Skip to main content
NCBI home page
ACS Medicinal Chemistry Letters logoLink to ACS Medicinal Chemistry Letters
. 2026 Jun 4;17(7):1601–1607. doi: 10.1021/acsmedchemlett.6c00159

Design of PKC-Targeting Benzolactams as Gli Inhibitors

Marianna Haddad 1, Jo Chvatal 1, Alexandria Kifer 1, Jayannah Herdrich 1, McCarthy Poljak 1, Kelvin L Billingsley 1,*
PMCID: PMC13358976  PMID: 42445003

Abstract

Dysregulated Hedgehog signaling, driven by oncogenic Gli transcription factors, plays a central role in many cancers and other Gli-dependent diseases. Inhibition of Gli through Smoothened-independent mechanisms represents a promising strategy to overcome resistance to clinical Hedgehog pathway inhibitors. Herein we report the synthesis and structure–activity relationship analysis of benzolactam analogues derived from TPPB, a potent inhibitor of Gli signaling that suppresses Gli activity via a protein kinase C (PKC)-mediated mechanism. Strategic chemical modifications were introduced across positions of the benzolactam core to assess their effects on biological activity using Gli-reporter cell-based assays. Our findings identified key structural features required for PKC-mediated Gli inhibition, and computational modeling revealed novel interactions between the PKC C1 domain and benzolactam analogues with nanomolar potency. Together, these studies provide a framework for designing therapeutics targeting Gli-driven diseases resistant to current treatments.

Keywords: Hedgehog signaling pathway, Gli transcription factors, Protein kinase C, Benzolactam, Inhibitor, Structure−activity relationship

graphic file with name ml6c00159_0007.jpg

graphic file with name ml6c00159_0006.jpg

Glioma-associated oncogenes (Gli) are transcription factors that function as central regulators of the Hedgehog (Hh) signaling pathway by controlling gene expression. − Gli proteins play a critical role during embryogenesis and development, but their activity is normally low in healthy adult tissues. Loss of Gli regulation in adult tissues is highly oncogenic, contributing to tumor formation and progression. , In turn, dysregulated Gli signaling is implicated in a range of cancers and diseases, including basal cell carcinoma, medulloblastoma, and tumors of the lung, stomach, and colon. − Consequently, therapeutic development efforts have focused on the strategic design of small-molecule inhibitors that can suppress aberrant Gli activity. To this end, multiple drugs, including vismodegib, sonidegib, and glasdegib, have been transitioned to clinical settings. , All of these FDA-approved therapeutics target the seven-transmembrane protein Smoothened (Smo), a G-protein-coupled receptor that serves as a key regulatory component in the Hh pathway. However, these agents are ineffective for cancers that rely on a Smo-independent mechanism of Gli activation. , Moreover, Smo-targeted therapies are prone to acquired resistance through mutations in the drug-binding pocket, leading to aggressive tumor recurrence with limited treatment options. , The development of Smo-independent therapeutic strategies therefore remains a critical unmet need for combating Gli-driven cancers.

Multiple studies have identified the unique regulatory roles of protein kinase C (PKC) enzymes on Gli transcription factors. − Conventional and novel PKC isozymes, in particular, have been shown to modulate Gli activity through dual mechanisms, both MEK/ERK-dependent and -independent. − Importantly, these isoforms are also amenable to small-molecule modulation, providing actionable pharmacological inroads. Despite these well-established connections, the systematic application of PKC modulators to models of Hh signaling has remained underexplored. Accordingly, our previous studies sought to exploit the PKC–Gli axis by identifying small-molecule PKC modulators capable of suppressing downstream Gli signaling. − To this end, small-molecule library screening efforts led to the identification of benzolactam-based TPPB (1), a potent PKC activator that effectively inhibited Gli activity at nanomolar concentrations in numerous Gli-driven cell lines (Figure ). In addition, 1 was found to function via a Smo-independent mechanism, offering a complementary therapeutic strategy to current clinical Hh pathway inhibitors. These findings were further validated across multiple in vitro models of Hh signaling and supported at the transcriptional level through modulation of canonical Hh target genes, including Gli1 and Ptch1. , These studies established a pharmacological framework for PKC-mediated inhibition of Gli signaling and provided the biological rationale for subsequent in vivo evaluation. To enable these studies, we designed and executed a novel 12-step synthetic route to access this lead compound, and 1 subsequently demonstrated effective suppression of tumor growth in an allograft model of basal cell carcinoma.

1.

1

Open in a new tab

Structure of TPPB (1), a dipeptide inhibitor of the Hedgehog signaling pathway, and the benzolactam scaffold. The pharmacophore is highlighted in purple, and positions used for structure–activity analysis are shown in red, green, and blue. TPPB = (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)­phenyl)-2,4-pentadienoylamino)­benzolactam.

The potent activity of 1, combined with our streamlined synthetic route, provides an ideal platform for the systematic investigation of structure–activity relationships (SARs). Importantly, 1 has not yet undergone medicinal chemistry optimization to enhance its efficacy as a Gli inhibitor. Furthermore, 1 diverges from several characteristics typical of conventional drug-like compounds. Specifically, this lead compound violates Lipinski’s rule of five due to its high molecular weight and contains Brenk structural alerts, including Michael acceptors and a conjugated polyene system (Table S1). In this study, we therefore present a detailed SAR analysis of the benzolactam scaffold with dual objectives: to identify structural features that govern Gli inhibitory potency and to explore modifications that improve its drug-like properties.

Benzolactams are well-established PKC activators, and numerous studies have demonstrated that these agents can exhibit nanomolar affinity for the PKC C1 domain. − Our previous function-oriented synthesis analysis with 1 revealed multiple positions on the scaffold that are advantageous for analogue development. The pharmacophore (purple in Figure ) was maintained to ensure high-affinity engagement with the PKC C1 domain. , Peripheral groups at N1, C2, and C8 mediate membrane interactions during PKC activation by stabilizing the PKC–ligand–membrane complex and promoting persistent PKC signaling (Figure ). , To exploit these tunable elements, our goal was to systematically vary the R1, R2, and R3 substituents, focusing on hydrophobic components that enhance membrane intercalation. To more closely mimic the structural features of 1, the C8-amide motif was retained to facilitate systematic variation of the carbonyl-bound hydrophobic groups.

Benzolactam analogues 4 featuring unique R1 substituents were initially targeted in our chemical synthesis efforts. This strategy employed our copper-catalyzed amino acid arylation methodology , using intermediate 2 and various hydrophobic amino acids (Scheme ). However, the steric environment of the ortho-substituted electrophile 2 necessitated reoptimization of the reaction conditions. Several hydrophobic amino acidsincluding l-valine, l-leucine, l-phenylalanine, l-alanine, and l-methionineproved to be successful under the updated protocol. For example, reaction with l-valine provided 3a in 63% yield, while l-leucine, l-alanine, and l-phenylalanine afforded 3b (30%), 3c (42%), and 3d (27%), respectively. l-Methionine displayed lower efficiency, giving 3e in only 9% yield. The resulting Cu-coupling products 3a–3e were deprotected under acidic conditions, followed by intramolecular peptide bond formation, generating the eight-membered macrocycle and installing the benzolactam pharmacophore. The cyclization efficiency varied among intermediates 3a–3e. The formation of 4a and 4b from 3a and 3b was highly efficient, with yields of 60% and 77%, respectively. Alanine-derived 3c afforded only trace amounts of cyclization product 4c, whereas phenylalanine-based 3d gave a moderate yield of 4d (49%) along with a 20% yield of the C2-epimer (epi-4d). Similarly, 4e was obtained in 20% yield from 3e, with 6% epi-4e also observed. Overall, this synthetic approach provided access to novel benzolactam analogues featuring diverse hydrophobic groups at the R1 position and provided building blocks for the integration of new substituents at R2 and R3.

1. Synthesis of Benzolactam Analogues 4a–4e .

1

Open in a new tab

a Reaction conditions: (a) CuI, Cs2CO3, l-amino acid, DMSO, 90 °C. (b) TFA, CH2Cl2, 0 °C or 4 M HCl in 1,4-dioxane, rt. (c) BOP, HOBt, NMM, DMF, rt.

* The C2 epimer (epi-4d) was isolated in 20% yield.

** The C2 epimer (epi-4e) was isolated in 6% yield.

Analogues 5 required derivatization of the N1 position to introduce novel R2 substituents. Previously, we employed a reductive amination protocol with formaldehyde to accomplish this transformation, allowing for the synthesis of an N-methylated product. This method could be directly applied to benzolactam 4 analogues to furnish 5a, 5f, 5k, and 5m in 43–83% yield (Scheme ). However, incorporation of other alkyl groups proved to be challenging, likely due to increased steric hindrance from the larger alkyl aldehydes. As an alternative approach, analogues 4 were treated with the respective alkyl halides under mild basic conditions to provide 5b–5e, 5g–5j, and 5l in 34–50% yield. These complementary protocols thus enabled rapid diversification of the R2 substituent.

2. Synthesis of Benzolactam Analogues 5a–5m .

2

Open in a new tab

a Reaction conditions: (a) CH2O, AcOH, NaBH3CN, CH3CN, 0 °C. (b) Alkyl bromide, Cs2CO3, acetonitrile, 70 °C.

To mimic the structure of lead compound 1, a C8-amide motif was used to introduce new R3 groups into the benzolactam scaffold. This approach required selected analogues 5 to be converted to intermediates 6, which possess a C8-amine (Scheme ). This transformation was performed through adaptation of our previous protocol, in which primary alcohol protection was first carried out, followed by nitration and subsequent reduction to generate the amine 6. The resulting intermediate was then acylated with the appropriate acid chloride to install the amide group, followed by base-mediated deprotection to provide the final set of analogues 7 evaluated in this study (Scheme ). Building on our previous findings, analogues 7a–7h were designed to expand modifications at the R3 position by systematically varying the alkyl chain length, branching, and saturation to further refine the structural requirements for Gli inhibition. Valine- and phenylalanine-derived cyclized intermediates (5a and 5k) were selected for these advanced SAR studies because 5a maintained key structural elements at N1 and C2 found in lead compound 1 whereas 5k offered increased hydrophobicity at C1 and was available in sufficient quantities via our designed route. Using this strategy, amides bearing isovaleric (7a), dimethylbutyryl (7b), octyl (7c), 5-(4-(trifluoromethyl)­phenyl)­pentanoyl (7d), oleoyl (7e and 7g), and stearoyl (7f and 7h) substituents were successfully synthesized in high yields (51–96%).

3. Synthesis of Benzolactam Analogues 7a–7h .

3

Open in a new tab

a Reaction conditions: (a) Ac2O, NEt3, rt. (b) HNO3, Ac2O, 0 °C. (c) Pd/C, H2, rt. (d) (i) 6, acid chloride, NEt3, THF, rt; (ii) KOH, EtOH, rt. Yields in parentheses are for step (d).

* An additional hydrogenation step (c) was required for the synthesis of 7d.

Benzolactam analogues 4a–7h were evaluated in Hh pathway reporter-based assays to determine how structural modifications influence Gli inhibitory activity (Table ). Initial screening was performed using Shh-LIGHT2 cells, an NIH-3T3-derived cell line that produces Gli-dependent luciferase activity. Because signaling in this model is Smo-dependent, pathway activation was induced by cotreatment with the Smo agonist SAG (200 nM) alongside each test compound. After a 30 h incubation period, firefly luciferase output driven by Gli transcription was normalized to constitutive Renilla luciferase expression, enabling quantification of pathway inhibition relative to the SAG-treated control condition. All benzolactam analogues were analyzed using dose–response experiments to determine their half-maximal inhibitory concentrations (IC50) (Table ). To evaluate activity under Smo-independent conditions, compounds were also tested in Sufu-KO-LIGHT cells, which lack the Hh pathway regulator Suppressor of Fused (Sufu) and therefore exhibit constitutive Gli-mediated transcription. Using an analogous assay format, compound potency was determined by calculating IC50 values relative to the vehicle control. To benchmark these assays, we initially used the clinically relevant Smo antagonist vismodegib (Table ). Consistent with the distinct signaling mechanism in these models, , this FDA-approved Hh inhibitor displays strong Gli suppression effects in Shh-LIGHT2 cells but shows minimal activity in the drug-resistant Sufu-KO-LIGHT assay (Table ).

1. Evaluation of Benzolactams in Shh-LIGHT2 and Sufu-KO-LIGHT Cell Lines .

  IC50 (nM)
compound Shh-LIGHT2 cell line Sufu-KO-LIGHT cell line
vismodegib 37 ± 3 >5000
TPPB (1) 52 ± 7 145 ± 11
4a >5000 >5000
4b >5000 >5000
4d 2442 ± 484 >5000
epi-4d >5000 >5000
4e >5000 >5000
5a >5000 >5000
5b >5000 >5000
5c >5000 >5000
5d >5000 >5000
5e >5000 >5000
5f >5000 >5000
5g >5000 >5000
5h >5000 >5000
5i 4709 ± 702 1184 ± 295
5j >5000 >5000
5k 388 ± 75 3459 ± 1585
5l >5000 >5000
5m 4981 ± 2243 >5000
7a 826 ± 220 922 ± 457
7b 191 ± 61 263 ± 161
7c 347 ± 108 586 ± 299
7d 216 ± 55 2131 ± 1256
7e 21 ± 16 24 ± 13
7f 28 ± 6 82 ± 68
7g 70 ± 19 125 ± 74
7h 106 ± 18 316 ± 280
Open in a new tab
a

IC50 values represent the mean of n > 3 biological replicates ± s.e.m.

b

For Shh-LIGHT2 cells, Gli luciferase levels are normalized to constitutive Renilla luciferase values and expressed relative to 200 nM SAG (100%).

c

For Sufu-KO-LIGHT cells, Gli luciferase levels are normalized to CellTiter viability values and expressed relative to those of DMSO (100%).

d

Values from ref .

Structure–activity analyses revealed key structural features within the benzolactam scaffold that influence Gli inhibitory activity (Table ). To further benchmark these assays using a PKC modulator, lead compound 1 was exposed to both cell lines and exhibited high potency, with IC50 values of 52 ± 7 nM in Shh-LIGHT2 cells and 145 ± 11 nM in Sufu-KO-LIGHT cells. These results confirmed that Gli inhibition occurs downstream of Smo, which is consistent with our previous reports. Initially, we examined analogues 4a–4e and 5a–5m, where the majority of the benzolactam derivatives satisfy Lipinski’s rules and do not possess structural alerts (Table S1). Analogues 4a–4e, which contain hydrophobic R1 substituents but lack R2 and R3 groups, were largely inactive. Among this series, only 4d produced a measurable response in the Shh-LIGHT2 assay, although inhibition was observed only at micromolar concentrations and was absent in the Sufu-KO-LIGHT model. Notably, the epimer epi-4d showed no inhibitory activity in either cell line, potentially suggesting that the use of l-amino acid-derived building blocks is an appropriate strategy for analogue development. Benzolactams 5a–5m, which incorporate additional substituents at the R2 position, also exhibited substantially lower potency than lead compound 1. Although several members of this series displayed modest Gli inhibitory activity at micromolar concentrations, these effects were not consistently maintained across both cellular models. Interestingly, while the R1 and R2 substituents have been proposed to promote PKC–membrane interactions and enhance PKC signaling, , increasing hydrophobicity at either position or varying the log P of the analogues (Table S1) did not yield a corresponding improvement in Gli suppression.

In contrast to the limited activity observed for analogues 4a–4e and 5a–5m, benzolactams 7a–7h, which incorporate hydrophobic substituents at the C8-amide position, exhibited robust Gli inhibitory activity (Table ). Compounds 7a and 7b, which contain short, branched alkyl chains, displayed triple-digit nanomolar potency in both cell lines. Analogues 7c and 7d, featuring medium-length alkyl chains or aryl-containing motifs, produced inhibitory effects within a similar activity range. Notably, incorporation of longer saturated or monounsaturated chains further increased Gli inhibition. In particular, 7e and 7f maintained double-digit nanomolar IC50 values in both cellular models and exhibited enhanced potency relative to lead compound 1. Collectively, these results indicate that the C8-amide motif present in 1 and analogues 7a–7h plays a critical role in mediating Gli inhibitory activity downstream of Smo. Moreover, these findings suggest that introducing hydrophobic substituents at the R3 position may maximize activity, as many of the most potent C8-amide-bearing analogues (7e–7h) also possessed higher log P values (Table S1). As suggested by previous reports on benzolactams, elongated alkyl chains likely enhance membrane association during PKC activation; , however, short- or branched-chain C8-amide analogues may also serve as promising leads, as they maintain favorable physicochemical properties while avoiding structural alerts (Table S1).

These analyses in the Shh-LIGHT2 and Sufu-KO-LIGHT cellular models identified analogue 7e as a highly potent inhibitor of Gli activity with results statistically superior to those with 1. To demonstrate that the effects of 7e remain mediated by PKC, this analogue was subjected to assays in the Shh-LIGHT2 and Sufu-KO-LIGHT cell lines in the presence of PKC inhibitor GF 109203X (1 μM). Under these conditions, a dramatic loss of compound potency was observed. The IC50 value shifted to 1650 ± 623 nM in Shh-LIGHT2 cells. Similarly, 7e displayed significantly reduced activity in the presence of the PKC inhibitor in the Sufu-KO-LIGHT cell line, with the IC50 decreasing in potency to >5000 nM. These results suggest that the Gli inhibitory effects of 7e, like other PKC agonists, remain dependent on PKC. To further evaluate activity in an independent cellular context, we examined the ability of 7e to inhibit Gli-dependent differentiation of C3H10T1/2 mesenchymal stem cells into alkaline phosphatase-positive osteoblasts, a physiologically relevant model of Hh-mediated osteogenic differentiation, where it exhibited an IC50 of 22 ± 4 nM. To estimate the toxicity of 7e, cell viability was also measured in each cell line, and in all cases the CC50 was >5000 nM, suggesting that 7e may possess an appropriate therapeutic window. Lastly, we examined the metabolic stability of 7e in human liver microsome assays, where it displayed an intrinsic clearance of 3.2 ± 0.8 μL min–1 mg–1, which supports favorable metabolic stability. Overall, these findings establish 7e as a potent, PKC-dependent Gli inhibitor with promising cellular activity, low toxicity, and encouraging metabolic stability.

To further interpret the observed SAR trends, molecular docking studies were conducted using the PKCδ C1b domain to examine the binding interactions of the benzolactam core found in 5a relative to lead compound 1 and the potent analogue 7e (Figure ). As previously reported, the pharmacophoric elements of the benzolactam scaffold form three key hydrogen-bonding interactions with residues T242, L251, and G253 (Figure A). , In addition, a CH−π interaction is observed between the P241 side chain and the aromatic ring of the benzolactam core. Importantly, both 1 and 7e adopt a similar conformation and preserve these interactions while also establishing an additional contact between the C8-amide and M239 within the PKC C1 domain (Figure B,C), an interaction thathas been previously described for clinically relevant PKC ligands such as bryostatin 1 and ATX. For each ligand, this interaction occurs within an appropriate bonding distance (2.2–2.4 Å) and favorable hydrogen-bonding orientation (155–171°). Therefore, the C8-amide may play a role in ligand engagement with the PKC C1 domain, which is in agreement with the enhanced binding affinity displayed by 1 for the PKC C1 domain (K i = 11.9 ± 1.1 nM) relative to benzolactam 5a (K i = 334 ± 14 nM). , In addition, the hydrophobic substituent attached to the C8-amide may further extend toward the membrane environment, helping to shield polar regions of the protein surface and facilitating membrane intercalation during PKC activation and signaling. These findings provide a docking-based framework that may help interpret the enhanced activity of C8-amide-substituted analogues and will be further evaluated experimentally in future studies to assess their mechanistic relevance.

2.

2

Open in a new tab

Docking simulations of (A) benzolactam 5a, (B) lead compound 1, and (C) analogue 7e with the C1b domain of protein kinase C δ (PKCδ). (A) The benzolactam scaffold 5a forms hydrogen-bonding interactions with G253, L251, and T242, and a CH−π interaction with P241. (B, C) The C8-amide of 1 and 7e forms an additional interaction with M239.

In summary, this study presents a comprehensive investigation of benzolactam derivatives as modulators of the PKC-driven suppression of aberrant Gli signaling. Efficient synthetic strategies were established to generate analogues with targeted diversification at key positions in the scaffold. These compounds were subsequently evaluated through SAR studies in cellular models representing both Smo-dependent and Smo-independent modes of Gli activation as well as in a physiologically relevant differentiation model of endogenous Hh signaling. The combined biological data and molecular modeling analyses highlight the C8-amide functionality as a feature that may facilitate productive interaction between the benzolactam scaffold and the PKC C1 domain. Furthermore, incorporation of hydrophobic substituents was found to significantly enhance activity when localized to the C8-amide position, while analogous modifications at other regions yielded comparatively modest effects. Together, these findings enabled the identification of highly potent inhibitors, including 7e, which surpasses the activity of the previously reported lead compound 1 while also exhibiting low cellular toxicity and favorable metabolic stability in human liver microsome assays. Importantly, optimization of substituent size and branching at the C8-amide may also provide a means to balance potency with favorable drug-like properties. Overall, this work establishes a foundation for the rational design of next-generation PKC modulators and highlights a viable strategy for Smo-independent suppression of Gli activity.

Safety Statement

No unexpected or unusually high safety hazards were encountered.

Supplementary Material

ml6c00159_si_001.pdf (5.2MB, pdf)

Acknowledgments

This research was supported by funds granted by the College of Arts and Sciences at Loyola University Chicago.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.6c00159.

  • Experimental procedures and characterization of synthesized compounds; methods for biological assays in all cell lines and microsomes; 1H and 13C NMR spectra (PDF)

M.H., A.K., and M.P. synthesized and characterized compounds. J.C. and M.H. performed biological assays. J.H. conducting modeling experiments. K.L.B. guided the experimentation. M.H. and K.L.B. prepared the manuscript.

The authors declare no competing financial interest.

References

  1. Zhang Y., Beachy P. A.. Cellular and molecular mechanisms of Hedgehog signalling. Nat. Rev. Mol. Cell Biol. 2023;24(9):668–687. doi: 10.1038/s41580-023-00591-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Wu F., Zhang Y., Sun B., McMahon A. P., Wang Y.. Hedgehog signaling: From basic biology to cancer therapy. Cell Chem. Biol. 2017;24(3):252–280. doi: 10.1016/j.chembiol.2017.02.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Sigafoos A. N., Paradise B. D., Fernandez-Zapico M. E.. Hedgehog/GLI signaling pathway: Transduction, regulation, and implications for disease. Cancers (Basel) 2021;13(14):3410. doi: 10.3390/cancers13143410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hui C. C., Angers S.. Gli proteins in development and disease. Annu. Rev. Cell Dev. Biol. 2011;27:513–537. doi: 10.1146/annurev-cellbio-092910-154048. [DOI] [PubMed] [Google Scholar]
  5. Jing J., Wu Z., Wang J., Luo G., Lin H., Fan Y., Zhou C.. Hedgehog signaling in tissue homeostasis, cancers, and targeted therapies. Signal Transduction Targeted Ther. 2023;8(1):315. doi: 10.1038/s41392-023-01559-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Pak E., Segal R. A.. Hedgehog signal transduction: Key players, oncogenic drivers, and cancer therapy. Dev. Cell. 2016;38(4):333–344. doi: 10.1016/j.devcel.2016.07.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Sari I., Phi L., Jun N., Wijaya Y., Lee S., Kwon H.. Hedgehog signaling in cancer: A prospective therapeutic target for eradicating cancer stem cells. Cells. 2018;7(11):208. doi: 10.3390/cells7110208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ling G., Ahmadian A., Persson Å., Undén A., Afink G., Williams C., Uhlén M., Toftgård R., Lundeberg J., Pontén F.. PATCHED and p53 gene alterations in sporadic and hereditary basal cell cancer. Oncogene. 2001;20(53):7770–7778. doi: 10.1038/sj.onc.1204946. [DOI] [PubMed] [Google Scholar]
  9. Huang S. Y., Yang J.-Y.. Targeting the Hedgehog pathway in pediatric medulloblastoma. Cancers (Basel) 2015;7(4):2110–2123. doi: 10.3390/cancers7040880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Rimkus T. K., Carpenter R. L., Qasem S., Chan M., Lo H.-W.. Targeting the Sonic Hedgehog signaling pathway: Review of Smoothened and GLI inhibitors. Cancers (Basel) 2016;8(2):22. doi: 10.3390/cancers8020022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jamieson C., Martinelli G., Papayannidis C., Cortes J.. Hedgehog pathway inhibitors: A new therapeutic class for the treatment of acute myeloid leukemia. Blood Cancer Discovery. 2020;1(2):134–145. doi: 10.1158/2643-3230.BCD-20-0007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Yauch R. L., Dijkgraaf G. J., Alicke B., Januario T., Ahn C. P., Holcomb T., Pujara K., Stinson J., Callahan C. A., Tang T., Bazan J. F., Kan Z., Seshagiri S., Hann C. L., Gould S. E., Low J. A., Rudin C. M., de Sauvage F. J.. Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma. Science. 2009;326(5952):572–574. doi: 10.1126/science.1179386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nguyen N., Cho J.. Hedgehog pathway inhibitors as targeted cancer therapy and strategies to overcome drug resistance. Int. J. Mol. Sci. 2022;23(3):1733. doi: 10.3390/ijms23031733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Atwood S. X., Li M., Lee A., Tang J. Y., Oro A. E.. GLI activation by atypical protein kinase C ι/λ regulates the growth of basal cell carcinomas. Nature. 2013;494(7438):484–488. doi: 10.1038/nature11889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Neill G. W., Ghali L. R., Green J. L., Ikram M. S., Philpott M. P., Quinn A. G.. Loss of protein kinase Cα expression may enhance the tumorigenic potential of Gli1 in basal cell carcinoma. Cancer Res. 2003;63(15):4692–4697. [PubMed] [Google Scholar]
  16. Riobo N. A., Haines G. M., Emerson C. P. Jr.. Protein kinase C-δ and mitogen-activated protein/extracellular signal-regulated kinase-1 control GLI activation in Hedgehog signaling. Cancer Res. 2006;66(2):839–845. doi: 10.1158/0008-5472.CAN-05-2539. [DOI] [PubMed] [Google Scholar]
  17. Lauth M., Bergström Å., Toftgård R.. Phorbol esters inhibit the Hedgehog signalling pathway downstream of suppressor of fused, but upstream of Gli. Oncogene. 2007;26(35):5163–5168. doi: 10.1038/sj.onc.1210321. [DOI] [PubMed] [Google Scholar]
  18. Cai Q., Li J., Gao T., Xie J., Evers B. M.. Protein kinase C delta negatively regulates Hedgehog signaling by inhibition of Gli1 activity. J. Biol. Chem. 2009;284(4):2150–2158. doi: 10.1074/jbc.M803235200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tran U., Zhang G., Eom R., Billingsley K. L., Ondrus A. E.. Small molecule intervention in a protein kinase C–Gli transcription factor axis. ACS Chem. Biol. 2020;15(6):1321–1327. doi: 10.1021/acschembio.0c00355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mendoza M., Tran U., Zhang G. C., Leister J., To K., Malepeai-Tofaeono T., Ondrus A. E., Billingsley K. L.. Indolactam dipeptides as nanomolar Gli inhibitors. ACS Med. Chem. Lett. 2022;13(7):1036–1042. doi: 10.1021/acsmedchemlett.1c00562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tran U., Billingsley K. L.. Targeting the PKC-Gli axis: Bryostatin 1 as a Hedgehog pathway inhibitor. J. Nat. Prod. 2025;88(11):2789–2794. doi: 10.1021/acs.jnatprod.5c01072. [DOI] [PubMed] [Google Scholar]
  22. Haddad M., Wilber C., MacQueen J., Billingsley K.. Benzolactam-based Gli inhibitors: Synthesis and structural analysis. ACS Med. Chem. Lett. 2025;16(8):1655–1661. doi: 10.1021/acsmedchemlett.5c00324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Properties were obtained from SwissADME:; https://www.swissadme.ch/ (accessed 2026-03-17).
  24. Endo Y., Takehana S., Ohno M., Driedger P. E., Stabel S., Mizutani M. Y., Tomioka N., Itai A., Shudo K.. Clarification of the binding mode of teleocidin and benzolactams to the Cys2 domain of protein kinase Cδ by synthesis of hydrophobically modified, teleocidin-mimicking benzolactams and computational docking simulation. J. Med. Chem. 1998;41(9):1476–1496. doi: 10.1021/jm970704s. [DOI] [PubMed] [Google Scholar]
  25. Kozikowski A. P., Wang S., Ma D., Yao J., Ahmad S., Glazer R. I., Bogi K., Acs P., Modarres S., Lewin N. E., Blumberg P. M.. Modeling, chemistry, and biology of the benzolactam analogues of indolactam V (ILV). 2. Identification of the binding site of the benzolactams in the CRD2 activator-binding domain of PKCδ and discovery of an ILV analogue of improved isozyme selectivity. J. Med. Chem. 1997;40(9):1316–1326. doi: 10.1021/jm960875h. [DOI] [PubMed] [Google Scholar]
  26. Mach U. R., Lewin N. E., Blumberg P. M., Kozikowski A. P.. Synthesis and pharmacological evaluation of 8- and 9-substituted benzolactam-v8 derivatives as potent ligands for protein kinase C, a therapeutic target for Alzheimer’s disease. ChemMedChem. 2006;1(3):307–314. doi: 10.1002/cmdc.200500068. [DOI] [PubMed] [Google Scholar]
  27. Ma D., Tang G., Kozikowski A. P.. Synthesis of 7-substituted benzolactam-V8s and their selectivity for protein kinase C isozymes. Org. Lett. 2002;4(14):2377–2380. doi: 10.1021/ol026125l. [DOI] [PubMed] [Google Scholar]
  28. Ma D., Zhang T., Wang G., Kozikowski A. P., Lewin N. E., Blumberg P. M.. Synthesis of 7,8-disubstituted benzolactam-V8 and its binding to protein kinase C. Bioorg. Med. Chem. Lett. 2001;11(2):99–101. doi: 10.1016/S0960-894X(00)00609-0. [DOI] [PubMed] [Google Scholar]
  29. Haynes-Smith J., Diaz I., Billingsley K. L.. Modular total synthesis of protein kinase C activator (−)-indolactam V. Org. Lett. 2016;18(9):2008–2011. doi: 10.1021/acs.orglett.6b00614. [DOI] [PubMed] [Google Scholar]
  30. Chen J. K., Taipale J., Young K. E., Maiti T., Beachy P. A.. Small molecule modulation of Smoothened activity. Proc. Natl. Acad. Sci. U.S.A. 2002;99(22):14071–14076. doi: 10.1073/pnas.182542899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Beck, B. ; Chen, Y.-F. ; Dere, W. ; Devanarayan, V. ; Eastwood, B. J. ; Farmen, M. W. ; Iturria, S. J. ; Iversen, P. W. ; Kahl, S. D. ; Moore, R. A. ; Sawyer, B. D. ; Weidner, J. . Assay operations for SAR support. In Assay Guidance Manual; Markossian, S. , Grossman, A. , Brimacombe, K. , et al., Eds.; Eli Lilly & Company and the National Center for Advancing Translational Sciences: Bethesda, MD, 2004; https://www.ncbi.nlm.nih.gov/books/NBK91994/ (accessed 2026-03-17). [Google Scholar]
  32. Hom M. E., Ondrus A. E., Sakata-Kato T., Rack P. G., Chen J. K.. Bicyclic imidazolium inhibitors of Gli transcription factor activity. ChemMedChem. 2020;15(12):1044–1049. doi: 10.1002/cmdc.202000169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Toullec D., Pianetti P., Coste H., Bellevergue P., Grand-Perret T., Ajakane M., Baudet V., Boissin P., Boursier E., Loriolle F., Duhamel L., Charon D., Kirilovsky J.. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 1991;266(24):15771–15781. doi: 10.1016/S0021-9258(18)98476-0. [DOI] [PubMed] [Google Scholar]
  34. Hyman J. M., Firestone A. J., Heine V. M., Zhao Y., Ocasio C. A., Han K., Sun M., Rack P. G., Sinha S., Wu J. J., Solow-Cordero D. E., Jiang J., Rowitch D. H., Chen J. K.. Small-molecule inhibitors reveal multiple strategies for Hedgehog pathway blockade. Proc. Natl. Acad. Sci. U.S.A. 2009;106(33):14132–14137. doi: 10.1073/pnas.0907134106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nakagawa Y., Irie K., Yanagita R. C., Ohigashi H., Tsuda K.. Indolactam-V is involved in the CH/π interaction with Pro-11 of the PKCδ C1B domain: Application for the structural optimization of the PKCδ ligand. J. Am. Chem. Soc. 2005;127(16):5746–5747. doi: 10.1021/ja050447d. [DOI] [PubMed] [Google Scholar]
  36. Katti S., Igumenova T. I.. Structural insights into C1-ligand interactions: Filling the gaps by in silico methods. Adv Biol Regul. 2021;79:100784. doi: 10.1016/j.jbior.2020.100784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kozikowski A. P., Nowak I., Petukhov P. A., Etcheberrigaray R., Mohamed A., Tan M., Lewin N., Hennings H., Pearce L. L., Blumberg P. M.. New amide-bearing benzolactam-based protein kinase C modulators induce enhanced secretion of the amyloid precursor protein metabolite sAPPα. J. Med. Chem. 2003;46(3):364–373. doi: 10.1021/jm020350r. [DOI] [PubMed] [Google Scholar]
  38. Katti S. S., Krieger I. V., Ann J., Lee J., Sacchettini J. C., Igumenova T. I.. Structural anatomy of protein kinase C C1 domain interactions with diacylglycerol and other agonists. Nat. Commun. 2022;13(1):2695. doi: 10.1038/s41467-022-30389-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml6c00159_si_001.pdf (5.2MB, pdf)

Articles from ACS Medicinal Chemistry Letters are provided here courtesy of American Chemical Society

RESOURCES