7.5 Role of Indoleamine 2,3-dioxygenase Inhibitor ...
Immunotherapy is a promising strategy for cancer therapy.Therapeutic vaccination of cancer patients is an approach to stimulate their immune system against cancer cells.This approach,however, showed limited efficacy in vivo.Cancer cells can develop enzymatic mechanisms allowing tumors to resist or escape immune rejection.Among the enzymes involved, indoleamine 2,3-dioxygenase (IDO) represents an important factor.IDO is an intracellular monomeric heme-containing enzyme that catalyzes the rapid degradation of tryptophan (Trp) in the initial step of the kynurenine pathway, the de novo biosynthetic route for nicotinamide adenine dinucleotide(NAD) production.This results in a local Trp depletion that severely affects the proliferation of T lymphocytes and is thereby profoundly immunosuppressive.Many studies have identified IDO as an enzyme that is silent in most tissues.However, it is highly active in the placenta and is required for the maternal immune tolerance of the fetus.Many types of human tumors express IDO in a constitutive manner.The expression of IDO in human carcinomas is also associated with poor prognosis and reduced survival.Because IDO expression is strongly induced by interferon-gamma,even tumors that do not constitutively express IDO may do so when exposed to inflammatory conditions, for example, resulting from an ongoing immune response.IDO thus noticeably protects foreign cells against immune rejection.Based on its implication in immunosuppression, and particularly in cancer, IDO clearly represents an attractive target for the development of inhibitors.
Indoleamine-2,3-dioxygenase 1 (IDO1) is a monomeric 45 kDa hemoglobin-containing oxidase, it is one key dioxygenase in the process of tryptophan-kynurenine metabolism, and usually acts as one immune escape strategy for cancer cells.Recent investigations have shown that IDO1 upregulation is associated with increased Treg numbers in malignant melanoma.It also has been identified that Treg can participate in the silencing of T cells response by acting to enforce a dominant negative regulation on T cells activation, proliferation and cytokine production.By coopting IDO1 activity, tumor cells can effectively mask their rapid cellular growth and evade the host immune surveillance.Therefore, inhibition of IDO1 activity by small-molecule drug was one valuable strategy for cancer patient to re-establish immunogenic response [21].
Since the participation of IDO1 in oncogenesis was first uncovered in 2003, thousands of bioactive small molecule inhibitors have been reported, nevertheless, only five compounds are undergoing clinical trials.Among these, 1-Methyl-D-tryptophan (D1MT) is in phase I/II clinical studies at NewLink Genetics Cop.for the treatment of metastatic prostate cancer, acute myeloid leukemia,primary malignant brain tumors, metastatic pancreatic cancer, metastatic breast cancer, metastatic melanoma, as well as NSCLC.The N-hydroxyamidine INCB024360 is in phase II/III clinical trials at Incyte Corp., used as a monotherapy as well as in combination with various antibodies, for the treatment of advanced or metastatic cancers.Another IDO1 inhibitor, the imidazole GDC-0901(navoximod) is in phase I clinical trials at NewLink Genetics Corp.in collaboration with Genentech Inc.in subjects with recurrent advanced solid tumors.The in vivo study revealed that treatment with GDC-0901 led to a significant reduction of tumor size which was highly relevant to its functional immune response.Two 2nd/3rd generation IDO1 inhibitors, PF-0684003(EOS-200271) and BMS-986205 (ONO-7701), have also entered clinical trials within the last few months.PF-0684003, from Pfizer Inc./iTeos Therapeutics SA, is in phase I clinical trials for the treatment of patients with grade IV glioblastoma or grade III anaplastic glioma.BMS-986205 is being evaluated at Bristol-Myers Squibb Co.in phase I/II advanced cancer [22].
Many of the first known IDO1 inhibitors are Trp or indole derivatives, the oldest one being 2,5-dihydro-L-phenylalanine (Figure 8.3B) and the most widely used being 1-methyl-L-tryptophan(L1MT).L1MT passes all structural filters, but its moderate activity (Ki = 19-53 μM) has been used in the past as justification for publishing equally weak inhibitors.
Compounds with quinone or iminoquinone functionalities display high activities in enzymatic IDO1 inhibition assays.Interestingly, for TDO already in 1961 the redox- active compounds catechol, hydroquinone, p-quinone, L-dihydroxyphenylalanine, and L-epinephrine were described as inhibitors.For IDO1, the activity of menadione and related quinone compounds was first reported in the patent literature in 2006.Quinones could either inhibit IDO1 by specific interaction with the enzyme active site or by redox-cycling with the reducing cofactors or by chemical reactivity toward nucleophilic amino acid side chains.
Besides enzymatic IDO1 inhibition results provided in all references, in some works also cellular inhibition results were reported.While in one article describing quinone inhibitors, activities reduced by 2-3 orders of magnitude were reported in cellular assays as compared to enzymatic assays,59 in later works similar activities were found in both contexts.In vivo data for menadione administered to a mouse melanoma model showed reduced tumor volume and IDO1- mediated efficacy.Some benzofuranoquinones were shown not to damage microtubules and the actin cytoskeleton, not to generate measurable levels of oxidative stress, and not to induce cytotoxicity, at variance with β-lapachone.
While the exact mode of action of the structurally highly diverse quinone compounds remains an open question, they fail most filters for promiscous compounds and it seems evident that they do not represent a suitable starting point for developing highly selective IDO1 inhibitors.(https://www.daowen.com)
The first known natural-product inhibitors of IDO1 are the moderately active norharman or β-carboline derivatives.Other natural-product inhibitors include benzomalvin E, halicloic acids A and B, and thielavin derivatives, which all showed activity in an IDO1 enzymatic assay, but no further analysis was carried out.Tryptanthrin derivatives showed activity both in enzymatic and in cellular IDO1 inhibition assays, in a T-cell proliferation assay, in a surface plasmon resonance binding assay, and in Lewis lung cancer tumor-bearing mice.The phytochemical galanal showed activity in enzymatic and in cellular IDO1 inhibition assays, but cell viability was not reported.
Most inhibitor scaffolds for IDO1 contain at least two aromatic rings, exceptions being the scaffolds, which in some cases seem to compensate small size by chemical reactivity.O-Benzylhydroxylamine and derivatives with activities in the single-digit micromolar range were described in a patent in 2009, reporting both enzymatic and cellular assay results.Phenylhydrazine was discovered by fragment screening and reported to be a potent enzymatic and cellular IDO1 inhibitor.However, its selectivity for IDO1 over other heme proteins and its reversibility remain to be investigated.The scaffolds benzyl mercaptan and S-benzylisothiourea were tested in enzymatic and in cellular assays.However, cell viability was not reported, and the compounds fail most cheminformatics filters for their reactivity.Modification of the earlier described 2(3H)-benzothiazolethione scaffold based on its docked conformation yielded a series of N-phenylthiosemicarbazides, the best one displaying an enzymatic IC50 value of 1.2 μM.Because of their different chemical functionalities, these compounds show different suitability profiles,spanning from an almost filled radar chart to an almost empty one.However, a common issue for these small compounds will be their selectivity.
4-Phenylimidazole (4PI), a known heme binder, was described early on as IDO1 inhibitor and gained additional attention due to its cocrystallization with IDO1 in 2006.4PI derivatives up to 10-fold more potent than the parent compound were designed by a structure-based approach.Fungistatic drugs of the imidazole type such as miconazole and econazole were discovered in two independent screens to be active against IDO1, while similar 1,2,4-triazole drugs such as fluconazole were completely inactive.Structure-based modifications of the 1-substituted imidazole antifungal scaffold led to more soluble but less potent compounds.NewLink Genetics developed further 4-substituted phenylimidazoles with nanomolar potency but a low LE because they featured a long extension into the B pocket.Fusion of the two aromatic rings of 4PI by an aliphatic carbon atom, however, enhanced potency with less impact on efficiency.Replacement of the imidazole motif in the fused compound by other heterocycles such as 1,2,3-triazoles led to much less active compounds, while the orientation of the fused imidazole ring seemed to be less important.
In summary, the phenylimidazole scaffold provides a promising starting point for the development of IDO1 inhibitors, as its binding mode to the active site is known through X-ray crystallography, it passes all structural filters, rational modifications have been shown to be feasible, and sensible structure-activity relationships are observed.The fact that 4PI and the fungistatic imidazoles inhibit various heme enzymes suggests that specificity for IDO1 needs to be achieved through optimized molecular recognition by the B pocket.
4-Aryl-1,2,3-triazoles were discovered by pharmacophore modeling in 2010, with the parent compound displaying an enzymatic IC50 value of 60 μM and passing all structural filters.In one subsequent work, only derivatives with similar or lower potency were reported.However, the rationally designed triazole 3 (MMG-0358) showed nanomolar activities in both enzymatic and cellular assays.Additionally, it demonstrated low cellular toxicity and a high selectivity for IDO1 over TDO.Nanomolar cellular IC50 values were also reported for the N-phenyl-1,2,3-triazol-4-amine compounds from Vertex.As mentioned above, bioisosteric replacement of imidazole by 1,2,3-triazole in the fused compounds from NewLink Genetics led to only moderately active compounds.
In conclusion, the 1,2,3-triazole scaffold provides an interesting alternative to the imidazole scaffold, as it could exhibit better specificity with respect to other heme proteins.
HTS of Incyte’s corporate collection led to the discovery of N-hydroxyamidines as potent,reversible, competitive IDO1 inhibitors.The most potent compound of this series, compound 4,displayed enzymatic and cellular IC50 values of 67 and 19 nM, suppressed kynurenine generation in vivo, and inhibited melanoma growth in a mouse model.Modeling of the binding of 4 to IDO1 assumed that the oxygen of the hydroxyamidine binds to the heme iron and forms a hydrogen bond with the aniline nitrogen.The phenyl ring was placed inside the A pocket, producing a tight fit,while the amino substituent on the furazan ring could form a hydrogen bond to the propanoic acid group on the heme ring.Compound 4 was used extensively as reference compound by other groups and was erroneously sold under the name of the clinical candidate 1 by several chemical vendors.The correct structure of 1 (epacadostat, CAS no.1204669-58-8) was disclosed and belongs to a further optimized series of N-hydroxyamidines with extension to the B-pocket.The scaffold fails the ALARM NMR filter because of its furazan group.
One IDO1 inhibitor from the NCI Diversity Set with a Ki of 1.5 μM was reported in 2006.The selenic anti-inflammatory antioxidant ebselen reportedly inhibits IDO1 by binding to several of its cysteine residues.The prodrug candesartan cilexetil was found to inhibit IDO1 with an enzymatic IC50 value of 12 μM.The active form of this angiotensin II receptor antagonist, obtained after ester hydrolysis, was reported to be a much weaker inhibitor.Replacing the ester by different amides led to 10-fold increase in enzymatic activity but did not improve the cellular IC50 value of 2.6 μM of candesartan cilexetil.A series of benzenesufonyl hydrazides were tested in enzymatic and cellular IDO1 inhibition assays, displaying nanomolar IC50 values in both tests.However, cell viability was not tested.Amgen reported compound 2 to inhibit IDO1 with an IC50 value of 3 μM, and its selectivity for IDO1 over IDO2 and TDO was demonstrated.This compound was later cocrystallized with IDO1 and used for rational compound optimization, which led to the discovery of imidazothiazole derivatives occupying both pocket A and pocket B.Nanomolar enzymatic inhibition activities were obtained with a urea linker, the best compound showing an enzymatic IC50 value of 77 nM.The pharma company Curadev reported aminonitriles as potent IDO1 inhibitors.However, most of these fail the PAINS filter as phenolic Mannich bases and the Lilly MedChem filter because of their cyanomethylamine functionality.In a second report, extensive enzymatic, cellular, and in vivo data were provided for a different scaffold, diamino substituted furopyridines.Here, the tolerance of bulky substituents in both 3 and 7 positions makes it difficult to imagine how this scaffold could fit into the IDO1 active site.A 2-aminophenylurea scaffold and related compounds for IDO1 inhibition yield low nanomolar to picomolar cellular IC50 values.These are the most potent reported compounds, but little data are available about their specificity and mode of action.In this case, their three-dimensional structure does not seem to be able to fill the IDO1 active site without clashing with surrounding protein residues.In addition to these patented compounds.