Dutogliptin, a selective DPP4 inhibitor, improves glycaemic control in patients with type 2 diabetes: a 12-week, double-blind, randomized, placebo-controlled, multicentre trial.

AIM: To determine efficacy and tolerability of dutogliptin, a dipeptidyl peptidase 4 (DPP4) inhibitor, in patients with type 2 diabetes mellitus. METHODS: This was a 12-week, multicentre, randomized, double-blind, placebo-controlled trial in 423 patients with type 2 diabetes with suboptimal metabolic control. Following a 2-week single-blind placebo run-in, patients aged 18-75 years with a body mass index of 25-48 kg/m(2) and baseline HbA1c of 7.3-11.0% were randomized 2:2:1 to receive once-daily oral therapy with either dutogliptin (400 or 200 mg) or placebo on a background medication of either metformin alone, a thiazolidinedione (TZD) alone or a combination of metformin plus a TZD. RESULTS: Average HbA1c at baseline was 8.4%. Administration of dutogliptin 400 and 200 mg for 12 weeks decreased HbA1c by -0.52% (p < 0.001) and -0.35% (p = 0.006), respectively (placebo-corrected values), with absolute changes in HbA1c for the 400 mg, 200 mg and placebo groups of -0.82, -0.64 and -0.3%, respectively. The proportion of patients achieving an HbA1c < 7% was 27, 21 and 12% at dutogliptin doses of 400 and 200 mg or placebo, respectively (p = 0.008 for comparison of 400 mg vs. placebo). Fasting plasma glucose (FPG) levels were significantly reduced in both active treatment groups compared to placebo: the placebo-corrected difference was -1.00 mmol/l (p < 0.001) for the 400 mg group and -0.88 mmol/l (p = 0.003) for the 200 mg group. Dutogliptin caused significantly greater reductions in postprandial glucose AUC (0-2h) in both the 400 and 200 mg groups (placebo corrected values -2.58 mmol/l/h, p < 0.001 and -1.63 mmol/l/h, p = 0.032, respectively). In general, patients tolerated the study drug well. There were minor, not clinically meaningful differences in adverse events (AEs) between dutogliptin-treated patients and placebo controls, and 60% of all reported AEs were mild. Vital signs and body weight were stable, and routine safety laboratory parameters did not change compared with placebo. Trough ex vivo DPP4 inhibition at the end of the 12-week treatment period was 80 and 70%, at the 400 and 200 mg doses of dutogliptin, respectively. CONCLUSIONS: Dutogliptin treatment for 12 weeks improved glycaemic control in patients with type 2 diabetes who were on a background medication of metformin, a TZD or metformin plus a TZD. Tolerability was favourable for both doses tested. The 400 mg dose of dutogliptin resulted in larger changes of HbA1c and FPG and more subjects reached an HbA1c target of < 7% than the 200 mg dose.

The dipeptidyl peptidase-4 inhibitor PHX1149 improves blood glucose control in patients with type 2 diabetes mellitus.

AIM: To determine the efficacy and tolerability of PHX1149, a novel dipeptidyl peptidase-4 (DPP4) inhibitor, in patients with type 2 diabetes. METHODS: This is a multicentre, randomized, double-blind, placebo-controlled, 4-week study in patients with type 2 diabetes with suboptimal metabolic control. Patients with a baseline haemoglobin A(1c) (HbA(1c)) of 7.3 to 11.0% were randomized 1 : 1 : 1 : 1 to receive once-daily oral therapy with either PHX1149 (100, 200 or 400 mg) or placebo; patients were on a constant background therapy of either metformin alone or metformin plus a glitazone. RESULTS: Treatment with 100, 200 or 400 mg of PHX1149 significantly decreased postprandial glucose area under the curve AUC(0-2 h) by approximately 20% (+0.11 +/- 0.50, -2.08 +/- 0.51, -1.73 +/- 0.49 and -1.88 +/- 0.48 mmol/l x h, respectively, for placebo and 100, 200 and 400 mg (p = 0.002, 0.008 and 0.004 vs. placebo). Postprandial AUC(0-2 h) of intact glucagon-like peptide-1, the principal mediator of the biological effects of DPP4 inhibitors, was increased by 3.90 +/- 2.83, 11.63 +/- 2.86, 16.42 +/- 2.72 and 15.75 +/- 2.71 pmol/l x h, respectively, for placebo and 100, 200 and 400 mg (p = 0.053, 0.001 and 0.002 vs. placebo). Mean HbA(1c) was lower in all dose groups; the placebo-corrected change in the groups receiving 400 mg PHX1149 was -0.28% (p = 0.02). DPP4 inhibition on day 28 was 53, 73 and 78% at 24 h postdose in the groups receiving 100, 200 and 400 mg PHX1149, respectively. There were no differences in adverse events between PHX1149-treated and placebo subjects. CONCLUSIONS: Addition of the DPP4 inhibitor PHX1149 to a stable regimen of metformin or metformin plus a glitazone in patients with type 2 diabetes was well tolerated and improved blood glucose control.

FLT3 K663Q is a novel AML-associated oncogenic kinase: Determination of biochemical properties and sensitivity to Sunitinib (SU11248).

Somatic mutations of FLT3 resulting in constitutive kinase activation are the most common acquired genomic abnormality found in acute myeloid leukemia (AML). The majority of these mutations are internal tandem duplications (ITD) of the juxtamembrane region (JM). In addition, a minority of cases of AML are associated with mutation of the FLT3 activation loop (AL), typically involving codons D835 and/or I836. We hypothesized that other novel mutations of FLT3 could also contribute to leukemogenesis. We genotyped 109 cases of AML and identified two novel gain-of-function mutations. The first mutation, N841 H, is similar to previously described mutations involving amino-acid substitutions of codon 841. The other novel mutation, FLT3 K663Q, is the first AML-associated gain-of-function mutation located outside the JM and AL domains. Of note, this mutation was potently inhibited by Sunitinib (SU11248), a previously described FLT3 kinase inhibitor. Sunitinib reduced the proliferation and induced apoptosis of transformed Ba/F3 cells expressing FLT3 K663Q. The potency of Sunitinib against FLT3 K663Q was similar to its potency against FLT3 ITD mutations. We conclude that FLT3 mutations in AML can involve novel regions of the TK1. Future studies are needed to define the incidence and prognostic significance of FLT3 mutations outside the well-established JM and AL regions.

New paradigms for the treatment of cancer: the role of anti-angiogenesis agents.

Angiogenesis, the sprouting of new blood vessels, plays a role in diverse disease states including cancer, diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, psoriasis, atherosclerosis, and restenosis. With regard to cancer, the clinical association of tumor vascularity with tumor aggressiveness has been clearly demonstrated in numerous tumor types. The observation of increased microvessel density in tumors not only serves as an independent prognostic indicator, but also suggests that anti-angiogenic therapy may be an important component of treatment regimens for cancer patients. The complexity of the angiogenic process, which involves both positive and negative regulators, provides a number of targets for therapy. Many positive regulators, including growth factor receptors, matrix metalloproteinases, and integrins, have been correlated with increased vascularity of tumors and poor prognosis for patient survival. Thus, these serve as ideal targets for anti-angiogenesis therapy. Many inhibitors of these targets are currently undergoing clinical evaluation as potential anti-cancer agents. In this article, we discuss the role of positive regulators in angiogenesis and tumor growth and describe the anti-angiogenic agents under development.

Effects of SU101 in combination with cytotoxic agents on the growth of subcutaneous tumor xenografts.

SU101 (leflunomide, N-[4-(trifluoromethyl)-phenyl] 5-methylisoxazole-4-carboxamide), an inhibitor of platelet-derived growth factor receptor signaling, has shown promising clinical activity in Phase I and II studies. Currently, SU101 in combination with cytotoxic agents is in late-stage clinical development for the treatment of cancers. In previous reports, efficacy in vivo versus varied tumor xenografts was observed. As part of the preclinical development of SU101 as a cancer therapy, the combination of SU101 with cytotoxic agents was studied in athymic mice bearing small, established, s.c. human tumor cell xenografts of glioblastoma (SF763T cells), lung (Calu-6 cells), or head and neck (KB cells) origin. In the SF763T model, the combination of SU101 with carmustine resulted in a statistically significant growth inhibition of 74% compared with the vehicle control; this combination was more effective than either agent alone. In the Calu-6 model, the combination of SU101, cisplatin, and etoposide resulted in a growth inhibition of 75% that was statistically greater than that of the vehicle-treated control group and groups treated with one or two agents. In the KB model, the combination of SU101, 5-fluorouracil, and cisplatin resulted in a statistically significant growth inhibition of 69% compared with the vehicle control. Treatment with one or two agents did not significantly inhibit growth in this model. Importantly, in addition to enhanced efficacy resulting from combination therapies, the combination treatments tested were well tolerated, as evidenced by lack of mortality. These data suggest that SU101 in combination with cytotoxic agents may provide clinical benefit and warrant further clinical investigation.

The angiogenesis inhibitor SU5416 has long-lasting effects on vascular endothelial growth factor receptor phosphorylation and function.

SU5416, a selective inhibitor of the tyrosine kinase activity of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR, is currently in Phase III clinical trials for the treatment of advanced malignancies. In cellular assays, SU5416 inhibits the VEGF-dependent mitogenic/proliferative response of human umbilical vein endothelial cells (HUVECs). In tumor xenograft models, SU5416 inhibits the growth of tumors from a variety of origins by inhibiting tumor angiogenesis. In three different human tumor xenograft models, infrequent (once or twice a week) administration of SU5416 is efficacious despite the fact that it has a short plasma half-life (30 min), which suggests that SU5416 has long-lasting inhibitory activity in vivo. The goal of the present study was to determine the basis for the prolonged activity of SU5416. The results indicate that a short (3 h) exposure to 5 microM SU5416 (to mimic plasma levels of the compound as measured in patients who were receiving SU5416 therapy) produced long-lasting (at least 72 h) inhibition of the VEGF-dependent proliferation of HUVECs in culture, which indicate that SU5416 has long-lasting inhibitory activity in vitro as well as in vivo. SU5416 treatment of HUVECs did not affect surface expression of Flk-1/KDR or the affinity of the receptor for VEGF. Instead, the durability of the in vitro activity of SU5416 was shown to be attributable to its long-lasting ability to specifically inhibit VEGF-dependent phosphorylation of Flk-1/KDR and subsequent downstream signaling, although SU5416 is not an irreversible inhibitor of Flk-1/KDR tyrosine kinase activity. The long-lasting inhibition of cellular responses to VEGF was attributable to the accumulation of SU5416 in cells, as shown using radiolabeled compound, such that inhibitory cellular concentrations of SU5416 are maintained long after the removal of the compound from the medium. The long-lasting inhibitory activity of SU5416 in vitro is consistent with the finding that SU5416 has demonstrated evidence of biological activity in clinical studies when administered twice a week despite a short plasma half-life.

High levels of HER-2 expression alter the ability of epidermal growth factor receptor (EGFR) family tyrosine kinase inhibitors to inhibit EGFR phosphorylation in vivo.

The epidermal growth factor receptor (EGFR) and HER-2 tyrosine kinases have been implicated in the development, progression, and severity of several human cancers and are attractive targets for therapeutic intervention. SU11925 was developed as a small molecule inhibitor of the tyrosine kinase activity of both EGFR and HER-2. In cellular assays, SU11925 exhibited similar potency against EGFR and HER-2, inhibiting EGF-stimulated EGFR autophosphorylation in A431 (human epidermoid carcinoma) cells with an IC(50) of 30 nM and HER-2 phosphorylation in SK-OV-3TP5 (human ovarian carcinoma) cells with an IC(50) of 38 nM. In contrast to its similar activity against the two targets in cellular assays, approximately 10-fold higher plasma concentrations of SU11925 were required to inhibit HER-2 phosphorylation in HER-2-overexpressing tumors compared with EGFR phosphorylation in EGFR-overexpressing tumors in vivo. Consistent with the proposed mechanism of action of this inhibitor, SU11925 inhibited the s.c. growth of EGFR- and HER-2-dependent tumors in athymic mice at doses that produced substantial inhibition of target receptor phosphorylation in vivo. An unexpected finding from these studies was that higher plasma concentrations of SU11925 were required to inhibit EGFR phosphorylation in vivo in tumors that also express high levels of HER-2 than in tumors that express EGFR alone. This observation, which suggests that it is more difficult to inhibit EGFR phosphorylation in vivo in cells that express high levels of HER-2, was confirmed with ZD1839 (Iressa), a selective EGFR inhibitor that also targets the tyrosine kinase catalytic site. The potential clinical implications of this observation are discussed.

In vitro selection and characterization of HIV-1 with reduced susceptibility to PMPA.

9-(2-phosphonomethoxypropyl)adenine (PMPA) has demonstrated remarkable anti-simian immunodeficiency virus (SIV) activity in macaque models of SIV infection and transmission prevention. Recently, PMPA and its oral prodrug, bis-POC PMPA, have also shown potent anti-human immunodeficiency virus type 1 (HIV-1) activity in Phase I clinical studies. In vitro experiments were performed to address the resistance properties of PMPA. After eight passages in increasing concentrations of PMPA, HIV-1IIIB was able to grow in the presence of 2 microM PMPA, fivefold above the IC50 of PMPA for wild-type parental virus. Sequence analysis of the reverse transcriptase (RT) genes from four of 15 RT clones demonstrated the presence of a K65R substitution in RT and recombinant HIV expressing the K65R RT mutation showed a threefold to fourfold increase in IC50 value for PMPA as compared to wild-type. Additional experiments demonstrated that viruses expressing other nucleoside-associated RT resistance mutations all showed wild-type or < threefold reduced susceptibility to PMPA in vitro. Interestingly, lamivudine-resistant viruses expressing the M184V RT mutation showed wild-type to slightly increased susceptibility to PMPA in vitro and addition of the M184V mutation to HIV with the K65R mutation resulted in reversion to wild-type susceptibility for PMPA. In agreement with the cell culture findings, Escherichia coli-expressed K65R RT showed fivefold reduced susceptibility to PMPA diphosphate, the active moiety of PMPA. Furthermore, in combination experiments, PMPA with hydroxyurea showed synergistic inhibition of HIV replication in vitro. The potent antiretroviral activity and favourable resistance profile of PMPA and bis-POC PMPA are being further investigated in ongoing clinical trials.

Kinetic analysis of the interaction between the diphosphate of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, ddCTP, AZTTP, and FIAUTP with human DNA polymerases beta and gamma.

The inhibitory effects of the diphosphate of (S)-1-(3-hydroxy-2-phosphonylmethoxy-propyl)cytosine (HPMPCpp) toward human DNA polymerases beta and gamma were studied. The Ki values of HPMPCpp were compared with the Ki values of the triphosphates of 3'-azidothymidine (AZTTP), 2',3'-dideoxycytidine (ddCTP) and 5-iodo-2'-fluoroarabinosyluridine (FIAUTP). The Ki values toward DNA polymerase beta in increasing order were 1.32, 1.43, 140, and 520 microM for ddCTP, FIAUTP, AZTTP and HPMPCpp, respectively. The Ki values toward DNA polymerase gamma in increasing order were 0.034, 0.031, 18.3 and 299 microM for ddCTP, FIAUTP, AZTTP and HPMPCpp, respectively. Therefore, HPMPC would be expected to have less inhibitory effects on DNA repair (DNA polymerase beta) and mitochondrial DNA synthesis (DNA polymerase gamma) than ddC, FIAU or AZT.

Anti-HIV activity of adefovir (PMEA) and PMPA in combination with antiretroviral compounds: in vitro analyses.

Adefovir (PMEA, 9-(2-phosphonomethoxyethyl)adenine), an acyclic nucleoside phosphonate analogue is active against retroviruses, hepadnaviruses and herpesviruses. Adefovir dipivoxil, an orally bioavailable prodrug of adefovir is currently in phase III clinical trials for the treatment of HIV and phase II clinical trials for the treatment of HBV infections. PMPA (9-(2-phosphonomethoxypropyl)adenine) is a related acyclic nucleoside phosphonate analogue that has demonstrated potent anti-SIV activity in rhesus macaques and recently has shown marked anti-HIV activity in a phase I clinical study. Since the standard of care for AIDS patients has become combination therapy, the effects of other antiretroviral compounds (d4T, ddC, AZT, ddI, 3TC, nelfinavir, ritonavir, indinavir, and saquinavir) on the anti-HIV activity of adefovir and PMPA were investigated in vitro. Adefovir and PMPA both demonstrated strong synergistic anti-HIV activity in combination with AZT. Adefovir demonstrated minor to moderate synergistic inhibition of HIV replication in combination with PMPA, d4T, ddC, nelfinavir, ritonavir, and saquinavir. PMPA demonstrated minor synergistic inhibition of HIV replication in combination with ddI and nelfinavir (and adefovir). All other combinations showed additive inhibition of HIV replication in vitro. Importantly, no antagonistic interactions were measured for any of the adefovir or PMPA combinations.

In vitro selection and molecular characterization of human immunodeficiency virus type 1 with reduced sensitivity to 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA).

9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA) is an acyclic nucleotide with potent in vitro activity against human immunodeficiency virus type 1 (HIV-1). The present study was undertaken to determine whether HIV-1 resistance to PMEA could be generated by in vitro selection and if so, to determine which mutations in reverse transcriptase (RT) were responsible. HIV-1LAI was serially passaged for 10 months in the presence of increasing concentrations of PMEA up to a maximum of 40 microM. After 40 passages, the 50% inhibitory concentration (IC50) of PMEA had increased almost 7-fold from 4.45 to 30.5 microM. Some cross-resistance to 2',3'-dideoxycytidine (ddC, zalcitabine), 2',3-dideoxyinosine (ddI, didanosine), and 3'-thiacytidine (3TC, lamivudine) was also observed, but no cross-reactive resistance to 3'-azido-3'-thymidine (AZT, zidovudine). Sequencing of the RT encoding region of each of eight pol clones from resistant isolates revealed a Lys-65-->Arg (K65R) substitution. HIV with the K65R mutation inserted by site-directed mutagenesis also had decreased sensitivity to PMEA in H9 cells and a similar cross-resistance profile. Thus, HIV can develop decreased sensitivity to PMEA after long-term in vitro exposure and this change is associated with a K65R substitution. Additional studies will be needed to determine whether a similar mutation in HIV RT develops in patients receiving PMEA or its orally bioavailable prodrug adefovir dipivoxil (bis-POM PMEA).

In vitro characterization of the anti-human cytomegalovirus activity of PMEA (Adefovir).

PMEA [9-[2-(phosphonomethoxy)ethyl]adenine; adefovir] has shown anti-cytomegalovirus activity in animal models and in preliminary human trials. PMEA diphosphate (PMEApp), the active antiviral metabolite of PMEA, is a potent inhibitor of human cytomegalovirus (HCMV) DNA polymerase. PMEA is efficiently taken up and phosphorylated to PMEApp in numerous human cell lines. In vitro replication of wild type and drug resistant HCMV clinical isolates is effectively inhibited by PMEA. PMEA in combination with other anti-HCMV agents shows additive inhibition of HCMV replication.

A rapid antiviral in situ enzyme-linked immunosorbent assay for feline immunodeficiency virus.

An in situ enzyme-linked immunosorbent assay (ELISA) was developed as a rapid alternative to the focal infectivity assay (FIA) for screening potential anti-retroviral molecules. The assay utilizes 96-well microtiter plates to allow for determination of antiviral effect and cytotoxicity of multiple compounds simultaneously. In contrast to the FIA which requires visual scoring of foci under low-power microscopy, the 96-well ELISA is read spectrophotometrically based on the soluble alkaline phosphatase substrate, p-nitrophenyl phosphate. The IC50 and CC50 values for several antiretroviral compounds were determined using the ELISA and results were confirmed by FIA. In all cases, compounds assayed by the newly described ELISA exhibited IC50 values in agreement with literature values derived from either the FIA or reverse transcriptase assays.

Virological, clinical and serological responses of sheep infected with tissue culture adapted bluetongue virus serotypes 10, 11, 13 and 17.

Sheep were experimentally infected with cloned strains of tissue culture adapted bluetongue virus (BTV) serotypes 10, 11, 13 and 17. All the infected animals developed viremia by Day 2 or 3 post-inoculation (P.I.) and reached maximum viremia on Day 7 P.I. The viremia lasted for 2 to 3 weeks. Animals infected with the different serotypes showed mild clinical bluetongue (BT) responses, characterized by pyrexia and leukopenia, which coincided with the peak of viremia. Antibodies appeared by Day 10 P.I. and reached maximum by Day 28 P.I. There was a temporal relationship between the increase in neutralizing antibody titer, the drop in titer and clearance of virus from the peripheral circulation. Recovery from primary infection protected the animals against secondary challenge with homologous virus.

Detection of viral antigens in bluetongue virus-infected ovine tissues, using the peroxidase-antiperoxidase technique.

An indirect immunoperoxidase procedure was developed to detect viral antigens in bluetongue virus (BTV)-infected tissues. Embryonating chicken eggs were infected with BTV serotypes 10, 11, 13, or 17, and the chorioallantoic membranes were subsequently fixed in formalin and embedded in paraffin. The peroxidase-antiperoxidase (PAP) system was used to examine the infected membranes for the presence of viral antigens. Sheep antisera raised against BTV serotypes 10, 11, 13, and 17 served as the primary antibodies in the PAP procedure. Specific staining was observed when each of these antisera was applied to membranes expressing antigens of homologous and heterologous BTV serotypes. The PAP method was rapid, reliable, and specific in its detection of BTV.

Human cytomegalovirus ie1 transactivates the alpha promoter-enhancer via an 18-base-pair repeat element.

The expression of alpha (immediate-early) genes of cytomegalovirus is regulated via a complex enhancer that consists of several different repeat elements. We describe here the autoinduction of expression from the alpha promoter-enhancer by the most abundant alpha gene product, a 491-amino-acid nuclear phosphoprotein referred to as ie1. We defined the 18-base-pair repeat element within the alpha enhancer as the signal through which ie1 acts to regulate gene expression. This element contains an NF kappa B site that may play an important role in ie1 autoregulation. Our analysis, which relied on deletions through the enhancer as well as reconstitution of responsiveness to a promoter with synthetic 18-base-pair repeats, strongly implicated ie1 in the transcriptional transactivation of the alpha promoter through its enhancer.

Expression of the catalytic subunit (UL54) and the accessory protein (UL44) of human cytomegalovirus DNA polymerase in a coupled in vitro transcription/translation system.

The catalytic subunit (UL54) and accessory protein (UL44) of human cytomegalovirus (HCMV) DNA polymerase have been cloned and expressed in an in vitro-coupled transcription/translation reticulocyte lysate system. The influence of the 5'-untranslated region (5'-UTR) on the efficiency of expression from the circular plasmids has been investigated. For expression of both UL54 and UL44, a truncated form of the alfalfa mosaic virus (AMV) RNA4 5'-UTR was found to be superior to the full-length AMV 5'-UTR or the original HCMV 5'-UTRs of different lengths. Protein products with Mr approximately 140 and 55 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in the UL54 and UL44 in vitro expression reactions, respectively. The properties of the expressed enzyme were compared with those of native HCMV DNA polymerase purified from HCMV-infected cells. DNA polymerase and 3'-5' exonuclease activities of the expressed UL54/UL44 complex were found to be dependent on salt concentration in the same manner as the activities of the native enzyme. The in vitro-expressed enzyme resembles the purified HCMV DNA polymerase in its affinity for deoxynucleoside triphosphates as well as in its sensitivity to known inhibitors (cidofovir diphosphate, ganciclovir triphosphate, and foscarnet). This straightforward method for protein expression may also be applicable to other enzymes where reproducible generation of fully functional products is desirable.

Characterization of drug resistance-associated mutations in the human cytomegalovirus DNA polymerase gene by using recombinant mutant viruses generated from overlapping DNA fragments.

A number of specific point mutations in the human cytomegalovirus (HCMV) DNA polymerase (UL54) gene have been tentatively associated with decreased susceptibility to antiviral agents and consequently with clinical failure. To precisely determine the roles of UL54 mutations in HCMV drug resistance, recombinant UL54 mutant viruses were generated by using cotransfection of nine overlapping HCMV DNA fragments into permissive fibroblasts, and their drug susceptibility profiles were determined. Amino acid substitutions located in UL54 conserved region IV (N408D, F412C, and F412V), region V (A987G), and delta-region C (L501I, K513E, P522S, and L545S) conferred various levels of resistance to cidofovir and ganciclovir. Mutations in region II (T700A and V715M) and region VI (V781I) were associated with resistance to foscarnet and adefovir. The region II mutations also conferred moderate resistance to lobucavir. In contrast to mutations in other UL54 conserved regions, those residing specifically in region III (L802M, K805Q, and T821I) were associated with various drug susceptibility profiles. Mutations located outside the known UL54 conserved regions (S676G and V759M) did not confer any significant changes in HCMV drug susceptibility. Predominantly an additive effect of multiple UL54 mutations with respect to the final drug resistance phenotype was demonstrated. Finally, the influence of selected UL54 mutations on the susceptibility of viral DNA replication to antiviral drugs was characterized by using a transient-transfection-plus-infection assay. Results of this work exemplify specific roles of the UL54 conserved regions in the development of HCMV drug resistance and may help guide optimization of HCMV therapy.