Identification of Potent Reverse Indazole Inhibitors for HPK1.

pubs.acs.org/acsmedchemlett Letter Identification of Potent Reverse Indazole Inhibitors for HPK1 Elsie C. Yu,* Joey L. Methot, Xavier Fradera, Charles A. Lesburg, Brian M. Lacey, Phieng Siliphaivanh, Ping Liu, Dustin M. Smith, Zangwei Xu, Jennifer A. Piesvaux, Shuhei Kawamura, Haiyan Xu, J. Richard Miller, Mark Bittinger, and Alexander Pasternak Cite This: ACS Med. Chem. Lett. 2021, 12, 459−466 ACCESS Metrics & More Read Online sı Supporting Information * Article Recommendations ABSTRACT: Hematopoietic progenitor kinase (HPK1), a negative regulator of TCR-mediated T-cell activation, has been recognized as a novel antitumor immunotherapy target. Structural optimization of kinase inhibitor 4 through a systematic twodimensional diversity screen of pyrazolopyridines led to the identification of potent and selective compounds. Crystallographic studies with HPK1 revealed a favorable water-mediated interaction with Asp155 and a salt bridge to Asp101 with optimized heterocyclic solvent fronts that were critical for enhanced potency and selectivity. Computational studies of model systems revealed differences in torsional profiles that allowed for these beneficial protein−ligand interactions. Further optimization of molecular properties led to identification of potent and selective reverse indazole inhibitor 36 that inhibited phosphorylation of adaptor protein SLP76 in human PBMC and exhibited low clearance with notable bioavailability in in vivo rat studies. KEYWORDS: HPK1 inhibitor, MAP4K1 inhibitor, reverse indazole, HPK1 crystal structure H with HPK1 wild-type (WT) mice. While certain mouse models showed improved responses with triggered protective immunity of anti-PD-L1, the HPK1 knockdown phenotype alone was able to suppress tumor formation in a select mouse model.7,11 These experimental findings suggest a small molecule HPK1 inhibitor may be able to promote antitumor immunity as a single agent or combination therapy with antiPD1 (Tables 1 and 2).10,12−14 Despite its discovery two decades ago, there are few selective small molecule inhibitors of HPK1 reported.7 Thus, a highthroughput screening (HTS) campaign was initiated to identify potent kinase inhibitor leads. Among the hits, numerous potent indazoles were identified (e.g., 1−2); however, the pharmacokinetic (PK) profiles of indazole 1 and azaindazole 2 were poor. The high rat plasma clearances greater than hepatic clearance suggested potential issues of glucuronidation of the exposed hydrogen bond donor.15,16 Among the hits, we also found reverse indazoles (RI) 3 and 4, that while greater than 75-fold less potent, showed appreciably ematopoietic progenitor kinase 1 (HPK1) is a member of a family of mitogen-activated protein kinase kinase kinase kinase (MAP4K) of Ste20 serine/threonine kinases and is also known as MAP4K1.1 HPK1 is exclusively expressed in the hematopoietic compartment, with the implication that HPK1 is involved in the regulation of signaling in hematopoietic lineages.2 In addition to negative T cell regulation via the T cell receptor (TCR), HPK1 also mediates T-cell suppression through PGE2 signaling.3,4 As a regulator of MAPK activity in T cells, HPK1 works by destabilization of the SLP76 adapter protein signaling complex via a proteasomal degradation pathway to transmit TCR signals to elicit downstream cellular events.5 At the same time, HPK1 may be triggered by various stimuli, such as growth factors, stress, inflammation, and differentiation cues, to transmit signals in different cell types including B-cells, dendritic cells (DC), and natural killer (NK) cells.6 Phosphorylation of SLP76 SH2 domain on S376 by HPK1 leads to dissociation of the LAT complex and proteasomal degradation of SLP76.7,8 This dissociation prevents downstream T-cell activation and proliferation. A recent study has noted that HPK1 kinase activity suppresses the immune functions of CD4+ T cells, CD8+ T cells, and DCs and inactivation of the HPK1 domain is capable of eliciting antitumor immune responses.9,10 Mouse genetic experiments suggest a kinase-activity-dependent combination benefit with anti-PD1 in a murine colon adenocarcinoma model. HPK1 kinase dead mice demonstrated improved antitumor response to anti-PD1 therapy, compared © 2021 American Chemical Society Received: December 21, 2020 Accepted: February 23, 2021 Published: March 1, 2021 459 https://dx.doi.org/10.1021/acsmedchemlett.0c00672 ACS Med. Chem. Lett. 2021, 12, 459−466 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett Table 1. Potency and In Vivo PK Profiles of HTS Hits Letter Table 2. SAR of C(6)-Aryl Analogues a Measured at [Tracer] ≈ Kd[Tracer]. bMeasured at [ATP] ≈ Km[ATP]. cSelectivity expressed as the ratio of JAK1 or JAK2 IC50 over HPK1 IC50. dMale rat Wistar Han at intravenous dose of 0.1 mg/kg as a solution in DMSO/PEG400/H2O. eMale rat Wistar Han at intravenous dose of 2 mg/kg as a solution in 30% captisol/70% water. CLp = total plasma clearance; Vd = volume of distribution; MRT = mean residence time. lower plasma clearance and represented a better starting point to achieve a desirable DMPK profile. Based on the promising PK of reverse indazole hits 3 and 4 and the absence of SAR from the screen, we generated an enriched two-dimensional diversity set to develop the N(1)and C(6)- substitution SAR off the pyrazolopyridine core,18 from which we discovered para-piperazine phenyl-containing RI 5 that contains the para-methoxy pyridine ring from hit 1. We counterscreened against a small panel of kinases, including JAK1 and JAK2, as they were commonly observed off-targets of the HTS hits. CDK2 was incorporated because of its essential role in cell proliferation. In addition to similarity sequence analyses, linear-regression plot analyses provided clarity as to which kinases could act as surrogates for other offtarget kinases that could pose significant toxicity or T-cell function issues. JAK1 correlated well with TYK2; JAK2 correlated well with JAK3; and CDK2 was a good surrogate for both CDK5 and GSK3A. Reverse indazole 5 showed an excellent improvement in potency by over a log unit versus 4 and, additionally, increased selectivity against JAK1 and JAK2 (83×, 83×). This C(6) pyridyl piece, though, proved to be unselective against CDK2 (4×). We then further developed the C(6) SAR with compounds with a static para-piperazine phenyl N(1) group to improve potency and selectivity. An unsubstituted phenyl group (6) led to a significant loss in potency (820 nM). oTolyl group replacement (7) further supported that ortho substituents are tolerated, but nonetheless, there was a 4-fold potency loss from 5 (410 nM). 2-Fluoro-6-methylphenyl (9) yielded a similar level of potency (121 nM) as the initial molecule 5 in a more lipophilic space (cLogD, 2.77). Measured at [Tracer] ≈ Kd[Tracer]. bMeasured at [ATP] ≈ Km[ATP]. cSelectivity expressed as the ratio of JAK1, JAK2, or CDK2 IC50 over HPK1 IC50. a Moreover, this group showed better selectivity against CDK2, (...truncated)