Structure-based optimisation of orally active & reversible MetAP-2 inhibitors maintaining a tight 'molecular budget'.

Structure-based led optimisation of orally active reversible Methionine Aminopeptidase-2 (MetAP-2) inhibitors utilising a 'molecular budget' medicinal chemistry strategy is described. The key physicochemical parameters of target molecules (cLogP, molecular size and H-bond donor count) were monitored through straightforward and intuitive use of atom count and distribution. The balance between structure-based design and an awareness of the physicochemical properties of the compounds synthesised enabled the rapid identification of a potent molecule with good oral pharmacokinetic (PK) characteristics by making fewer, higher quality compounds. The resulting candidate quality molecule was validated in a mechanistic cellular assay and a rodent secondary immunisation model.

Characterisation of phosphorylated nucleotides by collisional and electron-based tandem mass spectrometry.

RATIONALE: Tandem mass spectrometry of phosphorylated ions can often yield a limited number of product ions owing to the labile nature of phosphate groups. Developing techniques to improve dissociation for this type of ion has implications for the structural characterisation of many different phosphorylated ions, such as those from nucleotides, pharmaceutical compounds, peptides and polymers. METHODS: Solutions of adenosine monophosphate, diphosphate and triphosphate (AMP, ADP and ATP) were studied in a hybrid linear ion trap-Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Precursor ions with an overall single positive charge, including protonated nucleotides or nucleotide cations containing one, two or three sodium atoms, were isolated for tandem mass spectrometry. Collision-induced dissociation (CID) was performed in the linear ion trap, with electron-induced dissociation (EID) being conducted in the FTICR cell. RESULTS: EID resulted in many product ions not seen in CID. EID product ion spectra were seen to vary for AMP, ADP and ATP when the nucleotide cation contained zero, one, two or three sodiums. Precursor cations that contain two or three sodiums mainly formed product ions derived from the phosphate group. Conversely, when a precursor ion containing no sodium underwent EID, product ions mainly relating to the non-phosphate end of the ion were observed. The number of phosphate groups was not seen to greatly affect either CID or EID product ion spectra. CONCLUSIONS: The presence of sodium in a precursor ion directs electron-induced bond dissociation, thus enabling targeted, and therefore tuneable, fragmentation of groups within that precursor ion. For all precursor ions, the most useful product ion spectra were obtained by EID for a precursor ion containing one sodium, with bond dissociation occurring across the entire nucleotide cation. The findings of this study can be used to improve the structural elucidation of many phosphorylated molecules by broadening the range of product ions achievable. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

Applicability of gas chromatography/quadrupole-Orbitrap mass spectrometry in support of pharmaceutical research and development.

RATIONALE: Gas chromatography/mass spectrometry (GC/MS) is a fundamental tool used to identify impurities throughout the active pharmaceutical ingredients development process. The coupling of Orbitrap mass spectrometry with GC marks an exciting advance in capability for GC/MS, offering a significant step change in resolving power, mass accuracy, sensitivity and linear range. METHODS: A range of pharmaceutically relevant samples representing typical starting materials has been investigated with particular reference to impurity identification. The mass accuracy in Electron Ionisation (EI) and Chemical Ionisation (CI) was investigated for impurity identification. The linearity and mass accuracy over a wide dynamic range were evaluated. The number of scans obtained across chromatographic peaks was assessed at various resolution settings from 15,000 to 120,000 (full width at half maximum (FWHM) at m/z 200). RESULTS: All the accurate mass measurements for impurities were within <1 ppm of the theoretical m/z value. The scan speed at the highest resolution produced 11 scans across the peak, and the mass accuracy for all scans was consistently <1 ppm - sufficient for impurity investigations and quantitative analysis. Linearity was demonstrated for N,N,N'-trimethylethylenediamine over a concentration range of 0.0001 to 0.1250 µg/mL (w/v) with a correlation coefficient R(2) = 0.9996 and mass accuracy across all concentrations at <1.1 ppm. CONCLUSIONS: GC/Orbitrap MS has been evaluated for both qualitative and quantitative analysis of typical pharmaceutical precursors and impurities. Accurate mass measurement across a wide dynamic range, linearity and the ability to identify impurities in EI and CI illustrate that this instrument is a powerful tool of great benefit to pharmaceutical analysis.

Epigenetic Priming by Hypomethylation Enhances the Immunogenic Potential of Tolinapant in T-cell Lymphoma.

Programmed cell death mechanisms are important for the regulation of tumor development and progression. Evasion of and resistance to apoptosis are significant factors in tumorigenesis and drug resistance. Bypassing apoptotic pathways and eliciting another form of regulated cell death, namely necroptosis, an immunogenic cell death (ICD), may override apoptotic resistance. Here, we present the mechanistic rationale for combining tolinapant, an antagonist of the inhibitor of apoptosis proteins (IAP), with decitabine, a hypomethylating agent (HMA), in T-cell lymphoma (TCL). Tolinapant treatment alone of TCL cells in vitro and in syngeneic in vivo models demonstrated that ICD markers can be upregulated, and we have shown that epigenetic priming with decitabine further enhances this effect. The clinical relevance of ICD markers was confirmed by the direct measurement of plasma proteins from patients with peripheral TCL treated with tolinapant. We showed increased levels of necroptosis in TCL lines, along with the expression of cancer-specific antigens (such as cancer testis antigens) and increases in genes involved in IFN signaling induced by HMA treatment, together deliver a strong adaptive immune response to the tumor. These results highlight the potential of a decitabine and tolinapant combination for TCL and could lead to clinical evaluation. SIGNIFICANCE: The IAP antagonist tolinapant can induce necroptosis, a key immune-activating event, in TCL. Combination with DNA hypomethylation enhances tolinapant sensitivity and primes resistant cells by re-expressing necrosome proteins. In addition, this combination leads to increases in genes involved in IFN signaling and neoantigen expression, providing further molecular rationale for this novel therapeutic option.

Electron-induced dissociation of singly charged organic cations as a tool for structural characterization of pharmaceutical type molecules.

Collision-induced dissociation (CID) and electron-induced dissociation (EID) have been investigated for a selection of small, singly charged organic molecules of pharmaceutical interest. Comparison of these techniques has shown that EID carried out on an FTICR MS and CID performed on a linear ion trap MS produce complementary data. In a study of 33 molecule-cations, EID generated over 300 product ions compared to 190 product ions by CID with an average of only 3 product ions per precursor ion common to both tandem MS techniques. Even multiple stages of CID failed to generate many of the product ions observed following EID. The charge carrying species is also shown to have a very significant effect on the degree of fragmentation and types of product ion resulting from EID. Protonated species behave much like the ammonium adduct with suggestion of a hydrogen atom from the charge carrying species strongly affecting the fragmentation mechanism. Sodium and potassium are retained by nearly every product ion formed from [M + Na](+) or [M + K](+) and provide information to complement the EID of [M + H](+) or [M + NH(4)](+). In summary, EID is proven to be a fitting partner to CID in the structural elucidation of small singly charged ions and by studying EID of a molecule-ion holding different charge carrying species, an even greater depth of detail can be obtained for functional groups commonly used in synthetic chemistry.

Antagonism of inhibitors of apoptosis proteins reveals a novel, immune response-based therapeutic approach for T-cell lymphoma.

Tolinapant (ASTX660) is a potent, nonpeptidomimetic antagonist of cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2) and X-linked IAP, which is currently being evaluated in a phase 2 study in T-cell lymphoma (TCL) patients. Tolinapant has demonstrated evidence of single-agent clinical activity in relapsed/refractory peripheral TCL and cutaneous TCL. To investigate the mechanism of action underlying the single-agent activity observed in the clinic, we have used a comprehensive translational approach integrating in vitro and in vivo models of TCL confirmed by data from human tumor biopsies. Here, we show that tolinapant acts as an efficacious immunomodulatory molecule capable of inducing complete tumor regression in a syngeneic model of TCL exclusively in the presence of an intact immune system. These findings were confirmed in samples from our ongoing clinical study showing that tolinapant treatment can induce changes in gene expression and cytokine profile consistent with immune modulation. Mechanistically, we show that tolinapant can activate both the adaptive and the innate arms of the immune system through the induction of immunogenic forms of cell death. In summary, we describe a novel role for IAP antagonists as immunomodulatory molecules capable of promoting a robust antitumor immune response in TCL.

Ubiquitylation of MLKL at lysine 219 positively regulates necroptosis-induced tissue injury and pathogen clearance.

Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system.

The application of gas chromatography/atmospheric pressure chemical ionisation time-of-flight mass spectrometry to impurity identification in Pharmaceutical Development.

Accurate mass measurement (used to determine elemental formulae) is an essential tool for impurity identification in pharmaceutical development for process understanding. Accurate mass liquid chromatography/mass spectrometry (LC/MS) is used widely for these types of analyses; however, there are still many occasions when gas chromatography (GC)/MS is the appropriate technique. Therefore, the provision of robust technology to provide accurate mass GC/MS (and GC/MS/MS) for this type of activity is essential. In this report we describe the optimisation and application of a newly available atmospheric pressure chemical ionisation (APCI) interface to couple GC to time-of-flight (TOF) MS.To fully test the potential of the new interface the APCI source conditions were optimised, using a number of standard compounds, with a variety of structures, as used in synthesis at AstraZeneca. These compounds were subsequently analysed by GC/APCI-TOF MS. This study was carried out to evaluate the range of compounds that are amenable to analysis using this technique. The range of compounds that can be detected and characterised using the technique was found to be extremely broad and include apolar hydrocarbons such as toluene. Both protonated molecules ([M + H](+)) and radical cations (M(+.)) were observed in the mass spectra produced by APCI, along with additional ion signals such as [M + H + O](+).The technique has been successfully applied to the identification of impurities in reaction mixtures from organic synthesis in process development. A typical mass accuracy of 1-2 mm/zunits (m/z 80-500) was achieved allowing the reaction impurities to be identified based on their elemental formulae. These results clearly demonstrate the potential of the technique as a tool for problem solving and process understanding in pharmaceutical development. The reaction mixtures were also analysed by GC/electron ionisation (EI)-MS and GC/chemical ionisation (CI)-MS to understand the capability of GC/APCI-MS relative to these two firmly established techniques.

Using electron induced dissociation (EID) on an LC time-scale to characterize a mixture of analogous small organic molecules.

LC ESI FTICR MS of a sample of cediranib identified this pharmaceutical target molecule plus an additional 10 compounds of interest, all of which were less than 10% total ion current (TIC) peak intensity relative to cediranib. LC FTICR tandem mass spectrometry using electron induced dissociation (EID) has been achieved and has proven to be the best way to generate useful product ion information for all of these singly protonated molecules. Cediranib [M + H](+) fragmented by EID to give 29 product ions whereas QTOF-CID generated only one very intense product ion, and linear ion trap-CID, which generated 10 product ions, but all with poor S/N. Twenty-six of the EID product ions were unique to this fragmentation technique alone. By considering the complementary LC-EID and LC-CID data together, all 10 unknown compounds were structurally characterized and proven to be analogous to cediranib. Of particular importance, EID produced unique product ion information for one of the low level cediranib analogues that enabled full characterization of the molecule such that the presence of an extra propylpyrrolidine group was discovered and proven to be located on the pyrrolidine ring of cediranib, solving an analytical problem that could not be solved by collision induced dissociation (CID). Thus, it has been demonstrated that EID is in harmony with the chromatography duty-cycle and the dynamic concentration range of synthetic compounds containing trace impurities, providing crucial analytical information that cannot be obtained by more traditional methodologies.

Mechanistic medicine: novel strategies for clinical trials.

Autoimmune diseases affect a significant proportion of the population and the development of therapeutics able to manipulate the immune response to deliver effective treatment in these diseases is an accepted approach for drug discovery. This article will focus on recent strategies for achieving selectivity through target choice, thus reducing overall clinical immunosuppression. We review the use of mechanistic pharmacodynamic assays preclinically and in the clinic to assess target engagement and to build the relationship between target coverage and efficacy, to guide dosing. Finally, we review the use of monogenic diseases to deliver proof of mechanism clinical studies and to identify patient populations in larger autoimmune diseases that may be sensitive to intervention with a specific therapeutic.

N-4-Pyrimidinyl-1H-indazol-4-amine inhibitors of Lck: indazoles as phenol isosteres with improved pharmacokinetics.

2,4-Dianilino pyrimidines are well-known inhibitors of tyrosine kinases including lymphocyte specific kinase (Lck). Structure-activity relationships at the 4-position are discussed and rationalised. Examples bearing a 2-methyl-5-hydroxyaniline substituent at the 4-position were especially potent but showed poor oral pharmacokinetics. Replacement of this substituent by 4-amino(5-methyl-1H-indazole) yielded compounds with comparable enzyme potency and improved pharmacokinetic properties.

Ligation of CD28 stimulates the formation of a multimeric signaling complex involving grb-2-associated binder 2 (gab2), SRC homology phosphatase-2, and phosphatidylinositol 3-kinase: evidence that negative regulation of CD28 signaling requires the gab2 pleckstrin homology domain.

Grb-2-associated binder (Gab)2 is a scaffolding adaptor protein that has been reported to promote growth factor and cytokine receptor signal transduction, but inhibit TCR-mediated signaling events. In this study, we show that ligation of CD28 by its natural ligand B7-1/CD80, induces tyrosine phosphorylation of Gab2 and its coassociation with Src homology phosphatase (SHP)-2 and class IA PI3K in Jurkat cells. Overexpression of wild-type Gab2 revealed a negative role in regulation of CD3/CD28 induction of the transcription factors NF-kappaB and AP-1. To characterize this inhibitory function further, we used Gab2 mutants unable to bind either PI3K or SHP-2 and a PH domain deletion mutant. Although PI3K has previously been implicated as necessary for Gab2-mediated inhibition of TCR signaling, Gab2 mutants defective in their ability to bind PI3K or SHP-2 retained their inhibitory function, whereas deletion of the PH domain ablated the inhibitory effect of Gab2. Together, these data demonstrate that CD28 stimulation of T cells is sufficient to induce an inhibitory multimeric signaling complex involving Gab2, SHP-2, and PI3K. Furthermore, the inhibitory capacity of Gab2 is strictly dependent upon the integrity of its PH domain, suggesting phosphoinositide-mediated membrane recruitment is important to Gab2 function in T cells.