First-in-Human Study of the Reversible BTK Inhibitor Nemtabrutinib in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia and B-Cell Non-Hodgkin Lymphoma.

Nemtabrutinib is an orally bioavailable, reversible inhibitor of Bruton tyrosine kinase (BTK) and C481S mutant BTK. We evaluated the safety, pharmacology, and antitumor activity of nemtabrutinib in relapsed/refractory hematologic malignancies. Forty-eight patients with chronic lymphocytic leukemia (CLL), B-cell non-Hodgkin lymphoma (NHL), or Waldenström macroglobulinemia (WM), relapsed/refractory after ≥2 prior therapies were enrolled in the open-label, single-arm, phase I MK-1026-001 study (NCT03162536) to receive nemtabrutinib 5 to 75 mg once daily in 28-day cycles. Dose finding progressed using a 3 + 3 dose escalation design. Primary endpoints were safety and the recommended phase II dose (RP2D). Among 47 treated patients, 29 had CLL, 17 had NHL, and 1 had WM. Grade ≥3 treatment-emergent adverse events occurred in 37 (89%), most commonly neutropenia (11; 23.4%), febrile neutropenia (7; 14.9%), and pneumonia (7; 14.9%). The RP2D was 65 mg daily. An overall response rate of 75% was observed in patients with CLL at 65 mg daily. SIGNIFICANCE: This first-in-human phase I study demonstrates the safety and preliminary efficacy of nemtabrutinib in patients with relapsed/refractory B-cell malignancies. These data support further exploration of nemtabrutinib in larger clinical studies. This article is featured in Selected Articles from This Issue, p. 5.

Preclinical evaluation of combination nemtabrutinib and venetoclax in chronic lymphocytic leukemia.

Inhibitors of B cell receptor (BCR) signaling such as the Bruton's tyrosine kinase (BTK) inhibitors are effective therapeutics for chronic lymphocytic leukemia (CLL). The first-in-class covalent BTK inhibitor, ibrutinib, produces durable responses in most CLL patients; however, complete responses are only observed in a minority of patients. B cell lymphoma 2 (BCL2), an anti-apoptotic protein that contributes to CLL cell survival, has also been investigated as a therapeutic target. The BCL2 inhibitor venetoclax is effective in patients with CLL and can produce undetectable minimal residual disease, allowing discontinuation of therapy. In combination, ibrutinib and venetoclax have shown preclinical synergy and clinical efficacy. Nemtabrutinib is a next generation, reversible inhibitor of BTK that potently inhibits BCR signaling in treatment-naïve and ibrutinib-refractory CLL cells ex vivo. The clinical efficacy of combining BTK inhibitors with BCL2 inhibitors motivated us to evaluate the novel combination of nemtabrutinib and venetoclax. In vitro studies show that nemtabrutinib and venetoclax are not antagonistic to each other. In an adoptive transfer CLL mouse model, mice treated with nemtabrutinib and venetoclax had prolonged survival compared to mice treated with ibrutinib and venetoclax. Our preclinical studies further validate the combination of BTK inhibitors with venetoclax and justify further investigation of combining nemtabrutinib with venetoclax in CLL.

Inhibition of the BTK-IDO-mTOR axis promotes differentiation of monocyte-lineage dendritic cells and enhances anti-tumor T cell immunity.

Monocytic-lineage inflammatory Ly6c+CD103+ dendritic cells (DCs) promote antitumor immunity, but these DCs are infrequent in tumors, even upon chemotherapy. Here, we examined how targeting pathways that inhibit the differentiation of inflammatory myeloid cells affect antitumor immunity. Pharmacologic inhibition of Bruton's tyrosine kinase (BTK) and the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) or deletion of Btk or Ido1 allowed robust differentiation of inflammatory Ly6c+CD103+ DCs during chemotherapy, promoting antitumor T cell responses and inhibiting tumor growth. Immature Ly6c+c-kit+ precursor cells had epigenetic profiles similar to conventional DC precursors; deletion of Btk or Ido1 promoted differentiation of these cells. Mechanistically, a BTK-IDO axis inhibited a tryptophan-sensitive differentiation pathway driven by GATOR2 and mTORC1, and disruption of the GATOR2 in monocyte-lineage precursors prevented differentiation into inflammatory DCs in vivo. IDO-expressing DCs and monocytic cells were present across a range of human tumors. Thus, a BTK-IDO axis represses differentiation of inflammatory DCs during chemotherapy, with implications for targeted therapies.

The new small tyrosine kinase inhibitor ARQ531 targets acute myeloid leukemia cells by disrupting multiple tumor-addicted programs.

Tyrosine kinases have been implicated in promoting tumorigenesis of several human cancers. Exploiting these vulnerabilities has been shown to be an effective anti-tumor strategy as demonstrated for example by the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, for treatment of various blood cancers. Here, we characterize a new multiple kinase inhibitor, ARQ531, and evaluate its mechanism of action in preclinical models of acute myeloid leukemia. Treatment with ARQ531, by producing global signaling pathway deregulation, resulted in impaired cell cycle progression and survival in a large panel of leukemia cell lines and patient-derived tumor cells, regardless of the specific genetic background and/or the presence of bone marrow stromal cells. RNA-seq analysis revealed that ARQ531 constrained tumor cell proliferation and survival through Bruton's tyrosine kinase and transcriptional program dysregulation, with proteasome-mediated MYB degradation and depletion of short-lived proteins that are crucial for tumor growth and survival, including ERK, MYC and MCL1. Finally, ARQ531 treatment was effective in a patient-derived leukemia mouse model with significant impairment of tumor progression and survival, at tolerated doses. These data justify the clinical development of ARQ531 as a promising targeted agent for the treatment of patients with acute myeloid leukemia.

Preclinical efficacy for a novel tyrosine kinase inhibitor, ArQule 531 against acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is the most common type of adult leukemia. Several studies have demonstrated that oncogenesis in AML is enhanced by kinase signaling pathways such as Src family kinases (SFK) including Src and Lyn, spleen tyrosine kinase (SYK), and bruton's tyrosine kinase (BTK). Recently, the multi-kinase inhibitor ArQule 531 (ARQ 531) has demonstrated potent inhibition of SFK and BTK that translated to improved pre-clinical in vivo activity as compared with the irreversible BTK inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) models. Given the superior activity of ARQ 531 in CLL, and recognition that this molecule has a broad kinase inhibition profile, we pursued its application in pre-clinical models of AML. METHODS: The potency of ARQ 531 was examined in vitro using FLT3 wild type and mutated (ITD) AML cell lines and primary samples. The modulation of pro-survival kinases following ARQ 531 treatment was determined using AML cell lines. The effect of SYK expression on ARQ 531 potency was evaluated using a SYK overexpressing cell line (Ba/F3 murine cells) constitutively expressing FLT3-ITD. Finally, the in vivo activity of ARQ 531 was evaluated using MOLM-13 disseminated xenograft model. RESULTS: Our data demonstrate that ARQ 531 treatment has anti-proliferative activity in vitro and impairs colony formation in AML cell lines and primary AML cells independent of the presence of a FLT3 ITD mutation. We demonstrate decreased phosphorylation of oncogenic kinases targeted by ARQ 531, including SFK (Tyr416), BTK, and fms-related tyrosine kinase 3 (FLT3), ultimately leading to changes in down-stream targets including SYK, STAT5a, and ERK1/2. Based upon in vitro drug synergy data, we examined ARQ 531 in the MOLM-13 AML xenograft model alone and in combination with venetoclax. Despite ARQ 531 having a less favorable pharmacokinetics profile in rodents, we demonstrate modest single agent in vivo activity and synergy with venetoclax. CONCLUSIONS: Our data support consideration of the application of ARQ 531 in combination trials for AML targeting higher drug concentrations in vivo.

The BTK Inhibitor ARQ 531 Targets Ibrutinib-Resistant CLL and Richter Transformation.

Targeted inhibition of Bruton tyrosine kinase (BTK) with the irreversible inhibitor ibrutinib has improved outcomes for patients with hematologic malignancies, including chronic lymphocytic leukemia (CLL). Here, we describe preclinical investigations of ARQ 531, a potent, reversible inhibitor of BTK with additional activity against Src family kinases and kinases related to ERK signaling. We hypothesized that targeting additional kinases would improve global inhibition of signaling pathways, producing more robust responses. In vitro treatment of patient CLL cells with ARQ 531 decreases BTK-mediated functions including B-cell receptor (BCR) signaling, viability, migration, CD40 and CD86 expression, and NF-κB gene transcription. In vivo, ARQ 531 was found to increase survival over ibrutinib in a murine Eµ-TCL1 engraftment model of CLL and a murine Eµ-MYC/TCL1 engraftment model resembling Richter transformation. Additionally, ARQ 531 inhibits CLL cell survival and suppresses BCR-mediated activation of C481S BTK and PLCγ2 mutants, which facilitate clinical resistance to ibrutinib.Significance: This study characterizes a rationally designed kinase inhibitor with efficacy in models recapitulating the most common mechanisms of acquired resistance to ibrutinib. Reversible BTK inhibition is a promising strategy to combat progressive CLL, and multikinase inhibition demonstrates superior efficacy to targeted ibrutinib therapy in the setting of Richter transformation. Cancer Discov; 8(10); 1300-15. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1195.

In-vitro and in-vivo combined effect of ARQ 092, an AKT inhibitor, with ARQ 087, a FGFR inhibitor.

The PI3K/AKT pathway plays an important role in the initiation and progression of cancer, and the drug development efforts targeting this pathway with therapeutic interventions have been advanced by academic and industrial groups. However, the clinical outcome is moderate. Combination of inhibition of PI3K/AKT and other targeted agents became a feasible approach. In this study we assessed the combined effect of ARQ 092, a pan-AKT inhibitor, and ARQ 087, a pan-FGFR inhibitor, in vitro and in vivo. In a panel of 45 cancer cell lines, on 24% (11 out of 45) the compounds showed synergistic effect, on 62% (28 out of 45) additive, and on 13% (6 out of 45) antagonistic. The highest percentage of synergism was found on endometrial and ovarian cancer cell lines. Mutational analysis revealed that PIK3CA/PIK3R1 mutations and aberrant activation of FGFR2 predicted synergism, whereas Ras mutations showed a reverse correlation. Pathway analysis revealed that a combination of ARQ 092 and ARQ 087 enhanced the inhibition of both the AKT and FGFR pathways in cell lines in which synergistic effects were found (AN3CA and IGROV-1). Cell cycle arrest and apoptotic response occurred only in AN3CA cell, and was not seen in IGROV-1 cells. Furthermore, enhanced antitumor activity was observed in mouse models with endometrial cancer cell line and patient-derived tumors when ARQ 092 and ARQ 087 were combined. These results from in-vitro and in-vivo studies provide a strong rationale in treating endometrial and other cancers with the activated PI3K/AKT and FGFR pathways.

Preclinical Activity of ARQ 087, a Novel Inhibitor Targeting FGFR Dysregulation.

Dysregulation of Fibroblast Growth Factor Receptor (FGFR) signaling through amplifications, mutations, and gene fusions has been implicated in a broad array of cancers (e.g. liver, gastric, ovarian, endometrial, and bladder). ARQ 087 is a novel, ATP competitive, small molecule, multi-kinase inhibitor with potent in vitro and in vivo activity against FGFR addicted cell lines and tumors. Biochemically, ARQ 087 exhibited IC50 values of 1.8 nM for FGFR2, and 4.5 nM for FGFR1 and 3. In cells, inhibition of FGFR2 auto-phosphorylation and other proteins downstream in the FGFR pathway (FRS2α, AKT, ERK) was evident by the response to ARQ 087 treatment. Cell proliferation studies demonstrated ARQ 087 has anti-proliferative activity in cell lines driven by FGFR dysregulation, including amplifications, fusions, and mutations. Cell cycle studies in cell lines with high levels of FGFR2 protein showed a positive relationship between ARQ 087 induced G1 cell cycle arrest and subsequent induction of apoptosis. In addition, ARQ 087 was effective at inhibiting tumor growth in vivo in FGFR2 altered, SNU-16 and NCI-H716, xenograft tumor models with gene amplifications and fusions. ARQ 087 is currently being studied in a phase 1/2 clinical trial that includes a sub cohort for intrahepatic cholangiocarcinoma patients with confirmed FGFR2 gene fusions (NCT01752920).

Discovery of 3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine (ARQ 092): An Orally Bioavailable, Selective, and Potent Allosteric AKT Inhibitor.

The work in this paper describes the optimization of the 3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine chemical series as potent, selective allosteric inhibitors of AKT kinases, leading to the discovery of ARQ 092 (21a). The cocrystal structure of compound 21a bound to full-length AKT1 confirmed the allosteric mode of inhibition of this chemical class and the role of the cyclobutylamine moiety. Compound 21a demonstrated high enzymatic potency against AKT1, AKT2, and AKT3, as well as potent cellular inhibition of AKT activation and the phosphorylation of the downstream target PRAS40. Compound 21a also served as a potent inhibitor of the AKT1-E17K mutant protein and inhibited tumor growth in a human xenograft mouse model of endometrial adenocarcinoma.

Targeting AKT1-E17K and the PI3K/AKT Pathway with an Allosteric AKT Inhibitor, ARQ 092.

As a critical component in the PI3K/AKT/mTOR pathway, AKT has become an attractive target for therapeutic intervention. ARQ 092 and a next generation AKT inhibitor, ARQ 751 are selective, allosteric, pan-AKT and AKT1-E17K mutant inhibitors that potently inhibit phosphorylation of AKT. Biochemical and cellular analysis showed that ARQ 092 and ARQ 751 inhibited AKT activation not only by dephosphorylating the membrane-associated active form, but also by preventing the inactive form from localizing into plasma membrane. In endometrial PDX models harboring mutant AKT1-E17K and other tumor models with an activated AKT pathway, both compounds exhibited strong anti-tumor activity. Combination studies conducted in in vivo breast tumor models demonstrated that ARQ 092 enhanced tumor inhibition of a common chemotherapeutic agent (paclitaxel). In a large panel of diverse cancer cell lines, ARQ 092 and ARQ 751 inhibited proliferation across multiple tumor types but were most potent in leukemia, breast, endometrial, and colorectal cancer cell lines. Moreover, inhibition by ARQ 092 and ARQ 751 was more prevalent in cancer cell lines containing PIK3CA/PIK3R1 mutations compared to those with wt-PIK3CA/PIK3R1 or PTEN mutations. For both ARQ 092 and ARQ 751, PIK3CA/PIK3R1 and AKT1-E17K mutations can potentially be used as predictive biomarkers for patient selection in clinical studies.

Discovery and optimization of a series of 3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amines: orally bioavailable, selective, and potent ATP-independent Akt inhibitors.

This paper describes the implementation of a biochemical and biophysical screening strategy to identify and optimize small molecule Akt1 inhibitors that act through a mechanism distinct from that observed for kinase domain ATP-competitive inhibitors. With the aid of an unphosphorylated Akt1 cocrystal structure of 12j solved at 2.25 Å, it was possible to confirm that as a consequence of binding these novel inhibitors, the ATP binding cleft contained a number of hydrophobic residues that occlude ATP binding as expected. These Akt inhibitors potently inhibit intracellular Akt activation and its downstream target (PRAS40) in vitro. In vivo pharmacodynamic and pharmacokinetic studies with two examples, 12e and 12j, showed the series to be similarly effective at inhibiting the activation of Akt and an additional downstream effector (p70S6) following oral dosing in mice.

Discovery of a novel mode of protein kinase inhibition characterized by the mechanism of inhibition of human mesenchymal-epithelial transition factor (c-Met) protein autophosphorylation by ARQ 197.

A number of human malignancies exhibit sustained stimulation, mutation, or gene amplification of the receptor tyrosine kinase human mesenchymal-epithelial transition factor (c-Met). ARQ 197 is a clinically advanced, selective, orally bioavailable, and well tolerated c-Met inhibitor, currently in Phase 3 clinical testing in non-small cell lung cancer patients. Herein, we describe the molecular and structural basis by which ARQ 197 selectively targets c-Met. Through our analysis we reveal a previously undisclosed, novel inhibitory mechanism that utilizes distinct regulatory elements of the c-Met kinase. The structure of ARQ 197 in complex with the c-Met kinase domain shows that the inhibitor binds a conformation that is distinct from published kinase structures. ARQ 197 inhibits c-Met autophosphorylation and is highly selective for the inactive or unphosphorylated form of c-Met. Through our analysis of the interplay between the regulatory and catalytic residues of c-Met, and by comparison between the autoinhibited canonical conformation of c-Met bound by ARQ 197 to previously described kinase domains of type III receptor tyrosine kinases, we believe this to be the basis of a powerful new in silico approach for the design of similar inhibitors for other protein kinases of therapeutic interest.

A novel mode of protein kinase inhibition exploiting hydrophobic motifs of autoinhibited kinases: discovery of ATP-independent inhibitors of fibroblast growth factor receptor.

Protein kinase inhibitors with enhanced selectivity can be designed by optimizing binding interactions with less conserved inactive conformations because such inhibitors will be less likely to compete with ATP for binding and therefore may be less impacted by high intracellular concentrations of ATP. Analysis of the ATP-binding cleft in a number of inactive protein kinases, particularly in the autoinhibited conformation, led to the identification of a previously undisclosed non-polar region in this cleft. This ATP-incompatible hydrophobic region is distinct from the previously characterized hydrophobic allosteric back pocket, as well as the main pocket. Generalized hypothetical models of inactive kinases were constructed and, for the work described here, we selected the fibroblast growth factor receptor (FGFR) tyrosine kinase family as a case study. Initial optimization of a FGFR2 inhibitor identified from a library of commercial compounds was guided using structural information from the model. We describe the inhibitory characteristics of this compound in biophysical, biochemical, and cell-based assays, and have characterized the binding mode using x-ray crystallographic studies. The results demonstrate, as expected, that these inhibitors prevent activation of the autoinhibited conformation, retain full inhibitory potency in the presence of physiological concentrations of ATP, and have favorable inhibitory activity in cancer cells. Given the widespread regulation of kinases by autoinhibitory mechanisms, the approach described herein provides a new paradigm for the discovery of inhibitors by targeting inactive conformations of protein kinases.

Structural basis for Rab GTPase recognition and endosome tethering by the C2H2 zinc finger of Early Endosomal Autoantigen 1 (EEA1).

Regulation of endosomal trafficking by Rab GTPases depends on selective interactions with multivalent effectors, including EEA1 and Rabenosyn-5, which facilitate endosome tethering, sorting, and fusion. Both EEA1 and Rabenosyn-5 contain a distinctive N-terminal C(2)H(2) zinc finger that binds Rab5. How these C(2)H(2) zinc fingers recognize Rab GTPases remains unknown. Here, we report the crystal structure of Rab5A in complex with the EEA1 C(2)H(2) zinc finger. The binding interface involves all elements of the zinc finger as well as a short N-terminal extension but is restricted to the switch and interswitch regions of Rab5. High selectivity for Rab5 and, to a lesser extent Rab22, is observed in quantitative profiles of binding to Rab family GTPases. Although critical determinants are identified in both switch regions, Rab4-to-Rab5 conversion-of-specificity mutants reveal an essential requirement for additional substitutions in the proximal protein core that are predicted to indirectly influence recognition through affects on the structure and conformational stability of the switch regions.

Structural basis for Rab11-mediated recruitment of FIP3 to recycling endosomes.

The Rab11 GTPase regulates recycling of internalized plasma membrane receptors and is essential for completion of cytokinesis. A family of Rab11 interacting proteins (FIPs) that conserve a C-terminal Rab-binding domain (RBD) selectively recognize the active form of Rab11. Normal completion of cytokinesis requires a complex between Rab11 and FIP3. Here, we report the crystal structure and mutational analysis of a heterotetrameric complex between constitutively active Rab11 and a FIP3 construct that includes the RBD. Two Rab11 molecules bind to dyad symmetric sites at the C terminus of FIP3, which forms a non-canonical coiled-coiled dimer with a flared C terminus and hook region. The RBD overlaps with the coiled coil and extends through the C-terminal hook. Although FIP3 engages the switch and interswitch regions of Rab11, the mode of interaction differs significantly from that of other Rab-effector complexes. In particular, the switch II region undergoes a large structural rearrangement from an ordered but non-complementary active conformation to a remodeled conformation that facilitates the interaction with FIP3. Finally, we provide evidence that FIP3 can form homo-oligomers in cells, and that a critical determinant of Rab11 binding in vitro is necessary for FIP3 recruitment to recycling endosomes during cytokinesis.

TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism.

Rab GTPases regulate membrane trafficking by cycling between inactive (GDP-bound) and active (GTP-bound) conformations. The duration of the active state is limited by GTPase-activating proteins (GAPs), which accelerate the slow intrinsic rate of GTP hydrolysis. Proteins containing TBC (Tre-2, Bub2 and Cdc16) domains are broadly conserved in eukaryotic organisms and function as GAPs for Rab GTPases as well as GTPases that control cytokinesis. An exposed arginine residue is a critical determinant of GAP activity in vitro and in vivo. It has been expected that the catalytic mechanism of TBC domains would parallel that of Ras and Rho family GAPs. Here we report crystallographic, mutational and functional analyses of complexes between Rab GTPases and the TBC domain of Gyp1p. In the crystal structure of a TBC-domain-Rab-GTPase-aluminium fluoride complex, which approximates the transition-state intermediate for GTP hydrolysis, the TBC domain supplies two catalytic residues in trans, an arginine finger analogous to Ras/Rho family GAPs and a glutamine finger that substitutes for the glutamine in the DxxGQ motif of the GTPase. The glutamine from the Rab GTPase does not stabilize the transition state as expected but instead interacts with the TBC domain. Strong conservation of both catalytic fingers indicates that most TBC-domain GAPs may accelerate GTP hydrolysis by a similar dual-finger mechanism.

ESCRT complexes assembled and GLUEd.

The ESCRT-I, -II, and -III complexes act sequentially to sort monoubiquitinated transmembrane proteins into multivesicular bodies for targeted degradation in the lysosome. Two papers published in a recent issue of Cell provide insights into the structural organization and functional interactions of the ESCRT-I complex and ESCRT-II GLUE domain.

Structural basis of family-wide Rab GTPase recognition by rabenosyn-5.

Rab GTPases regulate all stages of membrane trafficking, including vesicle budding, cargo sorting, transport, tethering and fusion. In the inactive (GDP-bound) conformation, accessory factors facilitate the targeting of Rab GTPases to intracellular compartments. After nucleotide exchange to the active (GTP-bound) conformation, Rab GTPases interact with functionally diverse effectors including lipid kinases, motor proteins and tethering complexes. How effectors distinguish between homologous Rab GTPases represents an unresolved problem with respect to the specificity of vesicular trafficking. Using a structural proteomic approach, we have determined the specificity and structural basis underlying the interaction of the multivalent effector rabenosyn-5 with the Rab family. The results demonstrate that even the structurally similar effector domains in rabenosyn-5 can achieve highly selective recognition of distinct subsets of Rab GTPases exclusively through interactions with the switch and interswitch regions. The observed specificity is determined at a family-wide level by structural diversity in the active conformation, which governs the spatial disposition of critical conserved recognition determinants, and by a small number of both positive and negative sequence determinants that allow further discrimination between Rab GTPases with similar switch conformations.

Determinants of Rab5 interaction with the N terminus of early endosome antigen 1.

The Rab5 effector early endosome antigen 1 (EEA1) is a parallel coiled coil homodimer with an N-terminal C(2)H(2) Zn(2+) finger and a C-terminal FYVE domain. Rab5 binds to independent sites at the N and C terminus of EEA1. To gain further insight into the structural determinants for endosome tethering and fusion, we have characterized the interaction of Rab5C with truncation and site-specific mutants of EEA1 using quantitative binding measurements. The results demonstrate that the C(2)H(2) Zn(2+) finger is both essential and sufficient for the N-terminal interaction with Rab5. Although the heptad repeat C-terminal to the C(2)H(2) Zn(2+) finger provides the driving force for stable homodimerization, it does not influence either the affinity or stoichiometry of Rab5 binding. Hydrophobic residues predicted to cluster on a common face of the C(2)H(2) Zn(2+) finger play a critical role in the interaction with Rab5. Although the homologous C(2)H(2) Zn(2+) finger of the Rab5 effector Rabenosyn binds to Rab5 with comparable affinity, the analogous C(2)H(2) Zn(2+) finger of the yeast homologue Vac1 shows no detectable interaction with Rab5, reflecting non-conservative substitutions of critical residues. Large changes in the intrinsic tryptophan fluorescence of Rab5 accompany binding to the C(2)H(2) Zn(2+) finger of EEA1. These observations can be explained by a mode of interaction in which a partially exposed tryptophan residue located at the interface between the switch I and II regions of Rab5 lies within a hydrophobic interface with a cluster of non-polar residues in the C(2)H(2) Zn(2+) finger of EEA1.