Covalent fragment libraries in drug discovery-Design, synthesis, and screening methods.

As the development of drugs with a covalent mode of action is becoming increasingly popular, well-validated covalent fragment-based drug discovery (FBDD) methods have been comparatively slow to keep up with the demand. In this chapter the principles of covalent fragment reactivity, library design, synthesis, and screening methods are explored in depth, focussing on literature examples with direct applications to practical covalent fragment library design and screening. Further, questions about the future of the field are explored and potential useful advances are proposed.

Co-ordinated control of the Aurora B abscission checkpoint by PKCε complex assembly, midbody recruitment and retention.

A requirement for PKCε in exiting from the Aurora B dependent abscission checkpoint is associated with events at the midbody, however, the recruitment, retention and action of PKCε in this compartment are poorly understood. Here, the prerequisite for 14-3-3 complex assembly in this pathway is directly linked to the phosphorylation of Aurora B S227 at the midbody. However, while essential for PKCε control of Aurora B, 14-3-3 association is shown to be unnecessary for the activity-dependent enrichment of PKCε at the midbody. This localisation is demonstrated to be an autonomous property of the inactive PKCε D532N mutant, consistent with activity-dependent dissociation. The C1A and C1B domains are necessary for this localisation, while the C2 domain and inter-C1 domain (IC1D) are necessary for retention at the midbody. Furthermore, it is shown that while the IC1D mutant retains 14-3-3 complex proficiency, it does not support Aurora B phosphorylation, nor rescues division failure observed with knockdown of endogenous PKCε. It is concluded that the concerted action of multiple independent events facilitates PKCε phosphorylation of Aurora B at the midbody to control exit from the abscission checkpoint.

Multiparameter Kinetic Analysis for Covalent Fragment Optimization by Using Quantitative Irreversible Tethering (qIT).

Chemical probes that covalently modify cysteine residues in a protein-specific manner are valuable tools for biological investigations. Covalent fragments are increasingly implemented as probe starting points, but the complex relationship between fragment structure and binding kinetics makes covalent fragment optimization uniquely challenging. We describe a new technique in covalent probe discovery that enables data-driven optimization of covalent fragment potency and selectivity. This platform extends beyond the existing repertoire of methods for identifying covalent fragment hits by facilitating rapid multiparameter kinetic analysis of covalent structure-activity relationships through the simultaneous determination of Ki , kinact and intrinsic reactivity. By applying this approach to develop novel probes against electrophile-sensitive kinases, we showcase the utility of the platform in hit identification and highlight how multiparameter kinetic analysis enabled a successful fragment-merging strategy.

An Octahedral Cobalt(III) Complex with Axial NH3 Ligands that Templates and Selectively Stabilises G-quadruplex DNA.

Guanine-rich sequences of DNA are known to readily fold into tetra-stranded helical structures known as G-quadruplexes (G4). Due to their biological relevance, G4s are potential anticancer drug targets and therefore there is significant interest in molecules with high affinity for these structures. Most G4 binders are polyaromatic planar compounds which π-π stack on the G4's guanine tetrad. However, many of these compounds are not very selective since they can also intercalate into duplex DNA. Herein we report a new class of binder based on an octahedral cobalt(III) complex that binds to G4 via a different mode involving hydrogen bonding, electrostatic interactions and π-π stacking. We show that this new compound binds selectivity to G4 over duplex DNA (particularly to the G-rich sequence of the c-myc promoter). This new octahedral complex also has the ability to template the formation of G4 DNA from the unfolded sequence. Finally, we show that upon binding to G4, the complex prevents helicase Pif1-p from unfolding the c-myc G4 structure.

High-Throughput Kinetic Analysis for Target-Directed Covalent Ligand Discovery.

Cysteine-reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high-quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan-reactive compounds. Quantitative irreversible tethering (qIT), a general method for screening cysteine-reactive small molecules based upon the maximization of kinetic selectivity, is described. This method was applied prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and guide further optimization. The power of this technique is highlighted by the identification of a Cdk2-selective allosteric (type IV) kinase inhibitor whose novel mode-of-action could be exploited therapeutically.

Clickable 5'-γ-ferrocenyl adenosine triphosphate bioconjugates in kinase-catalyzed phosphorylations.

Clickable co-substrate: A tri-functional 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) derivative containing a clickable site was synthesized. This compound is an effective co-substrate in kinase-catalyzed phosphorylation reactions, which can be detected by both electrochemical and immunoassay detection methods. The clickable reaction site makes direct modification possible, which greatly expands its application.

Cellular senescence: a double-edged sword in the fight against cancer.

Over the last few decades, it has become apparent that oncogenic proliferative signals are coupled to a variety of growth inhibitory responses, such as the induction of apoptotic cell death or irreversible cell cycle arrest known as 'cellular senescence'. Thus, both apoptosis and cellular senescence are thought to act as important tumor suppression mechanisms. Unlike apoptotic cells, however, senescent cells remain viable for long periods of time and accumulate with increasing age in various organs and tissues. Moreover, recent studies reveal that although cellular senescence initially functions as a tumor suppressive process, it may eventually exhibit tumor-promoting effects. Therefore, it is conceivable that accumulation of senescent cells during the ageing process in vivo may contribute to the age-related increase in cancer incidence. In this review, we provide an update and perspective on recent advances made in understanding the deleterious side effects of cellular senescence.

A tri-copper(II) complex displaying DNA-cleaving properties and antiproliferative activity against cancer cells.

A new disubstituted terpyridine ligand and the corresponding tri-copper(II) complex have been prepared and characterised. The binding affinity and binding mode of this tri-copper complex (as well as the previously reported mono- and di-copper analogues) towards duplex DNA were determined by using UV/Vis spectroscopic titrations and fluorescent indicator displacement (FID) assays. These studies showed the three complexes to bind moderately (in the order of 10(4) M(-1)) to duplex DNA (ct-DNA and a 26-mer sequence). Furthermore, the number of copper centres and the nature of the substituents were found to play a significant role in defining the binding mode (intercalative or groove binding). The nuclease potential of the three complexes was investigated by using circular plasmid DNA as a substrate and analysing the products by agarose-gel electrophoresis. The cleaving activity was found to be dependent on the number of copper centres present (cleaving potency was in the order: tri-copper>di-copper>mono-copper). Interestingly, the tri-copper complex was able to cleave DNA without the need of external co-reductants. As this complex displayed the most promising nuclease properties, cell-based studies were carried out to establish if there was a direct link between DNA cleavage and cellular toxicity. The tri-copper complex displayed high cytotoxicity against four cancer cell lines. Of particular interest was that it displayed high cytotoxicity against the cisplatin-resistant MOLT-4 leukaemia cell line. Cellular uptake studies showed that the tri-copper complex was able to enter the cell and more importantly localise in the nucleus. Immunoblotting analysis (used to monitor changes in protein levels related to the DNA damage response pathway) and DNA-flow cytometric studies suggested that this tri-copper(II) complex is able to induce cellular DNA damage.

Arylstibonic acids are potent and isoform-selective inhibitors of Cdc25a and Cdc25b phosphatases.

Arylstibonates structurally resemble phosphotyrosine side chains in proteins and here we addressed the ability of such compounds to act as inhibitors of a panel of mammalian tyrosine and dual-specificity phosphatases. Two arylstibonates both possessing a carboxylate side chain were identified as potent inhibitors of the protein tyrosine phosphatase PTP-ß. In addition, they inhibited the dual-specificity, cell cycle regulatory phosphatases Cdc25a and Cdc25b with sub-micromolar potency. However, the Cdc25c phosphatase was not affected demonstrating that arylstibonates may be viable leads from which to develop isoform specific Cdc25 inhibitors.

Acrylamide fragment inhibitors that induce unprecedented conformational distortions in enterovirus 71 3C and SARS-CoV-2 main protease.

RNA viruses are critically dependent upon virally encoded proteases to cleave the viral polyproteins into functional proteins. Many of these proteases exhibit a similar fold and contain an essential catalytic cysteine, offering the opportunity to inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that target the active site cysteine of the 3C protease (3Cpro) of Enterovirus 71, the causative agent of hand, foot and mouth disease in humans, altering the substrate binding region. Further, we re-purpose these hits towards the main protease (Mpro) of SARS-CoV-2 which shares the 3C-like fold and a similar active site. The hit fragments covalently link to the catalytic cysteine of Mpro to inhibit its activity. We demonstrate that targeting the active site cysteine of Mpro can have profound allosteric effects, distorting secondary structures to disrupt the active dimeric unit.

Identification of the first structurally validated covalent ligands of the small GTPase RAB27A.

Rab27A is a small GTPase, which mediates transport and docking of secretory vesicles at the plasma membrane via protein-protein interactions (PPIs) with effector proteins. Rab27A promotes the growth and invasion of multiple cancer types such as breast, lung and pancreatic, by enhancing secretion of chemokines, metalloproteases and exosomes. The significant role of Rab27A in multiple cancer types and the minor role in adults suggest that Rab27A may be a suitable target to disrupt cancer metastasis. Similar to many GTPases, the flat topology of the Rab27A-effector PPI interface and the high affinity for GTP make it a challenging target for inhibition by small molecules. Reported co-crystal structures show that several effectors of Rab27A interact with the Rab27A SF4 pocket ('WF-binding pocket') via a conserved tryptophan-phenylalanine (WF) dipeptide motif. To obtain structural insight into the ligandability of this pocket, a novel construct was designed fusing Rab27A to part of an effector protein (fRab27A), allowing crystallisation of Rab27A in high throughput. The paradigm of KRas covalent inhibitor development highlights the challenge presented by GTPase proteins as targets. However, taking advantage of two cysteine residues, C123 and C188, that flank the WF pocket and are unique to Rab27A and Rab27B among the >60 Rab family proteins, we used the quantitative Irreversible Tethering (qIT) assay to identify the first covalent ligands for native Rab27A. The binding modes of two hits were elucidated by co-crystallisation with fRab27A, exemplifying a platform for identifying suitable lead fragments for future development of competitive inhibitors of the Rab27A-effector interaction interface, corroborating the use of covalent libraries to tackle challenging targets.

Cell cycle-dependent regulation of the forkhead transcription factor FOXK2 by CDK·cyclin complexes.

Several mammalian forkhead transcription factors have been shown to impact on cell cycle regulation and are themselves linked to cell cycle control systems. Here we have investigated the little studied mammalian forkhead transcription factor FOXK2 and demonstrate that it is subject to control by cell cycle-regulated protein kinases. FOXK2 exhibits a periodic rise in its phosphorylation levels during the cell cycle, with hyperphosphorylation occurring in mitotic cells. Hyperphosphorylation occurs in a cyclin-dependent kinase (CDK)·cyclin-dependent manner with CDK1·cyclin B as the major kinase complex, although CDK2 and cyclin A also appear to be important. We have mapped two CDK phosphorylation sites, serines 368 and 423, which play a role in defining FOXK2 function through regulating its stability and its activity as a transcriptional repressor protein. These two CDK sites appear vital for FOXK2 function because expression of a mutant lacking these sites cannot be tolerated and causes apoptosis.

A cancer-derived mutation in the PSTAIRE helix of cyclin-dependent kinase 2 alters the stability of cyclin binding.

Cyclin-dependent kinase 2 (cdk2) is a central regulator of the mammalian cell cycle. Here we describe the properties of a mutant form of cdk2 identified during large-scale sequencing of protein kinases from cancerous tissue. The mutation substituted a leucine for a proline in the PSTAIRE helix, the central motif in the interaction of the cdk with its regulatory cyclin subunit. We demonstrate that whilst the mutant cdk2 is considerably impaired in stable cyclin association, it is still able to generate an active kinase that can functionally complement defective cdks in vivo. Molecular dynamic simulations and biophysical measurements indicate that the observed biochemical properties likely stem from increased flexibility within the cyclin-binding helix.

The chemoselective one-step alkylation and isolation of thiophosphorylated cdk2 substrates in the presence of native cysteine.

The elucidation of signalling pathways relies heavily upon the identification of protein kinase substrates. Recent investigations have demonstrated the efficacy of chemical genetics using ATP analogues and modified protein kinases for specific substrate labelling. Here we combine N(6) -(cyclohexyl)ATPγS with an analogue-sensitive cdk2 variant to thiophosphorylate its substrates and demonstrate a pH-dependent, chemoselective, one-step alkylation to facilitate the detection or isolation of thiophosphorylated peptides.

A genetically-encoded crosslinker screen identifies SERBP1 as a PKCε substrate influencing translation and cell division.

The PKCε-regulated genome protective pathway provides transformed cells a failsafe to successfully complete mitosis. Despite the necessary role for Aurora B in this programme, it is unclear whether its requirement is sufficient or if other PKCε cell cycle targets are involved. To address this, we developed a trapping strategy using UV-photocrosslinkable amino acids encoded in the PKCε kinase domain. The validation of the mRNA binding protein SERBP1 as a PKCε substrate revealed a series of mitotic events controlled by the catalytic form of PKCε. PKCε represses protein translation, altering SERBP1 binding to the 40 S ribosomal subunit and promoting the assembly of ribonucleoprotein granules containing SERBP1, termed M-bodies. Independent of Aurora B, SERBP1 is shown to be necessary for chromosome segregation and successful cell division, correlating with M-body formation. This requirement for SERBP1 demonstrates that Aurora B acts in concert with translational regulation in the PKCε-controlled pathway exerting genome protection.

Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy.

Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with fluorescence lifetime imaging microscopy (FLIM) can identify G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

Cyclin D1 and cyclin D3 show divergent responses to distinct mitogenic stimulation.

D-type cyclins predominantly regulate progression through the cell cycle by their interactions with cyclin-dependent kinases (cdks). Here, we show that stimulating mitogenesis of Swiss 3T3 cells with phorbol esters or forskolin can induce divergent responses in the expression levels, localization and activation state of cyclin D1 and cyclin D3. Phorbol ester-mediated protein kinase C stimulation induces S phase entry which is dependent on MAPK activation and increases the levels and activation of cyclin D1, whereas forskolin-mediated cAMP-dependent protein kinase A stimulation induces mitogenesis that is independent of MAPK, but dependent upon mTor and specifically increases the level and activation of cyclin D3. These findings uncover additional levels of complexity in the regulation of the cell cycle at the level of the D-type cyclins and thus may have important therapeutic implications in cancers where specific D-cyclins are overexpressed.

Dose-dependent changes in cyclin D1 in response to 4-nitroquinoline 1-oxide-induced DNA damage.

Cells respond to DNA damage by either repairing the damage or committing to a death or senescence pathway, dependent on the level of damage sustained. In this study, we show that the protein levels of cyclin D1 and the CDK inhibitor, p21(CIP1), respond in a dose-dependent manner to the DNA damaging agent, 4-nitroquinoline 1-oxide (4NQO). Cyclin D1 responses were independent of p53 and resulted in a partial loss of Retinoblastoma protein phosphorylation. The differential responses of cyclin D1 and p21(CIP1) were associated with distinct cellular responses: in low dose treatments the cells recovered after a lag period whilst at medium and high doses, the cells died through seemingly distinct mechanisms. Our data suggest that the balance between cyclin D1 and p21(CIP1) following exposure to DNA damage may play a key role in determining the subsequent cellular responses.

Synthesis and O-phosphorylation of 3,3,4,4-tetrafluoroaryl-C-nucleoside analogues.

Enantioenriched tetrafluorinated aryl-C-nucleosides were synthesised in four steps from 1-benzyloxy-4-bromo-3,3,4,4-tetrafluorobutan-2-ol. The presence of the tetrafluorinated ethylene group is compatible with O-phosphorylation of the primary alcohol, as demonstrated by the successful preparation of the tetrafluorinated naphthyl-C-nucleotide.

Aziridinium ion ring formation from nitrogen mustards: mechanistic insights from ab initio dynamics.

Explicit solvent ab initio molecular dynamics simulations have been used to study the activation of the nitrogen mustards mechlorethamine and phosphoramide mustard into the aziridinium ion intermediates known to be involved in the alkylation of DNA. The simulations predict a concerted reaction occurring by way of neighboring-group participation with the nearby nucleophilic tertiary nitrogen, in agreement with previous theoretical studies. The calculated activation free energy of 20.4 kcal/mol for mechlorethamine agrees well with the value of 22.5 kcal/mol determined from available experimental data. Furthermore, these simulations indicate a dynamic transition state characterized by pronounced changes in the local water structure within the first hydration shell. Trajectories initiated from the transition-state region toward products reveal a reaction that proceeds directly to the solvent-separated ion pair. Failure to cross the transition state was observed in a small number of trajectories where the degree of coordination with the leaving group is less favorable for reaction. Direct vibrational excitation of mechlorethamine alone is not sufficient to induce reaction, even with energies far in excess of the activation energy. The simulations suggest that reactivity is strongly dependent on the degree of coordination and orientation of water molecules within the vicinity of the leaving group. The complete mechanism involving solvent reorganization, ionization of the C-Cl bond, and internal cyclization of the aziridinium ion ring was captured from elevated temperature simulations. Rate constants for aziridinium ion ring formation from both mechlorethamine and phosphoramide mustard (the alkylating moiety of cyclophosphamide) are calculated and compared with available pharmacokinetic data.