Polo-like kinase 4 inhibitor CFI-400945 suppresses liver cancer through cell cycle perturbation and eliciting antitumor immunity.

BACKGROUND AND AIMS: Prognosis of HCC remains poor due to lack of effective therapies. Immune checkpoint inhibitors (ICIs) have delayed response and are only effective in a subset of patients. Treatments that could effectively shrink the tumors within a short period of time are idealistic to be employed together with ICIs for durable tumor suppressive effects. HCC acquires increased tolerance to aneuploidy. The rapid division of HCC cells relies on centrosome duplication. In this study, we found that polo-like kinase 4 (PLK4), a centrosome duplication regulator, represents a therapeutic vulnerability in HCC. APPROACH AND RESULTS: An orally available PLK4 inhibitor, CFI-400945, potently suppressed proliferating HCC cells by perturbing centrosome duplication. CFI-400945 induced endoreplication without stopping DNA replication, causing severe aneuploidy, DNA damage, micronuclei formation, cytosolic DNA accumulation, and senescence. The cytosolic DNA accumulation elicited the DEAD box helicase 41-stimulator of interferon genes-interferon regulatory factor 3/7-NF-κß cytosolic DNA sensing pathway, thereby driving the transcription of senescence-associated secretory phenotypes, which recruit immune cells. CFI-400945 was evaluated in liver-specific p53/phosphatase and tensin homolog knockout mouse HCC models established by hydrodynamic tail vein injection. Tumor-infiltrated immune cells were analyzed. CFI-400945 significantly impeded HCC growth and increased infiltration of cluster of differentiation 4-positive (CD4 + ), CD8 + T cells, macrophages, and natural killer cells. Combination therapy of CFI-400945 with anti-programmed death-1 showed a tendency to improve HCC survival. CONCLUSIONS: We show that by targeting a centrosome regulator, PLK4, to activate the cytosolic DNA sensing-mediated immune response, CFI-400945 effectively restrained tumor progression through cell cycle inhibition and inducing antitumor immunity to achieve a durable suppressive effect even in late-stage mouse HCC.

Targeting Mitosis in Cancer: Emerging Strategies.

The cell cycle is an evolutionarily conserved process necessary for mammalian cell growth and development. Because cell-cycle aberrations are a hallmark of cancer, this process has been the target of anti-cancer therapeutics for decades. However, despite numerous clinical trials, cell-cycle-targeting agents have generally failed in the clinic. This review briefly examines past cell-cycle-targeted therapeutics and outlines how experience with these agents has provided valuable insight to refine and improve anti-mitotic strategies. An overview of emerging anti-mitotic approaches with promising pre-clinical results is provided, and the concept of exploiting the genomic instability of tumor cells through therapeutic inhibition of mitotic checkpoints is discussed. We believe this strategy has a high likelihood of success given its potential to enhance therapeutic index by targeting tumor-specific vulnerabilities. This reasoning stimulated our development of novel inhibitors targeting the critical regulators of genomic stability and the mitotic checkpoint: AURKA, PLK4, and Mps1/TTK.

Functional characterization of CFI-402257, a potent and selective Mps1/TTK kinase inhibitor, for the treatment of cancer.

Loss of cell-cycle control is a hallmark of human cancer. Cell-cycle checkpoints are essential for maintaining genome integrity and balanced growth and division. They are specifically deregulated in cancer cells and contain regulators that represent potential therapeutic targets. Monopolar spindle 1 (Mps1; also known as TTK protein kinase) is a core component of the spindle assembly checkpoint (SAC), a genome-surveillance mechanism that is important for cell survival, and has emerged as a candidate target for anticancer therapy. Here, we report the cellular and antitumor effects of CFI-402257, a potent (Mps1 Ki = 0.09 ± 0.02 nM; cellular Mps1 EC50 = 6.5 ± 0.5 nM), highly selective, and orally active small-molecule inhibitor of Mps1 that was identified through a drug-discovery program. Human cancer cells treated with CFI-402257 exhibit effects consistent with Mps1 kinase inhibition, specifically SAC inactivation, leading to chromosome missegregation, aneuploidy, and ultimately cell death. Oral administration of CFI-402257 in monotherapy or in combination with an anti-programmed cell death 1 (PD-1) antibody in mouse models of human cancer results in inhibition of tumor growth at doses that are well-tolerated. Our findings provide a rationale for the clinical evaluation of CFI-402257 in patients with solid tumors.

Safety and tolerability of CFI-400945, a first-in-class, selective PLK4 inhibitor in advanced solid tumours: a phase 1 dose-escalation trial.

BACKGROUND: CFI-400945 is a first-in-class oral inhibitor of polo-like kinase 4 (PLK4) that regulates centriole duplication. Primary objectives of this first-in-human phase 1 trial were to establish the safety and tolerability of CFI-400945 in patients with advanced solid tumours. Secondary objectives included pharmacokinetics, pharmacodynamics, efficacy, and recommended phase 2 dose (RP2D). METHODS: Continuous daily oral dosing of CFI-400945 was evaluated using a 3+3 design guided by incidence of dose-limiting toxicities (DLTs) in the first 28-day cycle. Safety was assessed by CTCAE v4.0. ORR and CBR were evaluated using RECIST v1.1. RESULTS: Forty-three patients were treated in dose escalation from 3 to 96 mg/day, and 9 were treated in 64 mg dose expansion. After DLT occurred at 96 and 72 mg, 64 mg was established as the RP2D. Neutropenia was a common high-grade (19%) treatment-related adverse event at ≥ 64 mg. Half-life of CFI-400945 was 9 h, with Cmax achieved 2-4 h following dosing. One PR (45 cycles, ongoing) and two SD ≥ 6 months were observed (ORR = 2%; CBR = 6%). CONCLUSIONS: CFI-400945 is well tolerated at 64 mg with dose-dependent neutropenia. Favourable pharmacokinetic profiles were achieved with daily dosing. Response rates were low without biomarker pre-selection. Disease-specific and combination studies are ongoing. TRIAL REGISTRATION: Clinical Trials Registration Number - NCT01954316 (Oct 1st, 2013).

A phase I trial of the aurora kinase inhibitor, ENMD-2076, in patients with relapsed or refractory acute myeloid leukemia or chronic myelomonocytic leukemia.

ENMD-2076 is a novel, orally-active molecule that inhibits Aurora A kinase, as well as c-Kit, FLT3 and VEGFR2. A phase I study was conducted to determine the maximum tolerated dose (MTD), recommended phase 2 dose (RP2D) and toxicities of ENMD-2076 in patients with acute myeloid leukemia (AML) and chronic myelomonocytic leukemia (CMML). Patients received escalating doses of ENMD-2076 administered orally daily [225 mg (n = 7), 375 mg (n = 6), 325 mg (n = 9), or 275 mg (n = 5)]. Twenty-seven patients were treated (26 AML; 1 CMML-2). The most common non-hematological toxicities of any grade, regardless of association with drug, were fatigue, diarrhea, dysphonia, dyspnea, hypertension, constipation, and abdominal pain. Dose-limiting toxicities (DLTs) consisted of grade 3 fatigue, grade 3 typhilitis, grade 3 syncope and grade 3 QTc prolongation). Of the 16 evaluable patients, one patient achieved a complete remission with incomplete count recovery (CRi), three experienced a morphologic leukemia-free state (MLFS) with a major hematologic improvement in platelets (HI-P), and 5 other patients had a reduction in marrow blast percentage (i.e. 11-65 %). The RP2D in this patient population is 225 mg orally once daily.

Discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as novel, highly potent and selective, orally bioavailable inhibitors of Tyrosine Threonine Kinase, TTK.

TTK/Mps1 is a key kinase controlling progression of cell division via participation in the mitotic spindle assembly checkpoint and is overexpressed in a number of human cancers. Herein we report the discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as a potent, novel class of TTK inhibitors. The series was identified by means of bioisosteric replacement of the related imidazopyrazine and imidazopyridazine scaffolds. Optimization led to the identification of compounds with excellent potency (Ki=0.8nM) and exceptional kinase selectivity. The SAR indicates a strong dependence of activity on the presence of the N-cyclopropyl-2-methylbenzamide moiety delineating the geometry for 1½ type kinase inhibitor. Molecular modeling indicates the extensive and optimal contacts, mediated through H-bonds and hydrophobic interactions, are responsible for the selectivity and potency of the inhibitors. The compounds demonstrate a strong anti-proliferative activity in a panel of human cancer cell lines (HCT116 GI50<15nM) and good rodent pharmacokinetics (oral %F 97%).

2-Methoxyestradiol inhibits experimental autoimmune encephalomyelitis through suppression of immune cell activation.

The endogenous metabolite of estradiol, 2-Methoxyestradiol (2ME2), is an antimitotic and antiangiogenic cancer drug candidate that also exhibits disease-modifying activity in animal models of rheumatoid arthritis (RA). We found that 2ME2 dramatically suppresses development of mouse experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). 2ME2 inhibits in vitro lymphocyte activation, cytokine production, and proliferation in a dose-dependent fashion. 2ME2 treatment of lymphocytes specifically reduced the nuclear translocation and transcriptional activity of nuclear factor of activated T-cells (NFAT) c1, whereas NF-κB and activator protein 1 (AP-1) activation were not adversely affected. We therefore propose that 2ME2 attenuates EAE through disruption of the NFAT pathway and subsequent lymphocyte activation. By extension, our findings provide a molecular rationale for the use of 2ME2 as a tolerable oral immunomodulatory agent for the treatment of autoimmune disorders such as MS in humans.

Preclinical characterization and clinical trial of CFI-400945, a polo-like kinase 4 inhibitor, in patients with relapsed/refractory acute myeloid leukemia and higher-risk myelodysplastic neoplasms.

CFI-400945 is a selective oral polo-like kinase 4 (PLK4) inhibitor that regulates centriole duplication. PLK4 is aberrantly expressed in patients with acute myeloid leukemia (AML). Preclinical studies indicate that CFI-400945 has potent in vivo efficacy in hematological malignancies and xenograft models, with activity in cells harboring TP53 mutations. In this phase 1 study in very high-risk patients with relapsed/refractory AML and myelodysplastic syndrome (MDS) (NCT03187288), 13 patients were treated with CFI-400945 continuously in dose escalation from 64 mg/day to 128 mg/day. Three of the 9 efficacy evaluable AML patients achieved complete remission (CR). Two of 4 AML patients (50%) with TP53 mutations and complex monosomal karyotype achieved a CR with 1 patient proceeding to allogenic stem cell transplant. A third patient with TP53 mutated AML had a significant reduction in marrow blasts by > 50% with an improvement in neutrophil and platelet counts. Responses were observed after 1 cycle of therapy. Dose-limiting toxicity was enteritis/colitis. A monotherapy and combination therapy study with a newer crystal form of CFI-400945 in patients with AML, MDS and chronic myelomonocytic leukemia (CMML) is ongoing (NCT04730258).

Identification of KIFC1 as a putative vulnerability in lung cancers with centrosome amplification.

Centrosome amplification (CA), an abnormal increase in the number of centrosomes in the cell, is a recurrent phenomenon in lung and other malignancies. Although CA promotes tumor development and progression by inducing genomic instability (GIN), it also induces mitotic stress that jeopardizes cellular integrity. CA leads to the formation of multipolar mitotic spindles that can cause lethal chromosome segregation errors. To sustain the benefits of CA by mitigating its consequences, malignant cells are dependent on adaptive mechanisms that represent therapeutic vulnerabilities. We aimed to discover genetic dependencies associated with CA in lung cancer. Combining a CRISPR/Cas9 functional genomics screen with tumor genomic analyses, we identified the motor protein KIFC1, also known as HSET, as a putative vulnerability specifically in lung adenocarcinoma (LUAD) with CA. KIFC1 expression was positively correlated with CA in LUAD and associated with worse patient outcomes, smoking history, and indicators of GIN. KIFC1 loss-of-function sensitized LUAD cells with high basal KIFC1 expression to potentiation of CA, which was associated with a diminished ability to cluster extra centrosomes into pseudo-bipolar mitotic spindles. Our work suggests that KIFC1 inhibition represents a novel approach for potentiating GIN to lethal levels in LUAD with CA by forcing cells to divide with multipolar spindles, rationalizing further studies to investigate its therapeutic potential.

Inhibition of Aberrantly Overexpressed Polo-like Kinase 4 Is a Potential Effective Treatment for DNA Damage Repair-Deficient Uterine Leiomyosarcoma.

PURPOSE: Uterine leiomyosarcoma (LMS) is an aggressive sarcoma and a subset of which exhibits DNA repair defects. Polo-like kinase 4 (PLK4) precisely modulates mitosis, and its inhibition causes chromosome missegregation and increased DNA damage. We hypothesize that PLK4 inhibition is an effective LMS treatment. EXPERIMENTAL DESIGN: Genomic profiling of clinical uterine LMS samples was performed, and homologous recombination (HR) deficiency scores were calculated. A PLK4 inhibitor (CFI-400945) with and without an ataxia telangiectasia mutated (ATM) inhibitor (AZD0156) was tested in vitro on gynecologic sarcoma cell lines SK-UT-1, SKN, and SK-LMS-1. Findings were validated in vivo using the SK-UT-1 xenograft model in the Balb/c nude mouse model. The effects of CFI-400945 were also evaluated in a BRCA2-knockout SK-UT-1 cell line. The mechanisms of DNA repair were analyzed using a DNA damage reporter assay. RESULTS: Uterine LMS had a high HR deficiency score, overexpressed PLK4 mRNA, and displayed mutations in genes responsible for DNA repair. CFI-400945 demonstrated effective antitumor activity in vitro and in vivo. The addition of AZD0156 resulted in drug synergism, largely due to a preference for nonhomologous end-joining DNA repair. Compared with wild-type cells, BRCA2 knockouts were more sensitive to PLK4 inhibition when both HR and nonhomologous end-joining repairs were impaired. CONCLUSIONS: Uterine LMS with DNA repair defects is sensitive to PLK4 inhibition because of the effects of chromosome missegregation and increased DNA damage. Loss-of-function BRCA2 alterations or pharmacologic inhibition of ATM enhanced the efficacy of the PLK4 inhibitor. Genomic profiling of an advanced-stage or recurrent uterine LMS may guide therapy.

DJ-1, a novel regulator of the tumor suppressor PTEN.

The phosphatidylinositol 3' kinase (PI3'K) pathway, which regulates cell survival, is antagonized by the PTEN tumor suppressor. The regulation of PTEN is unclear. A genetic screen of Drosophila gain-of-function mutants identified DJ-1 as a suppressor of PTEN function. In mammalian cells, DJ-1 underexpression results in decreased phosphorylation of PKB/Akt, while DJ-1 overexpression leads to hyperphosphorylation of PKB/Akt and increased cell survival. In primary breast cancer samples, DJ-1 expression correlates negatively with PTEN immunoreactivity and positively with PKB/Akt hyperphosphorylation. In 19/23 primary non-small cell lung carcinoma samples, DJ-1 expression was increased compared to paired nonneoplastic lung tissue, and correlated positively with relapse incidence. DJ-1 is thus a key negative regulator of PTEN that may be a useful prognostic marker for cancer.

The spindle assembly checkpoint is a therapeutic vulnerability of CDK4/6 inhibitor-resistant ER+ breast cancer with mitotic aberrations.

Inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6i) are standard first-line treatments for metastatic ER+ breast cancer. However, acquired resistance to CDK4/6i invariably develops, and the molecular phenotypes and exploitable vulnerabilities associated with resistance are not yet fully characterized. We developed a panel of CDK4/6i-resistant breast cancer cell lines and patient-derived organoids and demonstrate that a subset of resistant models accumulates mitotic segregation errors and micronuclei, displaying increased sensitivity to inhibitors of mitotic checkpoint regulators TTK and Aurora kinase A/B. RB1 loss, a well-recognized mechanism of CDK4/6i resistance, causes such mitotic defects and confers enhanced sensitivity to TTK inhibition. In these models, inhibition of TTK with CFI-402257 induces premature chromosome segregation, leading to excessive mitotic segregation errors, DNA damage, and cell death. These findings nominate the TTK inhibitor CFI-402257 as a therapeutic strategy for a defined subset of ER+ breast cancer patients who develop resistance to CDK4/6i.

Preclinical activity of a novel multiple tyrosine kinase and aurora kinase inhibitor, ENMD-2076, against multiple myeloma.

ENMD-2076 is a novel, orally-active molecule that has been shown to have significant activity against aurora and multiple receptor tyrosine kinases. We investigated the activity of ENMD-2076 against multiple myeloma (MM) cells in vitro and in vivo. ENMD-2076 showed significant cytotoxicity against MM cell lines and primary cells, with minimal cytotoxicity to haematopoietic progenitors. ENMD-2076 inhibited the phosphoinositide 3-kinase/AKT pathway and downregulated survivin and X-linked inhibitor of apoptosis as early as 6 h after treatment. With longer treatment (24-48 h), ENMD-2076 also inhibited aurora A and B kinases, and induced G(2)/M cell cycle arrest. In non-obese diabetic/severe combined immunodeficient mice implanted with H929 human plasmacytoma xenografts, oral treatment with ENMD-2076 (50, 100, 200 mg/kg per day) resulted in a dose-dependent inhibition of tumour growth. Immunohistochemical staining of excised tumours showed significant reduction in phospho-Histone 3 (pH3), Ki-67, and angiogenesis, and also a significant increase in cleaved caspase-3 at all dose levels compared to tumours from vehicle-treated mice. In addition, a significant reduction in p-FGFR3 was observed on Western blot. ENMD-2076 shows significant activity against MM cells in vitro and in vivo, and acts on several pathways important for myeloma cell growth and survival. These results provide preclinical rationale for clinical investigation of ENMD-2076 in MM.

Fcmr regulates mononuclear phagocyte control of anti-tumor immunity.

Myeloid cells contribute to tumor progression, but how the constellation of receptors they express regulates their functions within the tumor microenvironment (TME) is unclear. We demonstrate that Fcmr (Toso), the putative receptor for soluble IgM, modulates myeloid cell responses to cancer. In a syngeneic melanoma model, Fcmr ablation in myeloid cells suppressed tumor growth and extended mouse survival. Fcmr deficiency increased myeloid cell population density in this malignancy and enhanced anti-tumor immunity. Single-cell RNA sequencing of Fcmr-deficient tumor-associated mononuclear phagocytes revealed a unique subset with enhanced antigen processing/presenting properties. Conversely, Fcmr activity negatively regulated the activation and migratory capacity of myeloid cells in vivo, and T cell activation by bone marrow-derived dendritic cells in vitro. Therapeutic targeting of Fcmr during oncogenesis decreased tumor growth when used as a single agent or in combination with anti-PD-1. Thus, Fcmr regulates myeloid cell activation within the TME and may be a potential therapeutic target.

Author Correction: Fcmr regulates mononuclear phagocyte control of anti-tumor immunity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Overexpression of AKT2/protein kinase Bbeta leads to up-regulation of beta1 integrins, increased invasion, and metastasis of human breast and ovarian cancer cells.

To determine how AKT2 might contribute to tumor cell progression, a full-length, wild-type, human AKT2/protein kinase B (PKB)beta cDNA was transfected into a panel of eight human breast and ovarian cancer cells. AKT2 transfectants demonstrated increased adhesion and invasion through collagen IV because of up-regulation of beta1 integrins. In addition, AKT2 cells were more metastatic than control cells in vivo. Increased invasion by AKT2 was blocked by preincubation with an anti-beta1 integrin function blocking antibody, exposure to wortmannin, and by expression of phosphatase and tensin homologue tumor suppressor (PTEN). Confocal microscopy performed on transfected human breast cancer cells showed that unlike AKT1, AKT2 protein predominantly localized adjacent to the collagen IV matrix during cellular attachment. Overexpression of AKT2, but not AKT1 or AKT3, was sufficient to duplicate the invasive effects of phosphoinositide 3-OH kinase (PI3-K) transfected in breast cancer cells. Furthermore, expression of kinase dead AKT2(181 amino acid methionine [M]), and not kinase dead AKT1(179M) or AKT3(177M), was capable of blocking invasion induced by either human epidermal growth factor receptor-2 (HER-2) overexpression or by activation of PI3-K. Taken together, these data indicate that AKT2 mediates PI3-K-dependent effects on adhesion, motility, invasion, and metastasis in vivo.

Soluble interleukin-13Ralpha2 decoy receptor inhibits Hodgkin's lymphoma growth in vitro and in vivo.

Recent studies have demonstrated that the malignant Reed-Sternberg cells of Hodgkin's lymphoma (HL) secrete and are responsive to interleukin (IL)-13. We hypothesized that overexpression of a soluble IL-13 decoy receptor (sIL-13Ralpha2) via adenoviral-mediated gene transfer would inhibit IL-13-induced Reed-Sternberg cell proliferation. Western blot and ELISA analysis verified expression of sIL-13Ralpha2 in cell lysates and supernatants of AdsIL-13Ralpha2-transduced COS-7 cells. Treatment of two IL-13-responsive HL-derived cell lines, HDLM-2 and L-1236, with AdsIL-13Ralpha2-conditioned medium, resulted in the inhibition of cell proliferation, and down-regulated the phosphorylation of signal transducer and activator of transcription 6 (STAT6), an important mediator of IL-13 signaling. i.v. delivery of AdsIL-13Ralpha2 in NOD/SCID mice with s.c. implanted HDLM-2 cells delayed tumor onset and growth while enhancing survival compared with control mice. Intratumoral administration of AdsIL-13Ralpha2 led to the regression or stabilization of established tumors and was associated with diminished STAT6 phosphorylation. Our data demonstrate that AdsIL-13Ralpha2 can suppress HL growth in vitro and in vivo.

Discovery of Pyrazolo[1,5-a]pyrimidine TTK Inhibitors: CFI-402257 is a Potent, Selective, Bioavailable Anticancer Agent.

This work describes a scaffold hopping exercise that begins with known imidazo[1,2-a]pyrazines, briefly explores pyrazolo[1,5-a][1,3,5]triazines, and ultimately yields pyrazolo[1,5-a]pyrimidines as a novel class of potent TTK inhibitors. An X-ray structure of a representative compound is consistent with 1(1)/2 type inhibition and provides structural insight to aid subsequent optimization of in vitro activity and physicochemical and pharmacokinetic properties. Incorporation of polar moieties in the hydrophobic and solvent accessible regions modulates physicochemical properties while maintaining potency. Compounds with enhanced oral exposure were identified for xenograft studies. The work culminates in the identification of a potent (TTK K i = 0.1 nM), highly selective, orally bioavailable anticancer agent (CFI-402257) for IND enabling studies.

The Discovery of Orally Bioavailable Tyrosine Threonine Kinase (TTK) Inhibitors: 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides as Anticancer Agents.

The acetamido and carboxamido substituted 3-(1H-indazol-3-yl)benzenesulfonamides are potent TTK inhibitors. However, they display modest ability to attenuate cancer cell growth; their physicochemical properties, and attendant pharmacokinetic parameters, are not drug-like. By eliminating the polar 3-sulfonamide group and grafting a heterocycle at the 4 position of the phenyl ring, potent inhibitors with oral exposure were obtained. An X-ray cocrystal structure and a refined binding model allowed for a structure guided approach. Systematic optimization resulted in novel TTK inhibitors, namely 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides. Compounds incorporating the 3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl bicyclic system were potent (TTK IC50 < 10 nM, HCT116 GI50 < 0.1 µM), displayed low off-target activity (>500×), and microsomal stability (T(1/2) > 30 min). A subset was tested in rodent PK and mouse xenograft models of human cancer. Compound 75 (CFI-401870) recapitulated the phenotype of TTK RNAi, demonstrated in vivo tumor growth inhibition upon oral dosing, and was selected for preclinical evaluation.

A Density Functional Study of Active Site Models for Xanthine Oxidase.

The suggestion that hydroxide is coordinated to the oxidised molybdenum site in xanthine oxidase (XnO) is tested theoretically by computing the structures of a range of four-, five-, and six-coordinate active site models. The local density approximation of density functional theory has been used with the two experimentally verified singly bonded sulfur ligands modeled by both dithiolene, [SRCCRS](2-) (R = H and CH(3)), and thiolate, [CH(3)S](-) groups. Both ligand types give virtually identical results for analogous species. Based on a comparison of the computed M-L distances and those reported in recent EXAFS studies, it is concluded that both four- and six-coordination are unlikely since the optimized Mo-S contacts are too short or too long respectively. Of the five-coordinate MoOS(SR)(2)X models, the ones with X = [OH](-) give computed M-L bond lengths in excellent agreement with the reported EXAFS data while X = H(2)O, NH(3), [CH(3)S](-), and O(2-) give relatively poor agreement. The theoretical results imply that the active site represents a stable, preferred geometry rather than some imposed entatic state.