Design and synthesis of small-molecule inhibitors of two cancer targets :mTor and MDM2/p53

PhD ThesisCancer is a disease in which cellular control over growth and differentiation has been lost. Targeted therapies for the treatment of cancer are becoming an increasing area of focus within the pharmaceutical industry and academia, due to the increasing understanding of the biology behind tumourgenesis. There are 518 protein kinases within the human genome and they play a significant role within cellular signalling. Aberrant signalling of kinases can contribute to the development of cancer, and inhibition of kinase targets can result in either a cytostatic or cytotoxic effort. Kinases have a discrete ATP-binding domain, which presents an ideal target for small- molecule inhibitors. mTOR (mammalian target of rapamycin) is a serine/threonine protein kinase, which forms two complexes, mTORC1 and mTORC2, as part of the PI 3-K/Akt pathway, a growth/survival pathway which has aberrant signalling in a number of cancers. The development of ATP-competitive inhibitors of mTOR has been based on a series of 2,6- diaminosubstituted pyrimidines, with modest activity against mTOR, as exemplified by NU6027 and NU6227 originally designed as CDK2 inhibitors. Structure-activity relationships for inhibition of mTOR have been explored. The 4-substituent was either modified to a smaller alkoxy group or completely removed, giving reduced activity. At the 5- position compounds with other substituents were then synthesised. Modifications of the 2- and 6-amino groups were also investigated. The pyrimidine heterocycle was also replaced with two pyridine regioisomers. None of the synthesised compounds showed improved mTOR inhibitory activity over NU6227 and NU6027. The tumour suppressor p53 is activated as a response to DNA-damage, oncogene activation and cellular stress. Once activated p53 acts as a transcription initiator, inducing thetranscription of a number of genes, including those involved in halting the cell cycle, repairing DNA-damage and initiating apoptosis. A further transcriptional target of p53 is MDM2, a negative regulator of p53. Inhibitors of the MDM2-p53 interaction have been reported including the isoindolinones e.g. NCL-00008406. Structure-activity relationships (SAR) studies for the identification of replacement of the 4- nitro group showed that a 4-ethynyl substituent had a similar level of potency, along with the 4-bromo, 3-fluoro substituents. SARs around the isoindolinone A-ring identified the 6-tert- butyl substituent as equipotent to the parent. Synthesis of an oxetane derivative such as NCL- 00018327 has demonstrated that replacing the cyclopropyl group with a 3,3-oxetane substituent either maintained or improved activity against MDM2. Synthetic efforts have identified highly potent, low nanomolar, isoindolinone-based inhibitors of the MDM2-p53 protein-protein interaction. Optimal substituents for the benzyl group have been identified, avoiding the use of a nitro group which is toxic within drugs. Synthesis of an oxetane derivative has been shown to be either equi- or more potent than cyclopropyl derivatives, and this modification is predicted to improved the aqueous solubility by reducing the clogP. Synthesis of a tert-butyl analogues using an alternative synthetic route has developed the SAR around the A-ring and resulted in an improved synthetic scheme. Purification of a range of MDM2 mutant proteins has identified a crystallisable form of MDM2 and the recently solved crystal structure of an isoindolinone bound to MDM2 should guide further improvements to potency and aid the incorporation of groups to improve physical properties. Cancer is a disease in which control over cellular growth is deregulated. Synthetic efforts have been directed toward the development of two targeted anti-cancer agents; the kinase mTOR and the protein-protein interaction MDM2/p53. 2,6-diaminopyrimidine were identified as most inhibitors of mTOR, but demonstrated a flat SAR and no increase in potency was observed. Isoindolinones have been identified as a valuable scaffold to inhibit the protein-protein interaction and have demonstrated excellent potencies.Cancer Research U

Inhibition of plasmin-mediated TAFI activation may affect development but not progression of abdominal aortic aneurysms

Objective: Thrombin-activatable fibrinolysis inhibitor (TAFI) reduces the breakdown of fibrin clots through its action as an indirect inhibitor of plasmin. Studies in TAFI-deficient mice have implicated a potential role for TAFI in Abdominal Aortic Aneurysm (AAA) disease. The role of TAFI inhibition on AAA formation in adult ApoE-/- mice is unknown. The aim of this paper was to investigate the effects of TAFI inhibition on AAA development and progression. Methods: Using the Angiotensin II model of AAA, male ApoE-/- mice were infused with Angiotensin II 750ng/kg/min with or without a monoclonal antibody inhibitor of plasmin-mediated activation of TAFI, MA-TCK26D6, or a competitive small molecule inhibitor of TAFI, UK-396082. Results: Inhibition of TAFI in the Angiotensin II model resulted in a decrease in the mortality associated with AAA rupture (from 40.0% to 16.6% with MA-TCK26D6 (log-rank Mantel Cox test p = 0.16), and 8.3% with UK-396082 (log-rank Mantel Cox test p = 0.05)). Inhibition of plasmin-mediated TAFI activation reduced the incidence of AAA from 52.4% to 30.0%. However, late treatment with MA-TCK26D6 once AAA were already established had no effect on the progression of AAA in this model. Conclusions: The formation of intra-mural thrombus is responsible for the dissection and early rupture in the angiotensin II model of AAA, and this process can be prevented through inhibition of TAFI. Late treatment with a TAFI inhibitor does not prevent AAA progression. These data may indicate a role for inhibition of plasmin-mediated TAFI activation in the early stages of AAA development, but not in its progression

Identification of GSK3186899/DDD853651 as a Preclinical Development Candidate for the Treatment of Visceral Leishmaniasis

The leishmaniases are diseases that affect millions of people across the world, in particular visceral leishmaniasis (VL) which is fatal unless treated. Current standard of care for VL suffers from multiple issues and there is a limited pipeline of new candidate drugs. As such, there is a clear unmet medical need to identify new treatments. This paper describes the optimization of a phenotypic hit against Leishmania donovani, the major causative organism of VL. The key challenges were to balance solubility and metabolic stability while maintaining potency. Herein, strategies to address these shortcomings and enhance efficacy are discussed, culminating in the discovery of preclinical development candidate GSK3186899/DDD853651 (<b>1</b>) for VL

ESI-IMS-MS: A method for rapid analysis of protein aggregation and its inhibition by small molecules.

Electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is a powerful method for the study of conformational changes in protein complexes, including oligomeric species populated during protein self-aggregation into amyloid fibrils. Information on the mass, stability, cross-sectional area and ligand binding capability of each transiently populated intermediate, present in the heterogeneous mixture of assembling species, can be determined individually in a single experiment in real-time. Determining the structural characterisation of oligomeric species and alterations in self-assembly pathways observed in the presence of small molecule inhibitors is of great importance, given the urgent demand for effective therapeutics. Recent studies have demonstrated the capability of ESI-IMS-MS to identify small molecule modulators of amyloid assembly and to determine the mechanism by which they interact (positive, negative, non-specific binding, or colloidal) in a high-throughput format. Here, we demonstrate these advances using self-assembly of Aβ40 as an example, and reveal two new inhibitors of Aβ40 fibrillation

Combined PI3K and CDK2 inhibition induces cell death and enhances in vivo antitumour activity in colorectal cancer

Background: The phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway is commonly deregulated in human cancer, hence many PI3K and mTOR inhibitors have been developed and have now reached clinical trials. Similarly, CDKs have been investigated as cancer drug targets. Methods: We have synthesised and characterised a series of 6-aminopyrimidines identified from a kinase screen that inhibit PI3K and/or mTOR and/or CDK2. Kinase inhibition, tumour cell growth, cell cycle distribution, cytotoxicity and signalling experiments were undertaken in HCT116 and HT29 colorectal cancer cell lines, and in vivo HT29 efficacy studies. Results: 2,6-Diaminopyrimidines with an O4-cyclohexylmethyl substituent and a C-5-nitroso or cyano group (1,2,5) induced cell cycle phase alterations and were growth inhibitory (GI50<20 μM). Compound 1, but not 2 or 5, potently inhibits CDK2 (IC50=0.1 nM) as well as PI3K, and was cytotoxic at growth inhibitory concentrations. Consistent with kinase inhibition data, compound 1 reduced phospho-Rb and phospho-rS6 at GI50 concentrations. Combination of NU6102 (CDK2 inhibitor) and pictilisib (GDC-0941; pan-PI3K inhibitor) resulted in synergistic growth inhibition, and enhanced cytotoxicity in HT29 cells in vitro and HT29 tumour growth inhibition in vivo. Conclusions: These studies identified a novel series of mixed CDK2/PI3K inhibitors and demonstrate that dual targeting of CDK2 and PI3K can result in enhanced antitumour activity

An in vivo platform for identifying inhibitors of protein aggregation

Protein aggregation underlies an array of human diseases, yet only one small molecule therapeutic has been successfully developed to date. Here, we introduce an in vivo system, based on a β-lactamase tripartite fusion construct, capable of identifying aggregation-prone sequences in the periplasm of Escherichia coli and inhibitors that prevent their aberrant self-assembly. We demonstrate the power of the system using a range of proteins, from small unstructured peptides (islet amyloid polypeptide and amyloid β) to larger, folded immunoglobulin domains. Configured in a 48-well format, the split β-lactamase sensor readily differentiates between aggregation-prone and soluble sequences. Performing the assay in the presence of 109 compounds enabled a rank ordering of inhibition and revealed a new inhibitor of IAPP aggregation. This platform can be applied to both amyloidogenic and other aggregation-prone systems, independent of sequence or size, and can identify small molecules or other factors able to ameliorate or inhibit protein aggregation

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry.

The search for therapeutic agents that bind specifically to precursor protein conformations and inhibit amyloid assembly is an important challenge. Identifying such inhibitors is difficult because many protein precursors of aggregation are partially folded or intrinsically disordered, which rules out structure-based design. Furthermore, inhibitors can act by a variety of mechanisms, including specific or nonspecific binding, as well as colloidal inhibition. Here we report a high-throughput method based on ion mobility spectrometry-mass spectrometry (IMS-MS) that is capable of rapidly detecting small molecules that bind to amyloid precursors, identifying the interacting protein species and defining the mode of inhibition. Using this method we have classified a variety of small molecules that are potential inhibitors of human islet amyloid polypeptide (hIAPP) aggregation or amyloid-beta 1-40 aggregation as specific, nonspecific, colloidal or non-interacting. We also demonstrate the ability of IMS-MS to screen for inhibitory small molecules in a 96-well plate format and use this to discover a new inhibitor of hIAPP amyloid assembly

Aortic distensibility decreases with AAA formation, but is not affected by TAFI-inhibition, in the Angiotensin II model of AAA.

<p>The aorta was imaged in longitudinal section using the Vevo2100 scanner, and the distensibility measured using ECG-gated images and VevoVasc software. Panel A shows aortic wall distensibility in all mice with AAA (MA-TCK26D6 treated and sham treated). One week following initiation of Ang II infusion, mice received either MA-TCK26D6 or control (NaCl 0.9%) as an IV injection via the femoral vein. Panel B and C There was no difference in the change of distensibility by week 3 in mice receiving treatment with MA-TCK26D6 at 1 week compared with controls (data shown is change in distensibility between week 1 and week 3). Data is shown as mean±standard deviation, *** p = 0.001 compared to baseline by student t-test.</p

There is no effect of TAFI inhibition of the growth of an established AAA in the Angiotensin II model.

<p>AAA were induced in hyperlipidaemic mice by infusion of Ang II 750 ng/kg/min. After 1 week, mice were treated with a single injection of either MA-TCK26D6 or NaCl control. AAA progression in both groups was evaluated at 2 weeks post injection using Vevo2100 pre-clinical ultrasound scanning, and 3D reconstructions of the aortic segment at risk of AAA formation were created. Panel A Aortic diameter, Panel B Aortic volume, Panel C The process of creating the 3D aortic reconstruction. Panel D Example 3D reconstruction showing AAA progression over time (0, 1 and 3 weeks post Angiotensin II infusion). Data is shown as mean±standard deviation.</p