TGCT is a morbid benign tumour that rarely metastasizes in lungs and lymph nodes resulting in death within a median of 21.5 months post-diagnosis of malignancy. Where pexidartinib has received FDA approval for TGCT, it is implicated with drug-limiting liver toxicity. This led to drug screening and the discovery of DCC-3014 which inhibits CSF1R with an IC₅₀ of 3.7 nmol/L [44]. Human osteoclast precursor cells require CSF1 or receptor activator of nuclear factor kappa B ligand for differentiation [44]. In an in vitro assay, DCC-3014 blocked osteoclast differentiation and maturation with an IC₅₀ of 9.3 nmol/L [44]. The drug exhibits a safe and efficacious pharmacokinetic profile. It is highly aqueous with minimal inhibition of drugs interacting with cytochrome P450 (CYP) enzymes or human Ether-à-go-go-Related Gene (hERG) channel, marking its stability in liver microsomes and low risk of QT interval (Q and T electrical rhythm of the heart) prolongation [44]. Proven pre-clinical efficacy has encouraged further investigation where it has shown a decline in the tumour burden of TGCT patients [45]. It disrupts autocrine and paracrine signalling between inflammatory and TGCT cells [45].

A dose escalation study for TGCT has shown 30 mg for 5 days as a loading dose with a repetition of 30 mg twice weekly in cycles of 28 days to decrease non-classical monocytes [45]. Symptomatic improvements were observed in terms of pain, swelling, and range of motion [45]. Vimseltinib is currently being evaluated in phase I/II as a single agent for TGCT and solid tumours and in combination with anti-programmed death ligand 1 (PD-L1) antibodies against advanced or metastatic sarcomas [45].

Amongst all 7 kinase inhibitors, DCC-3014 binds with the lowest binding energy, having the most stable conformation and proving to be the most promising lead compound in the venture of drug development (Figure 4). Named vimseltinib, findings of phase I clinical trials demonstrate it to be a well-tolerated oral drug in patients with advanced sarcoma and TGCT [44]. DCC-3014 interacts with Met637, exhibiting exceptional target specificity. The kinase selectivity pocket differs by a single residue at Met637 for CSF1R and Leu644 for tyrosine-protein kinase KIT (c-KIT) [14]. The drug targets the JMD and maintains the kinase in an autoinhibitory conformation by binding with Trp550 which stabilizes the activation loop in a DFG-out conformation [44]. Vimseltinib (DCC-3014) is thus termed a selective switch-control CSF1R inhibitor [44]. Moreover, it is not a substrate of the P-glycoprotein efflux pump as it strongly binds with Cys666 which is a residue of the ATP binding site, thus evading one of the most significant drug resistance mechanisms, making it a promising drug candidate against cancers overexpressing CSF1R.

ARRY-382 is being evaluated for ovarian cancer, triple-negative breast cancer, head and neck squamous cell cancer, bladder cancer, metastatic colorectal cancer, pancreatic ductal adenocarcinoma, gastric cancer, advanced unresectable melanoma, advanced PD-L1 positive non-small cell lung cancer (NSCLC). Having a maximum tolerated dose (MTD) of 300 mg in combination with pembrolizumab, the trial has reported limited clinical benefits in addition to patients experiencing dose-limiting toxicities exhibiting an increase in transaminases (10.5–83.3%) and increased creatine phosphokinase (18.2–50.0%) [46].

ARRY-382 exhibits promising pharmaceutical potential (IC₅₀ = 9 nmol/L) [47] as it interacts with Cys666, Thr663, and Met637. Where Met637 interaction indicates high target specificity, strong interaction with Cys666 shows that it also evades the p-glycoprotein efflux pump. Thr663 is known to be a gatekeeper residue [26, 42]. The drug interacts with Trp550 as well as Tyr665, exhibiting similar efficacy as well as the mechanism of action of the prototypic CSF1R inhibitor—pexidartinib [39]. Additionally, it binds with Asp796 which stabilizes the DFG-out conformation of the activation loop, further stabilizing the autoinhibitory conformation of CSF1R kinase. The van der Waals forces are generated by the hydrophobic pocket formed by Val596, Ala614, Lys616, Val647, Thr663, and Leu785 [47]. The salt bridge is not conserved amongst Glu633 and Lys616, which causes CSF1R to be catalytically inactive (Figure 5).

Sorafenib interacts with Met637, exhibiting ligand-binding specificity. However, it maintains an autoinhibitory conformation by interacting with different residues forming a hydrophobic pocket of Val647, Val596, and Ala614. It forms a strong Pi-sigma bond with Leu588 and Met637 which are residues determining surface specificity. It regulates the activation loop to maintain CSF1R kinase in an autoinhibitory conformation by forming a conventional hydrogen bond with the Asp670. It evades the P-glycoprotein efflux pump, exhibiting great cellular potency, which is determined via its Pi-Pi interactions with Tyr665 (Figure 6).

Being a multikinase inhibitor, sorafenib inhibits cell proliferation as well as angiogenesis by inhibiting rapidly accelerated fibrosarcoma (RAF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) pathways in addition to CSF1R. Associated with wide-ranging adverse effects, it showed 3 months longer median survival for hepatocellular carcinoma (HCC) than those given placebo [48], however, recent phase III trial has given camrelizumab (C) plus rivoceranib precedence over sorafenib for HCC based on significantly prolonged progression-free survival (PFS), overall survival (OS) and overall response rate (ORR) [49]. Nevertheless, it received FDA approval in 2006 for renal cell carcinoma after exhibiting a two-fold increase in PFS [50].

Sotuletinib (BLZ945) is a brain-penetrant, oral CSF1R inhibitor that has demonstrated a reduction in TAM recruitment, tumour growth, and resistance to programmed cell death 1 (PD-1) inhibitors in animal models of intracranial glioblastoma multiforme (GBM). Recommended phase II dose (RP2D) is 1,200 mg/day (4 days on/10 days off) for single-agent BLZ945. The MTD was 700 mg/day (4 days on/10 days off) for BLZ945 + spartalizumab (anti-PD-1 antibody) in patients having cancers with upregulated TAMs including GBM and pancreatic cancer [51]. Dose-limiting toxicities (DLTs) included elevated hepatic enzymes whereas grade 3 adverse events increased in the combination arm as opposed to monotherapy [51]. The drug causes TAM depletion concomitant with CD8⁺ infiltration which has been observed in breast cancer cell lines as well as cervical cancer cell lines [51].

The most potent CSF1R inhibitor BLZ-945 (IC₅₀ = 1.2 nmol/L) exhibits carbon-hydrogen (C-H) interaction with gatekeeper residue Thr663 and ATP-binding site residue Cys666. An in vivo study demonstrated that CSF1R inhibitors show exceptional cellular potency [50% effective concentration (EC₅₀) = 0.104–0.245 µmol/L] [52]. These drugs have a p-glycoprotein efflux ratio < 2, suggesting that these are not substrates of p-glycoprotein [multidrug resistance protein 1 (MDR-1)]. Therefore, the drug is being analyzed in a positron emission tomography (PET) imaging technique of CSF1R in the brain [52]. BLZ-945 forms a Pi-sulphur bond with Met637, highlighting its distinct selectivity and inhibitory potential. However, prolonged exposure has shown resistance that develops due to hyperactivation of PI3K which can be overcome using combination therapy [53]. Moreover, it interacts within the hydrophobic pocket formed by Val596, Ala614, Val647, Leu785, as well as Lys616. The Glu633 and Lys616 salt bridge is not conserved which maintains CSF1R in an inactive conformation. Moreover, it forms an alkyl bond with Phe797 of the DFG motif which stabilizes the activation loop in an inactive state (Figure 7).

Chiauranib inhibits tumor angiogenesis, tumor cell mitosis, and chronic inflammatory microenvironment by targeting angiogenesis-related kinases [VEGF receptor 2 (VEGFR2), VEGFR1, VEGFR3, PDGF receptor alpha (PDGFRα), and c-KIT], mitosis-related kinase Aurora B and chronic inflammation related kinase CSF1R [54]. Chiauranib binds with Met637 and Leu588, exhibiting specificity, and Cys666, depicting evasion of drug resistance via p-glycoprotein efflux pump. Exhibiting exceptional specificity, it has limited off-kinase effects demonstrating better clinical safety and efficacy against relapsed or refractory small cell lung cancer (SCLC). Currently, in the phase 3 trial for SCLC, the drug is being examined at an oral dose of 500 mg once daily, 21 days as a cycle until disease progression (NCT04830813). It interacts within the hydrophobic pocket formed by Val596, Phe797, Leu785, Glu633, and Val647 and maintains CSF1R kinase in a DFG-out conformation by binding with Phe797, stabilizing it in an autoinhibitory conformation. Pi-Pi interactions with Trp550 stabilize the activation loop in an inactive conformation, making it a potent inhibitor (Figure 8). Recent phase III trials suggest a single-digit nanomolar range of IC₅₀ [55].

Dovitinib is a multi-kinase inhibitor that interacts with Met637, indicating its avidity for CSF1R. It forms a hydrogen bond with the activation loop residue Asp796, thus stabilizing the DFG-out conformation (Figure 9) which is essential for maintaining an autoinhibitory state of the protein. Moreover, interactions with Thr663 and Cys666 have been observed, indicative of its promising therapeutic potential in terms of exhibiting better cellular potency as well as evading resistance mechanisms. It exhibits similar interactions of the aforementioned kinase inhibitors forming a hydrophobic pocket by Leu785, Ala614, Val695, Val647, and Ile636. Moreover, the salt bridge interaction is hindered rendering the catalytic activity ineffective whereas the hydrophobic interaction aids prolonged drug-receptor binding.

Recent evidence shows dovitinib inhibits topoisomerase I and II by adjusting within the minor groove of the DNA and preventing decatenation elicited by topoisomerase [56]. The findings have been confirmed via molecular docking as well as DNA cleavage assays. Currently, in the phase III trial for renal cell carcinoma and phase II trial for breast cancer, dovitinib has shown promising potential for solid tumours with an IC₅₀ of 36 nmol/L against CSF1R [57]. In vitro studies declared dovitinib as an intense anti-proliferative agent against glioma [57]. However, clinical trials have reported various adverse effects due to which the drug has not been FDA-approved yet.

Edicotinib (JNJ-40346527), chiauranib, and sorafenib are carboxamides that are in clinical trials for various cancers. Chiauranib and sorafenib are multikinase inhibitors whereas edicotinib is a selective CSF1R inhibitor. Surprisingly, edicotinib interacts via alkyl bonds (Figure 10) with no interaction with residues determining specificity (Met637) or therapeutic potential (Thr663, Cys666). Edicotinib has an IC₅₀ of 3.2 nmol/L which is contrary to the results exhibited by molecular docking [58]. This suggests that the brain-penetrant CSF1R inhibitor edicotinib binds at a site other than the kinase domain of CSF1R warranting further investigation.

Targeted receptor inhibition and drug efficacy is determined via various pharmacokinetic factors including binding energy and IC₅₀. Binding energy highlights drug-receptor binding, structural affinity, and/or lead-target conformational stability. It determines the strength of interaction between the lead molecule and target receptor, thereby playing an essential role in lead optimization and subsequently, drug development. It is calculated via various computational methods and requires apt simulations to derive accurate results.

IC₅₀ is validated by in vitro methods using cell lines. Determining the correlation between in silico binding energies and IC₅₀ values is governed by the accuracy of computational methods employed as well as cell line authenticity. Generally, a strong correlation between IC₅₀ and binding energies is suggestive of the accuracy of computational methods employed in predicting binding energies, which conforms with the findings of this study, thus validating the methodology. DCC-3014 (vimseltinib), ARRY-382, and BLZ-945 are selective CSF1R inhibitors having the lowest binding energies which elucidates their strong structural affinity and conformational stability with the target kinase. The concomitant IC₅₀ values are in concordance with the predicted binding energies which confirm the reliability of the docking methodology elicited.

In conclusion, molecular docking comprises three major steps, namely target identification, lead generation elicited via high throughput screening, and lead optimization. Similar to the results of in vitro and in vivo analysis, in silico analysis cannot be solely used for determining drug efficacy, however, computational modelling can predict the promising potential of leads in a time-efficient and cost-effective manner.

The in silico findings evaluate target receptor CSF1R kinase binding sites and subsequently elucidate the downstream effects emerging due to drug inhibition by interacting with particular amino acid residues forming these sites. The authenticity and reliability of the computational docking employed have been refined via docking protocols, as well as by a comparative analysis elicited between in silico findings and results reported by peer-reviewed in vitro studies as well as clinical trials. The study analysis concludes that Met637-bound drugs with the lowest binding energies show exceptional conformational stability and have lesser off-site adverse effects. Similarly, molecular drug interactions with the hydrophobic pocket of CSF1R formed by Val596, Ala614, Val647, Leu785, and Lys616 help determine pharmacodynamic parameters including potency, dosing and administration. According to this study, the leads DCC-3014, ARRY-382 and BLZ-945 exhibit promising potential as pharmaceutical drugs based on their pharmacokinetic and pharmacodynamic properties analysed computationally.

This study contributes to a better understanding of CSF1R kinase aberrancy in cancer, the promising potential in targeting it, and subsequently reprogramming TAMs to disrupt the tumour microenvironment.