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EGFR is a protein kinase whose aberrant activity is frequently involved in the development of non-small lung cancer (NSCLC) drug resistant forms. The allosteric inhibition of this enzyme is currently one among the most attractive approaches to design and develop anticancer drugs. In a previous study, we reported the identification of a hit compound acting as type III allosteric inhibitor of the L858R/T790M double mutant EGFR. Herein, we report the design, synthesis and in vitro testing of a series of analogues of the previously identified hit with the aim of exploring the structure-activity relationships (SAR) around this scaffold. The performed analyses allowed us to identify two compounds 15 and 18 showing improved inhibition of double mutant EGFR with respect to the original hit, as well as interesting antiproliferative activity against H1975 NSCLC cancer cells expressing double mutant EGFR. The newly discovered compounds represent promising starting points for further hit-to-lead optimisation.

Effective inactivation of the HER2-HER3 tumor driver has remained elusive because of the challenging attributes of the pseudokinase HER3. We report a structure-function study of constitutive HER2-HER3 signaling to identify opportunities for targeting. The allosteric activation of the HER2 kinase domain (KD) by the HER3 KD is required for tumorigenic signaling and can potentially be targeted by allosteric inhibitors. ATP binding within the catalytically inactive HER3 KD provides structural rigidity that is important for signaling, but this is mimicked, not opposed, by small molecule ATP analogs, reported here in a bosutinib-bound crystal structure. Mutational disruption of ATP binding and molecular dynamics simulation of the apo KD of HER3 identify a conformational coupling of the ATP pocket with a hydrophobic AP-2 pocket, analogous to EGFR, that is critical for tumorigenic signaling and feasible for targeting. The value of these potential target sites is confirmed in tumor growth assays using gene replacement techniques.

Effective inactivation of the HER2-HER3 tumor driver has remained elusive because of the challenging attributes of the pseudokinase HER3. We report a structure-function study of constitutive HER2-HER3 signaling to identify opportunities for targeting. The allosteric activation of the HER2 kinase domain (KD) by the HER3 KD is required for tumorigenic signaling and can potentially be targeted by allosteric inhibitors. ATP binding within the catalytically inactive HER3 KD provides structural rigidity that is important for signaling, but this is mimicked, not opposed, by small molecule ATP analogs, reported here in a bosutinib-bound crystal structure. Mutational disruption of ATP binding and molecular dynamics simulation of the apo KD of HER3 identify a conformational coupling of the ATP pocket with a hydrophobic AP-2 pocket, analogous to EGFR, that is critical for tumorigenic signaling and feasible for targeting. The value of these potential target sites is confirmed in tumor growth assays using gene replacement techniques.

Kinases are among the most successful drug targets. To date, 72 small-molecule kinase inhibitors (SMKIs) have been approved by the US FDA, together with ~500 SMKIs in clinical trials. Although the topic has been heavily reviewed in recent years, an overview that focused on the currently approved SMKIs in combination with the emerging kinase-targeting bifunctional molecules is absent. Herein, we first provide an updated overview of the approved SMKIs, with an emphasis on their binding modes, classified in groups of type I and II ATP-competitive inhibitors, type III and IV allosteric inhibitors, and covalent inhibitors. We then highlight the novel chemical modalities in kinase targeting by using different types of proximity-inducing bifunctional molecules for kinase degradation and modifications.

The heat shock protein 70 (Hsp70) family of molecular chaperones are highly expressed in tumors. Inhibitors containing a pyridinium-modified benzothiazole, such as JG-98, bind to a conserved, allosteric site in Hsp70, showing promising anti-proliferative activity in cancer cells. When bound to Hsp70, the charged pyridinium makes favorable contacts; however, this moiety also increases the inhibitor's fluorescence, giving rise to undesirable interference in biochemical and cell-based assays. Here, we explore whether the pyridinium can be replaced with a neutral pyridine. We report that pyridine-modified benzothiazoles, such as compound 17h (JG2-38), have reduced fluorescence, yet retain promising anti-proliferative activity (EC50 values ~0.1 to 0.07 µM) in breast and prostate cancer cell lines. These chemical probes are expected to be useful in exploring the roles of Hsp70s in tumorigenesis and cell survival.

Background: Chronic myeloid leukaemia is in principle a treatable malignancy but drug resistance is lowering survival. Recent drug discoveries have opened up new options for drug combinations, which is a concept used in other areas for preventing drug resistance. Two of these are (I) Axitinib, which inhibits the T315I mutation of BCR-ABL1, a main source of drug resistance, and (II) Asciminib, which has been developed as an allosteric BCR-ABL1 inhibitor, targeting an entirely different binding site, and as such does not compete for binding with other drugs. These drugs offer new treatment options. Methods: We measured the proliferation of KCL-22 cells exposed to imatinib–dasatinib, imatinib–asciminib and dasatinib–asciminib combinations and calculated combination index graphs for each case. Moreover, using the median–effect equation we calculated how much axitinib can reduce the growth advantage of T315I mutant clones in combination with available drugs. In addition, we calculated how much the total drug burden could be reduced by combinations using asciminib and other drugs, and evaluated which mutations such combinations might be sensitive to. Results: Asciminib had synergistic interactions with imatinib or dasatinib in KCL-22 cells at high degrees of inhibition. Interestingly, some antagonism between asciminib and the other drugs was present at lower degrees on inhibition. Simulations revealed that asciminib may allow for dose reductions, and its complementary resistance profile could reduce the risk of mutation based resistance. Axitinib, however, had only a minor effect on T315I growth advantage. Conclusions: Given how asciminib combinations were synergistic in vitro, our modelling suggests that drug combinations involving asciminib should allow for lower total drug doses, and may result in a reduced spectrum of observed resistance mutations. On the other hand, a combination involving axitinib was not shown to be useful in countering drug resistance.

Optimization of the side-chain of a phenyl indole scaffold identified from a high-throughput screening campaign for inhibitors of the AAA+ ATPase p97 is reported. The addition of an N-alkyl piperazine led to high potency of this series in a biochemical assay, activity in cell-based assays, and excellent pharmaceutical properties. Molecular modeling based on a subsequently obtained cryo-EM structure of p97 in complex with a phenyl indole was used to rationalize the potency of these allosteric inhibitors.

Optimization of the side-chain of a phenyl indole scaffold identified from a high-throughput screening campaign for inhibitors of the AAA+ ATPase p97 is reported. The addition of an N-alkyl piperazine led to high potency of this series in a biochemical assay, activity in cell-based assays, and excellent pharmaceutical properties. Molecular modeling based on a subsequently obtained cryo-EM structure of p97 in complex with a phenyl indole was used to rationalize the potency of these allosteric inhibitors.

Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.

Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.

Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.

Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.

The strategy of applying kinase inhibitors as effective therapeutic agents has achieved unparalleled success in the past decade, while it is still a daunting challenge to develop kinase inhibitors with desirable selectivity among different kinase families. This chapter describes the basic conceptions in the field of kinase selectivity, available profiling technologies, and quantification of selectivity levels. The chapter highlights the different selectivity of approved kinase inhibitors, which include non-covalent type I and II ATP-competitive inhibitors, allosteric inhibitors, one lipid kinase inhibitor, and covalent inhibitors. Most approved kinase inhibitors are multitarget or unselective inhibitors that bind with the highly conserved ATP pocket, high selectivity is more likely to be achieved among kinases with only few closely related homologs or unique structural features in binding sites.

Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.

A high-throughput screen to discover inhibitors of p97 ATPase activity identified an indole amide that bound to an allosteric site of the protein. Medicinal chemistry optimization led to improvements in potency and solubility. Indole amide 3 represents a novel uncompetitive inhibitor with excellent physical and pharmaceutical properties that can be used as a starting point for drug discovery efforts.

Development of allosteric inhibitors into efficient drugs is hampered by their indirect mode-of-action and complex structure-kinetic relationships. To enablethe design of efficient allosteric drugs targeting the polymerase of hepatitis C virus(NS5B), the interaction characteristics of three non-nucleoside compounds (filibuvir, VX-222, and tegobuvir) inhibiting HCV replication via NS5B have been analyzed. Since there was no logical correlation between the anti-HCV replicative and enzyme inhibitory effects of the compounds, surface plasmon resonance biosensor technology was used to resolve the mechanistic, kinetic, thermodynamic and chemodynamic features of their interactions with their target and their effect on itsinteraction with RNA. Tegobuvir could not be seen to interact with NS5B at all while filibuvir interacted in a single reversible step (except at low temperatures) and VX-222 in two serial steps, interpreted as an induced fit mechanism. Both filibuvir and VX-222 interfered with the interaction between NS5B and RNA. They competed for binding to the enzyme, suggesting that they had a common inhibition mechanism and identical or overlapping binding sites. The greater anti-HCV replicative activityof VX-222 over filibuvir is hypothesized to be due to a greater allosteric conformational effect, resulting in the formation of a less catalytically competent complex. In addition, the induced fit mechanism of VX-222 gives it a kinetic advantage over filibuvir, exhibited as a longer residence time. These insights have important consequences for the selection and optimization of new allosteric NS5Binhibitors.

Human herpesviruses (HHVs) make up one of the most prevalent viral families and are the etiological agents of a variety of devastating human illnesses that lack safe and effective treatments. All HHVs express a dimeric serine protease that is essential to the viral life cycle. Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr), a gamma subfamily member, was chosen as the model system for developing broad-spectrum herpesviral inhibitors. Of particular interest are KSHV Pr inhibitors that act allosterically, either by dimer dissociation or other mechanisms. A small-molecule KSHV Pr inhibitor has been identified that traps a transient allosteric pocket at the protease dimer interface. The binding of this molecule (DD2) has been shown to prevent the dimerization of KSHV Pr, which is required for its activation. This novel binding pocket is bioinformatically predicted to be conserved across all HHV proteases and DD2 has been shown to have activity against the serine protease from cytolomegalovirus (CMV) a member of the beta herpesvirus subfamily. Additionally, methodologies have been developed to identify new inhibitors via a high throughput screen, elucidate the mechanism and binding modes of screening hits, and test for efficacy in a cell culture viral replication assay. This work presents methodologies and demonstrates the feasibility for identifying and using small-molecule allosteric inhibitors to target a conserved enzyme family that is conserved across a viral family.

Human herpesviruses (HHVs) make up one of the most prevalent viral families and are the etiological agents of a variety of devastating human illnesses that lack safe and effective treatments. All HHVs express a dimeric serine protease that is essential to the viral life cycle. Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr), a gamma subfamily member, was chosen as the model system for developing broad-spectrum herpesviral inhibitors. Of particular interest are KSHV Pr inhibitors that act allosterically, either by dimer dissociation or other mechanisms. A small-molecule KSHV Pr inhibitor has been identified that traps a transient allosteric pocket at the protease dimer interface. The binding of this molecule (DD2) has been shown to prevent the dimerization of KSHV Pr, which is required for its activation. This novel binding pocket is bioinformatically predicted to be conserved across all HHV proteases and DD2 has been shown to have activity against the serine protease from cytolomegalovirus (CMV) a member of the beta herpesvirus subfamily. Additionally, methodologies have been developed to identify new inhibitors via a high throughput screen, elucidate the mechanism and binding modes of screening hits, and test for efficacy in a cell culture viral replication assay. This work presents methodologies and demonstrates the feasibility for identifying and using small-molecule allosteric inhibitors to target a conserved enzyme family that is conserved across a viral family.