nitro groups of the dinitrophenyl ring. The resulting prodrugs are activated by glutathione in a reaction accelerated by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancers. This metabolism generates NO plus a PARP-1 inhibitor simultaneously, consuming reducing equivalents, leading to DNA damage concomitant with inhibition of DNA repair, and in the case of 13 inducing cross-linking glutathionylation of proteins. Compounds 5b and 13 reduced the growth rates of A549 human lung adenocarcinoma xenografts with no evidence of systemic toxicity.

Co-transcriptional production of RNA-DNA hybrids for simultaneous release of multiple split functionalities.

Control over the simultaneous delivery of different functionalities and their synchronized intracellular activation can greatly benefit the fields of RNA and DNA biomedical nanotechnologies and allow for the production of nanoparticles and various switching devices with controllable functions. We present a system of multiple split functionalities embedded in the cognate pairs of RNA-DNA hybrids which are programmed to recognize each other, re-associate and form a DNA duplex while also releasing the split RNA fragments which upon association regain their original functions. Simultaneous activation of three different functionalities (RNAi, Förster resonance energy transfer and RNA aptamer) confirmed by multiple in vitro and cell culture experiments prove the concept. To automate the design process, a novel computational tool that differentiates between the thermodynamic stabilities of RNA-RNA, RNA-DNA and DNA-DNA duplexes was developed. Moreover, here we demonstrate that besides being easily produced by annealing synthetic RNAs and DNAs, the individual hybrids carrying longer RNAs can be produced by RNA polymerase II-dependent transcription of single-stranded DNA templates.

Nitric oxide-releasing prodrug triggers cancer cell death through deregulation of cellular redox balance.

JS-K is a nitric oxide (NO)-releasing prodrug of the O (2)-arylated diazeniumdiolate family that has demonstrated pronounced cytotoxicity and antitumor properties in a variety of cancer models both in vitro and in vivo. The current study of the metabolic actions of JS-K was undertaken to investigate mechanisms of its cytotoxicity. Consistent with model chemical reactions, the activating step in the metabolism of JS-K in the cell is the dearylation of the diazeniumdiolate by glutathione (GSH) via a nucleophilic aromatic substitution reaction. The resulting product (CEP/NO anion) spontaneously hydrolyzes, releasing two equivalents of NO. The GSH/GSSG redox couple is considered to be the major redox buffer of the cell, helping maintain a reducing environment under basal conditions. We have quantified the effects of JS-K on cellular GSH content, and show that JS-K markedly depletes GSH, due to JS-K's rapid uptake and cascading release of NO and reactive nitrogen species. The depletion of GSH results in alterations in the redox potential of the cellular environment, initiating MAPK stress signaling pathways, and inducing apoptosis. Microarray analysis confirmed signaling gene changes at the transcriptional level and revealed alteration in the expression of several genes crucial for maintenance of cellular redox homeostasis, as well as cell proliferation and survival, including MYC. Pre-treating cells with the known GSH precursor and nucleophilic reducing agent N-acetylcysteine prevented the signaling events that lead to apoptosis. These data indicate that multiplicative depletion of the reduced glutathione pool and deregulation of intracellular redox balance are important initial steps in the mechanism of JS-K's cytotoxic action.

Activation of different split functionalities on re-association of RNA-DNA hybrids.

Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of protein functions and improved detection sensitivity. Here we report a similar technique based on a pair of RNA-DNA hybrids that can be used generally for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept, this work mainly focuses on the activation of RNA interference. However, the release of other functionalities (such as resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumours together with specific gene silencing. This split-functionality approach presents a new route in the development of 'smart' nucleic acid-based nanoparticles and switches for various biomedical applications.

Cross-linking protein glutathionylation mediated by O2-arylated bis-diazeniumdiolate "Double JS-K".

Attachment of glutathione (GSH) to cysteine residues in proteins (S-glutathionylation) is a reversible post-translational modification that can profoundly alter protein structure and function. Often serving in a protective role, for example, by temporarily saving protein thiols from irreversible oxidation and inactivation, glutathionylation can be identified and semiquantitatively assessed using anti-GSH antibodies, thought to be specific for recognition of the S-glutathionylation modification. Here, we describe an alternate mechanism of protein glutathionylation in which the sulfur atoms of the GSH and the protein's thiol group are covalently bound via a cross-linking agent, rather than through a disulfide bond. This form of thiol cross-linking has been shown to occur and has been confirmed by mass spectrometry at the solution chemistry level, as well as in experiments documenting the potent antiproliferative activity of the bis-diazeniumdiolate Double JS-K in H1703 cells in vitro and in vivo. The modification is recognized by the anti-GSH antibody as if it were authentic S-glutathionylation, requiring mass spectrometry to distinguish between them.

Structural modifications modulate stability of glutathione-activated arylated diazeniumdiolate prodrugs.

JS-K, a diazeniumdiolate-based nitric oxide (NO)-releasing prodrug, is currently in late pre-clinical development as an anti-cancer drug candidate. This prodrug was designed to be activated by glutathione (GSH) to release NO. To increase the potency of JS-K, we are investigating the effect of slowing the reaction of the prodrugs with GSH. Herein, we report the effect of replacement of nitro group(s) by other electron-withdrawing group(s) in JS-K and its homo-piperazine analogues on GSH activation and the drugs' biological activity. We show that nitro-to-cyano substitution increases the half-life of the prodrug in the presence of GSH without compromising the compound's in vivo antitumor activity.

Diazeniumdiolated carbamates: a novel class of nitric oxide donors.

We report an indirect method for synthesis of previously inaccessible diazeniumdiolated carbamates. Synthesis involves use of previously reported triisopropylsilyloxymethylated isopropylamine diazeniumdiolate (TOM-ylated IPA/NO). These novel diazeniumdiolated carbamate prodrugs upon activation release nitric oxide (NO) similar to their secondary amine counterparts. They are also efficient sources of intracellular NO. These prodrugs may have potential applications as therapeutic NO-donors.

Thiol Modification By Pharmacologically Active Agents of the Diazeniumdiolate Class.

Promising drug candidates of the diazeniumdiolate (NONOate) chemical family include several types of thiol modification among their mechanisms of action: 1) drugs designed to release nitric oxide (NO) on reaction with the thiol group of glutathione (GSH) arylate the GSH, a step that removes reducing equivalents from the cell; (2) a similar reaction of the drug with the thiol group of a protein changes its structure, leading to potentially impaired function and cell death; (3) the NO generated as a byproduct in the above reactions can undergo oxidation, leading to S-nitrosylation and S-glutathionylation; and (4) diazeniumdiolates can also generate nitroxyl, which reacts with thiol groups to form disulfides or sulfinamides.

Activation of the c-Jun N-terminal kinase/activating transcription factor 3 (ATF3) pathway characterizes effective arylated diazeniumdiolate-based nitric oxide-releasing anticancer prodrugs.

Improved therapies are needed for nonsmall cell lung cancer. Diazeniumdiolate-based nitric oxide (NO)-releasing prodrugs are a growing class of promising NO-based therapeutics. Recently, we have shown that O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, 1) is effective against nonsmall cell lung cancer (NSCLC) cells in culture and in vivo. Here we report mechanistic studies with compound 1 and its homopiperazine analogue and structural modification of these into more stable prodrugs. Compound 1 and its homopiperazine analogue were potent cytotoxic agents against NSCLC cells in vitro and in vivo, concomitant with activation of the SAPK/JNK stress pathway and upregulation of its downstream effector ATF3. Apoptosis followed these events. An aryl-substituted analogue, despite extended half-life in the presence of glutathione, did not activate JNK or have antitumor activity. The data suggest that rate of reactivity with glutathione and activation of JNK/ATF3 are determinants of cancer cell killing by these prodrugs.

The nitric oxide prodrug JS-K is effective against non-small-cell lung cancer cells in vitro and in vivo: involvement of reactive oxygen species.

Non-small-cell lung cancer is among the most common and deadly forms of human malignancies. Early detection is unusual, and there are no curative therapies in most cases. Diazeniumdiolate-based nitric oxide (NO)-releasing prodrugs are a growing class of promising NO-based therapeutics. Here, we show that O(2)-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) is a potent cytotoxic agent against a subset of human non-small-cell lung cancer cell lines both in vitro and as xenografts in mice. JS-K treatment led to 75% reduction in the growth of H1703 lung adenocarcinoma cells in vivo. Differences in sensitivity to JS-K in different lung cancer cell lines seem to be related to their endogenous levels of reactive oxygen species (ROS)/reactive nitrogen species (RNS). Other related factors, levels of peroxiredoxin 1 (PRX1) and 8-oxo-deoxyguanosine glycosylase (OGG1), also correlated with drug sensitivity. Treatment of the lung adenocarcinoma cells with JS-K resulted in oxidative/nitrosative stress in cells with high basal levels of ROS/RNS, which, combined with the arylating properties of the compound, was reflected in glutathione depletion and alteration in cellular redox potential, mitochondrial membrane permeabilization, and cytochrome c release. Inactivation of manganese superoxide dismutase by nitration was associated with increased superoxide and significant DNA damage. Apoptosis followed these events. Taken together, the data suggest that diazeniumdiolate-based NO-releasing prodrugs may have application as a personalized therapy for lung cancers characterized by high levels of ROS/RNS. PRX1 and OGG1 proteins, which can be easily measured, could function as biomarkers for identifying tumors sensitive to the therapy.

Macrophage-dependent nitric oxide expression regulates tumor cell detachment and metastasis after IL-2/anti-CD40 immunotherapy.

Using an orthotopic model of renal cell carcinoma, we showed previously that IL-2/anti-CD40 immunotherapy resulted in synergistic anti-tumor responses, whereas IL-2 or α-CD40 alone mediated partial transient anti-tumor effects. We now show that treatment of tumor-bearing mice with IL-2/α-CD40, but not IL-2 or α-CD40, induced significant nitric oxide synthase (NOS) 2 expression in tumor-associated macrophages. In control-treated mice (low NO), NOS2 inhibition reduced tumor burden. However, during immunotherapy (high NO), NOS2 inhibition or macrophage depletion reversed the ability of IL-2/α-CD40 treatment to reduce lung metastases but had no effect on primary tumor burden. Furthermore, IL-2/α-CD40 induced the IFN-γ- and NO-dependent decrease in matrix metalloproteinase (MMP) expression and activity, concomitant with increases in tissue inhibitor of metalloproteinase (TIMP) 1 and E-cadherin expression within tumors. Finally, treatment of tumor-bearing mice with the NO donor JS-K significantly reduced metastases. These data differentiate the mechanism for primary anti-tumor effects of IL-2/α-CD40 immunotherapy, which are independent of NO, from the NO-dependent inhibition of metastases. Furthermore, reduced MMP9 activity implicates M1-polarized macrophages within the tumor microenvironment as critical components of therapeutic response. Our data demonstrate the mechanistic basis for IL-2/α-CD40-mediated control of metastases and suggest that the context-dependent application of NO donors may hold promise for prevention of metastatic disease.

"Click" reaction in conjunction with diazeniumdiolate chemistry: developing high-load nitric oxide donors.

The use of Cu(I)-catalyzed "click" reactions of alkyne-substituted diazeniumdiolate prodrugs with bis- and tetrakis-azido compounds is described. The "click" reaction for the bis-azide using CuSO(4)/Na-ascorbate predominantly gave the expected bis-triazole. However, CuI/diisopropylethylamine predominantly gave uncommon triazolo-triazole products as a result of oxidative coupling. Neither set of "click" conditions showed evidence of compromising the integrity of the diazeniumdiolate groups. The chemistry developed has applications in the synthesis of polyvalent and dendritic nitric oxide donors.

Glycosylated PROLI/NO derivatives as nitric oxide prodrugs.

GlcNAc-PROLI/NO prodrugs that are activated by N-acetylglucosaminidase to release nitric oxide (NO) are described. A classical acid-amine coupling is used to bifunctionalize these PROLI/NO prodrugs, which on activation generate up to 4 mol of NO, a peptide residue, and an N-acetylglucosamine residue. Many of the prodrugs synthesized are efficient sources of intracellular NO.