Kinase Bypass Research Articles

EXTH-12. INHIBITION OF EPIDERMAL GROWTH FACTOR RECEPTOR AND PLATELET-DERIVED GROWTH FACTOR RECEPTOR-ALPHA EXERTS SYNERGISTIC EFFICACY IN GLIOBLASTOMA

Abstract Epidermal growth factor receptor (EGFR) alterations, including amplification and activating mutations, occur in more than half of GBM cases. EGFR is located on Chr. 7, and Chr. 7 gain is one of the earliest events precipitating gliomagenesis. EGFR inhibitors, monoclonal antibodies, vaccines, and CAR-T cells have failed in GBM due to intrinsic heterogeneity and receptor tyrosine kinase (RTK) bypass pathways that mediate therapeutic resistance. New targeted therapeutic approaches to leverage synergistic combinations are desperately needed to improve GBM prognosis. Using the TCGA and other GBM databases, we previously demonstrated that PDGFRA amplification in patients with EGFR-amplified GBM carries significantly worse survival. EGFR and PDGFRA co-expression occur in more than one-third of GBM patients. The PDGFRA ligand PDGFA is also located on Chr. 7, and its expression is significantly increased with Chr. 7 gain and EGFR copy number increase. Therefore, Chr. 7 gain inherently leads to co-activation of both EGFR and PDGFRA signaling. We used patient-derived glioblastoma cells with Chr. 7 gain to test combined inhibition of EGFR and PDGFRA in vitro. We found that combined inhibition of both EGFR and PDGFRA using FDA-approved EGFR-targeted agents (Erlotinib, Gefitinib, Dacomitinib, Neratinib, and Osimertinib) and Crenolanib, respectively, leads to synergistic cytotoxicity in vitro. Inhibition of either EGFR or PDGFRA led to receptor cross-activation, and EGF and PDGF-AA-induced RTK activation was blocked by Neratinib and Crenolanib. Immunoprecipitation experiments and proximity ligation assays demonstrated that combined inhibition prevents EGFR and PDGFRA heterodimerization and pathways of therapeutic resistance. This combined inhibition led to decreased activation of downstream signaling pathways, including PI3K and MAPK. We show that combined inhibition of EGFR and PDGFRA exerts synergistic cytotoxicity in GBM and prevents resistance pathways that emerge during single-agent targeted therapy. These pathways are targetable with FDA-approved agents that could be used in patients with GBM with Chr. 7 gain.

P13.18 Inhibition of epidermal growth factor receptor and platelet-derived growth factor receptor-alpha exerts synergistic efficacy in glioblastoma

Abstract BACKGROUND Despite our understanding of the genetic changes that precipitate gliomagenesis, targeted therapy has failed in glioblastoma (GBM) with median survival not significantly improved over the past two decades. Epidermal growth factor receptor (EGFR) alterations, including amplification and activating mutations, are among the most common genetic changes in GBM, occurring in more than half of cases. EGFR is located on Chr. 7, and Chr. 7 gain is one of the earliest events precipitating gliomagenesis. Various EGFR inhibitors, including tyrosine kinase inhibitors, monoclonal antibodies, vaccines, and CAR-T cells have failed in GBM due to intrinsic heterogeneity and receptor tyrosine kinase bypass pathways that mediate therapeutic resistance. New targeted therapeutic approaches to leverage synergistic combinations are desperately needed to improve GBM prognosis. Using the TCGA and other GBM databases, we have previously demonstrated that the presence of PDGFRAamplification in patients with EGFR-amplified GBM carries significantly worse survival. EGFR and PDGFRA co-expression occur in more than one-third of GBM patients. The PDGFRA ligand PDGFA is also located on Chr. 7, and its expression is significantly increased with Chr. 7 gain and EGFR copy number increase. Therefore, Chr. 7 gain inherently leads to co-activation of both EGFR and PDGFRA signaling pathways. MATERIALS AND METHODS We used models of patient-derived glioblastoma cells to test combined inhibition of epidermal growth factor receptor and platelet-derived growth factor receptor-alpha in vitro. RESULTS Using patient-derived GBM models with Chr. 7 gain, we found that combined inhibition of both EGFR and PDGFRA using a variety of FDA-approved EGFR-targeted agents (Erlotinib, Gefitinib, Dacomitinib, Neratinib, and Osimertinib) and Crenolanib, respectively, leads to synergistic cytotoxicity in vitro. We found that inhibition of either EGFR or PDGFRA alone led to receptor cross-activation, and EGF and PDGF-AA-induced receptor tyrosine kinase activation was blocked by Neratinib and Crenolanib. Immunoprecipitation experiments and proximity ligation assays demonstrated that combined inhibition prevents EGFR and PDGFRA heterodimerization and pathways of therapeutic resistance. This combined inhibition led to decreased activation of downstream signaling pathways, including phosphatidylinositol 3-kinase and mitogen-activated protein kinase. CONCLUSIONS We show that combined inhibition of EGFR and PDGFRA exerts synergistic cytotoxicity in GBM and prevents resistance pathways that emerge during single-agent targeted therapy against these receptor tyrosine kinases. These pathways are targetable with FDA-approved agents that could be used in patients with GBM with Chr. 7 gain.

Evaluating Cancer of the Central Nervous System Through Next-Generation Sequencing of Cerebrospinal Fluid

PurposeCancer spread to the central nervous system (CNS) often is diagnosed late and is unresponsive to therapy. Mechanisms of tumor dissemination and evolution within the CNS are largely unknown because of limited access to tumor tissue.Materials and MethodsWe sequenced 341 cancer-associated genes in cell-free DNA from cerebrospinal fluid (CSF) obtained through routine lumbar puncture in 53 patients with suspected or known CNS involvement by cancer.ResultsWe detected high-confidence somatic alterations in 63% (20 of 32) of patients with CNS metastases of solid tumors, 50% (six of 12) of patients with primary brain tumors, and 0% (zero of nine) of patients without CNS involvement by cancer. Several patients with tumor progression in the CNS during therapy with inhibitors of oncogenic kinases harbored mutations in the kinase target or kinase bypass pathways. In patients with glioma, the most common malignant primary brain tumor in adults, examination of cell-free DNA uncovered patterns of tumor evolution, including temozolomide-associated mutations.ConclusionThe study shows that CSF harbors clinically relevant genomic alterations in patients with CNS cancers and should be considered for liquid biopsies to monitor tumor evolution in the CNS.

Abstract 3582: Epidermal growth factor receptor and anaplastic lymphoma kinase bypass signaling in oral squamous cell carcinoma in vivo

Abstract Oral squamous cell carcinoma (OSCC) is an insidious disease with poor prognosis. This is due to invasive disease that is refractive to conventional therapies. We reported a gene-set of hypomethylated loci in metastatic OSCC involved in Epidermal Growth Factor Receptor (EGFR) signaling. EGFR over-expression is an early event in OSCC however therapies targeting EGFR have had dismal results. Our studies showed hypomethylation of Anaplastic Lymphoma Kinase (ALK) correlates with metastatic OSCC. ALK and EGFR signaling drive glioblastoma and non-small cell lung cancers (NSCLC). Furthermore, NSCLC that develop resistance to ALK inhibition demonstrate activation of EGFR bypass signaling. Based upon these findings, we hypothesize that simultaneous targeting of both ALK and EGFR in OSCC will yield greater anti-tumor activity than single drug therapies. Our objective was to assess expression and activation of EGFR and ALK signaling pathways in response to therapies targeting EGFR and/or ALK in vitro and in mouse OSCC xenograft models. Expression of total and activated EGFR and ALK was analyzed in HSC3 cells (invasive) and SCC4 cells (non-invasive) using western blot analysis. Effects on downstream signaling molecules (AKT, STAT3, and RAS) were also evaluated following treatments with TAE684 (ALK inhibitor) and/or Gefitinib (EGFR inhibitor). Anti-tumor effects of TAE684 and/or Gefitinib were evaluated in mouse OSCC xenograft models using HSC3 and SCC4 cell lines. This study revealed that HSC3 and SCC4 cell lines have unique growth, invasion, and signaling profiles resulting in varying responses to treatments targeting ALK and/or EGFR. HSC3 cells express ALK and have active EGFR whereas SCC4 cells have low ALK expression and low EGFR activity. Subsequently, HSC3 xenografts were sensitive to Gefitinib and co-targeting ALK resulted in an additive effect. Targeting ALK alone had no effect on tumor growth. Similar to NSCLC, western blot analysis revealed that ALK inhibition alone induces increased activation of EGFR bypass signaling in both HSC3 and SCC4 cells. In contrast SCC4 xenografts failed to respond to TAE684 and/or Gefitinib treatments. Co-treatment with TAE684 and Gefitinib down regulated the already low EGFR activity. Downstream effector molecules AKT and STAT3 had reduced activity in response to co-treatments in both HSC3 and SCC4 cell lines. Conversely, MAPK activity increased with all treatments in both cell lines. Therefore, we conclude that OSCC with active EGFR and ALK signaling are responsive to combination treatments targeting ALK and EGFR resulting in more efficacious anti-tumor effects than single drug therapies. Furthermore, single anti-ALK therapy induces compensatory EGFR activation and has no effect on tumor growth. Additional parallel pathways may account for elevated MAPK activity in response to all treatments and warrants further investigation. Citation Format: Cara B. Gonzales, Heping Chen, Jorge J. De La Chapa, Nameer B. Kirma. Epidermal growth factor receptor and anaplastic lymphoma kinase bypass signaling in oral squamous cell carcinoma in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3582. doi:10.1158/1538-7445.AM2015-3582

IGF-1R Inhibition Activates a YES/SFK Bypass Resistance Pathway: Rational Basis for Co-Targeting IGF-1R and Yes/SFK Kinase in Rhabdomyosarcoma

The insulin-like growth factor 1 receptor (IGF-1R) has surfaced as a significant target in multiple solid cancers due to its fundamental roles in pro-survival and anti-apoptotic signaling. However, development of resistance to IGF-1R blockade represents a significant hindrance and limits treatment efficacy in the clinic. In this study, we identified acquired resistance to IGF-1R blockade with R1507, an antibody against IGF-1R, and with BMS-754807, a small molecular inhibitor of IGF-1R/insulin receptor (IR). We showed that treatment with an IGF-IR antibody, R1507, or an IR/IGF-IR kinase inhibitor, BMS-754807, was associated with increased activation of YES/SRC family tyrosine kinase (SFK) in rhabdomyosarcoma (RMS). Combining anti–IGF-1R agents with SFK inhibitors resulted in blockade of IGF-1R inhibition–induced activation of YES/SFK and displayed advantageous antitumor activity in vitro and in vivo. Our data provide evidence that IGF-1R blockade results in activation of the YES/SRC family kinase bypass resistance pathway in vitro and in vivo. This may be of particular clinical relevance since both Yes and IGF components are overexpressed in RMS. Increased YES/SFK activation might serve as a clinical biomarker for predicting tumor resistance to IGF-1R inhibition. Dual inhibition of IGF-1R and SFK may have a broader and enhanced clinical benefit for patients with RMS.

Role of IGF-1R inhibition on an Src-family kinase bypass resistance pathway: A rational basis for cotargeting IGF-1R and Src kinase in pediatric sarcomas?

10025 Background: Dysregulation of IGF signaling plays a fundamental role in oncogenesis in pediatric sarcomas. We recently completed a Phase II study targeting the IGFI receptor signaling pathway in refractory Ewing’s and other sarcomas. We demonstrated an objective response rate of 16 percent, but most responses were transient lasting less than 18 weeks. The majority of patients, even those with initial responses, do not have long term benefit from IGFIR blockade, indicating the presence of an innately resistant tumor mass or the recruitment of compensatory pathways allowing for continued growth. To improve on these responses, we have been probing these tumors to identify other critical pathways that might allow combined targeting approaches. Methods: Multiple RMS and ES cell lines were treated with IGF1R kinase inhibitors and assayed for up-regulation of various signaling pathways. Combination treatment with IGF1R inhibitors and inhibitors of additional signaling pathways were then tested in vitro and in vivo using standard techniques. For in vivo xenograft studies, treatments began 11 days following orthotopic injection of tumor cells. Results: We have identified repid up-regulation of Src family kinase (SFK) signaling within 4 hours of IGF1R blockade in both RMS and ES cell lines. Of note, combined treatment with IGF1R Ab plus IGF1R kinase inhibitors most potently upregulated SFK signaling. Based on these findings, we tested combined IGF1R blockade with SFK inhibition using the commercially available drug, dasatinib. We show that dual blockade of IGF1R and SFK pathways were synergistic in vitro. Furthermore, in xenograft models of RMS, the combination IGF1R and SFK inhibition led to long-term disease free status for at least 90 days in some mice, never seen in our hands previously using these models. Conclusions: This work identified that IGF-1R inhibition induced activation of Src kinase that may act as a by-pass pathway. Synergistic activity of IGF-1R and SFK kinase inhibitors was observed in vitro and in vivo. Dual IGFI and SFK kinase inhibition may lead to improved therapeutic outcomes.

Application of kinase bypass strategies to nucleoside antivirals

Nucleoside and nucleotide analogs have served as the cornerstones of antiviral therapy for many viruses. However, the requirement for intracellular activation and side-effects caused by distribution to off-target sites of toxicity still limit the efficacy of the current generation of drugs. Kinase bypass strategies, where phosphorylated nucleosides are delivered directly into cells, thereby, removing the requirement for enzyme catalyzed phosphorylation steps, have already changed the face of antiviral therapy in the form of the acyclic nucleoside phosphonates, cidofovir, adefovir (given orally as its dipivoxil prodrug) and tenofovir (given orally as its disoproxil prodrug), currently used clinically. These strategies hold further promise to advance the field of antiviral therapy with at least 10 kinase bypass and tissue targeted prodrugs, representing seven distinct prodrug classes, currently in clinical trials. This article reviews the history of kinase bypass strategies applied to nucleoside antivirals and the evolution of different tissue targeted prodrug strategies, highlighting clinically relevant examples.

Evaluation of Novel Phosphoramidate ProTides of the 2′-Fluoro Derivatives of a Potent Anti-Varicella Zoster Virus Bicyclic Nucleoside Analogue

Recently, the synthesis and antiviral activity of a series of 2'-fluoro derivatives of the most potent anti-varicella zoster virus (VZV) agent reported to date, the bicyclic nucleoside analogue Cf1743, have been reported. Here, we present molecular modelling studies for the interaction of these compounds with VZV-encoded thymidine kinase (TK) and we report the synthesis of a series of phosphoramidate ProTides of these compounds designed to bypass the nucleoside kinase dependence of the parent nucleoside analogues. The phosphoramidate prodrugs were equally effective as their parent compounds against VZV in cell culture, but lost antiviral potency against TK-deficient VZV strains. ProTide-based kinase bypass is not successful in this case.

The Application of Phosphoramidate Protide Technology to Acyclovir Confers Anti-HIV Inhibition

Recently, it has been reported that phosphorylated acyclovir (ACV) inhibits human immunodeficiency virus type 1 (HIV-1) reverse transcriptase in a cell-free system. To deliver phosphorylated ACV inside cells, we designed ACV monophosphorylated derivatives using ProTide technology. We found that the L-alanine derived ProTides show anti-HIV activity at noncytotoxic concentrations; ester and aryl variation was tolerated. ACV ProTides with other amino acids, other than L-phenylalanine, showed no detectable activity against HIV in cell culture. The inhibitory activity of the prodrugs against herpes simplex virus (HSV) types -1 and -2 and thymidine kinase-deficient HSV-1 revealed different structure-activity relationships but was again consistent with successful nucleoside kinase bypass. Enzymatic and molecular modeling studies have been performed in order to better understand the antiviral behavior of these compounds. ProTides showing diminished carboxypeptidase lability translated to poor anti-HIV agents and vice versa, so the assay became predictive.

The application of the phosphoramidate ProTide approach confers micromolar potency against Hepatitis C virus on inactive agent 4′-azidoinosine: Kinase bypass on a dual base/sugar modified nucleoside

Novel phosphoramidate ProTides derived from 4′-azidoinosine have been prepared and evaluated in the replicon assay against hepatitis C Virus (HCV). The parent nucleoside analogue is inactive in this assay, while the ProTides are active at low μM levels in some cases. This is a rare example of an inosine nucleoside analogue with potent antiviral activity and further supports the notion of ProTides as a drug discovery motif.

Successful kinase bypass with new acyclovir phosphoramidate prodrugs

Novel phosphoramidates of acyclovir have been prepared and evaluated in vitro against acyclovir-sensitive and -resistant herpes simplex virus (HSV) types 1 and 2 and varicella-zoster virus (VZV). Unlike the parent nucleoside these novel phosphate prodrugs retain antiviral potency versus the ACV-resistant virus strain, suggesting an efficient bypass of the viral thymidine kinase.

HINT as Putative Phosphoramidase Responsible of ProTides Activation: Molecular Modelling Studies

The ProTide approach was developed by McGuigan et al. as a method to improve the biological activity of poorly active nucleoside analogues by “kinase bypass” in which lipophilic nucleotide pro-drugs act as intracellular phosphate delivery motifs and achieve the generation of free nucleotides by nucleoside kinase-independent means. The activation of phosphoramidates is considered to be based upon two enzymatic cleavages: the hydrolysis of the amino acid ester moiety as the trigger of the process, and the Psingle bondN bond cleavage as the final step that would release the corresponding nucleoside analogue monophosphate. The latter step is carried out by an enzyme with phosphoramidase activity and it has been proposed that the protein responsible of the cleavage of the Psingle bondN bond belongs to the HINT family: adenosine monophosphoramidates AMP-NH2, AMP-N-ɛ-(N-α-acetyl lysine methyl ester) and AMP-para-nitroaniline were identified as rabbit Hint and yeast Hint1 substrates. Notably, AMP-N-alanine methyl ester was also reported as a substrate for the same enzymes, which might indicate their role in the activation of our phosphoramidates. We have recently reported some initial docking studies on a series BVDU phosphoramidate analogues in the human HINT-1 active site, which showed that indeed this enzyme could be able to bind and process the phosphoramidates. In the work presented here, we have extended the modelling simulations to different antiviral nucleoside analogues and examined their binding to HINT-1, as well as to the homology models of HINT-2 and HINT-3, with the aim of developing a predictive computational model which could be used in the design of more potent and selective phosphoramidate analogues (Fig. 1).

Kinase Profiling as a Tool in Molecular Dissection of Imatinib Resistant Chronic Myeloid Leukemia (CML).

Imatinib (Gleevec) resistance (IR) in CML is a multifactorial phenomenon related to ineffective blockage of the bcr-abl kinase through point mutation and/or gene amplification, or alternatively kinase bypass through clonal evolution or presently unknown mechanisms. The newer generation of bcr-abl small molecule inhibitors (dasatinib, nilotinib) may be useful in overcoming ineffective bcr-abl blockage in IR-CML but may not be effective in other mechanisms of resistance. As a tool in classifying the pattern of disease resistance, we profiled kinase levels in IR-CML following switch to new kinase inhibitors or with imatinib dose escalation. In Ficoll-purified blood and bone marrow samples, we examined levels of BCR-ABL autophosphorylation (Tyr245), and phosphorylation of two bcr-abl targets STAT5 (Tyr694/699) and CRKL (Tyr207) by Western blot. Kinase profiling was compared to hematologic responses, cytogenetic analysis, FISH for the bcr-abl locus using dual fusion probes and quantitative(q)PCR for bcr-abl transcript. We profiled 26 patients with IR-CML undergoing therapy shifts as well as serial samples from 10 patients receiving frontline dasatinib therapy for newly diagnosed CML. In all but 1 of 68 analyzed samples, detection of phospho(p)-CRKL in unsorted white blood cells was associated with bcr-abl/abl qPCR ratios above 1%. Among all 68 samples, pSTAT5 and pBCR-ABL levels declined in parallel to pCRKL but were absent in more samples than pCRKL. Persistence of detectable pCRKL despite switch to nilotinib, dasatinib or high dose imatinib was seen in 5 patients with bcr-abl kinase domain (KD) mutations including 2 with G250E and 1 each with L248V, F311L and T315I, 2 patients with bcr-abl gene amplification and 3 patients without KD mutations including 2 with cytogenetic clonal evolution. Among these 10 patients with IR-CML and persistent pCRKL, complete hematologic responses were noted in 4 and complete cytogenetic response in 3. Response to therapy switch and absence of pCRKL was seen in tumors with E355G, L387M and E459K KD mutations and in 12 patients with no mutation or genomic amplification. Among the patients receiving frontline therapy with dasatinib, effective hematologic responses correlated well with loss of detectable pCRKL and pSTAT5. However, detectable pCRKL did not disappear until at least 8 weeks following initiation of therapy, and lagged hematologic response. The 2 patients treated with frontline dasatinib therapy who had persistent pCRKL after 20 weeks of treatment also showed amplification of the bcr-abl locus in tumor cells by FISH. Kinase profiling, particularly for pCRKL, is useful for monitoring molecular response in the therapy switch period in IR-CML and correlates well with the predicted pattern of response in tumors with KD mutations. The value of monitoring kinase levels frequently following initiation of frontline kinase inhibitor therapy in newly diagnosed CML remains to be established.

Heat Shock Protein 70 over Expression Is Associated to Imatinib Resistance in Chronic Myelogenous Leukemia.

Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative syndrome characterised by the expression of the recombinant oncoprotein Bcr-Abl. Imatinib inhibits Bcr-Abl tyrosine kinase activity leading to apoptosis of leukemic cells sparing normal hematopoiesis. Several mechanisms of resistance to imatinib have been identified both in vitro and in vivo: Bcr-Abl mutations, an over-expression of the Bcr-Abl kinase itself or other tyrosine kinase bypass. To identify unknown mechanism, we used an imatinib resistant cell line (K562-R) generated from the erythroblastic cell line K562 (K562-S) (Blood , 2000; 93: 1070–1079) for which all described mechanisms of resistance have been previously invalidated. Previous results from a proteomic study identified some chaperon proteins such as heat shock proteins with an increased expression level in K562-R. One of them, the heat shock protein 70, Hsp70, has a 3 fold increase expression level in K562-R cells, results which have been confirmed by western-blot analysis. To characterise the role of Hsp 70 in imatinib resistance, we inhibit Hsp 70 expression by RNA silencing (siRNA) in K562-R cells and over-express it in K562-S cells. Inhibition of Hsp70 protein expression by siRNA decrease Hsp70 expression rapidly over 90% at day 4 which is associated with a significant reduction of viability (66 ± 6%, n = 5, p < 0.03). Over expression of Hsp 70 in K562-S cells induced a significant increase of resistance to imatinib since the addition of imatinib only increases mortality by 27 ± 5 % in comparison to 52 ± 4 % for K562-S cells (n = 4, p < 0.001). Detection of HSF-1 phosphorylation, the major transcription factor involved in Hsp 70 expression, did not show significant differences between K562-S and K562-R cells although over a 3 fold increase is detected in the mRNA level of Hsp 70 in K562-R cells by quantitative PCR. Furthermore, the comparison of Hsp70 expression in mononuclear cells of 7 CML patients before imatinib treatment and at the relapse time shows that Hsp 70 is increased in imatinib resistant patients suggesting it could also play a role in resistance in vivo. Present study confirmed that over expression of Hsp 70 in the cell line K562-R is involved in the mechanism of imatinib resistance in vitro. Moreover, the correlation between the increase of Hsp 70 in CML patient cells and resistance suggests it could be an interesting marker and potentially a therapeutic target.

Second Generation of cycloSal‐Pronucleotides with Esterase‐Cleavable Sites: The ”Lock‐In”‐Concept †

A conceptual extension of the cycloSal‐pronucleotide approach is presented. The characteristic feature of the new cycloSal‐derivatives of the anti‐HIV active nucleoside analogue d4T 1 is the incorporation of an enzymatically cleavable carboxylic ester moiety with the intention to trap the triesters inside cells (”lock‐in”‐concept). CycloSal‐triesters bearing different ester groups in the 3‐or 5‐position of the cycloSal‐moiety are described. Surprisingly, only acetyl‐and levulinyl esters are cleaved readily in CEM cell extracts while alkyl esters were found to be stable. Nevertheless, in in‐vitro anti‐HIV assays most of the compounds achieve the thymidine–kinase bypass, thus proving that they act at least as nucleotide delivery systems. †In honor and celebration of the 70th birthday of Professor Leroy B. Townsend.

Synthesis and antiviral evaluation of phosphoramidate derivatives of (E)-5-(2-bromovinyl)-2'-deoxyuridine.

We report the design, synthesis and antiviral evaluation of a number of lipophilic, masked phosphoramidate derivatives of the antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), designed to act as membrane soluble prodrugs of the free nucleotide. The phosphoramidate derivatives of BVDU that contain L-alanine exhibited potent anti herpes simplex virus type 1 and varicella-zoster virus activity but lost marked activity against thymidine kinase-deficient virus strains. The phosphoramidate derivative bearing the amino acid alpha,alpha-dimethylglycine showed poor activity in all cell lines tested. It appears that successful kinase bypass by phosphoramidates is highly dependent on the nucleoside analogue, amino acid and ester structure, as well as the cell line to which the drugs are exposed.

Synthesis and Evaluation of 5-Halo 2′,3′-Didehydro-2′,3′-Dideoxynucleosides and their Blocked Phosphoramidates as Potential Anti-Human Immunodeficiency virus Agents: An Example of ‘Kinase Bypass’

A series of 5-halo derivatives of the anti-human immunodeficiency virus (HIV) nucleoside analogue d4T have been prepared by a general and highly stereoselective route. A key step is the electrophilic addition of N-iodosuccinimide to furanoid glycols. The 2′-iodo nucleosides thus obtained produced the corresponding 2′/3′-didehydro-2′/3′-dideoxynucleoside in a two-step elimination reaction upon treatment with potassium t-butoxide followed by sodium methoxide. The derivatives were tested for their ability to inhibit the replication of HIV-1 and HIV-2 in cell culture. Replacement of the thymine 5-methyl group of the parent nucleoside analogue (d4T) by I, CI or F resulted in compounds that showed poor antiviral activity. In view of this, we studied the application of blocked phosphate (phosphoramidate) technology to probe the efficacy of intracellular monophosphate delivery in these systems. In one case, significant antiviral potency was obtained in this way.

6 Kinase by-pass: A new strategy for antiviral drug discovery: C. McGuigan, R.N. Pathirana, P. Bellevergue and M. Davies [Dept. of Chemistry, University of Southampton, Southampton, UK], N. Mahmood [MRC Collaborative Centre, Mill Hill, UK] and A.J. Hay [National Institute for Medical Research, London, UK

6 Kinase by-pass: A new strategy for antiviral drug discovery: C. McGuigan, R.N. Pathirana, P. Bellevergue and M. Davies [Dept. of Chemistry, University of Southampton, Southampton, UK], N. Mahmood [MRC Collaborative Centre, Mill Hill, UK] and A.J. Hay [National Institute for Medical Research, London, UK

Kinase bypass: A new strategy for anti-HIV drug design

The 5′-bis(trichloroethyl) phosphate derivatives of several 3′-O-acyl thymidines were prepared from the thymidine phosphate. Even though the parent nucleosides are inactive as antiviral agents, the phosphates are selective inhibitors of the proliferation of HIV. This activity is attributed to a new mechanism of action, we herein describe as “kinase by-pass”.

Nucleoside analogues previously found to be inactive against HIV may be activated by simple chemical phosphorylation

Nucleoside analogues previously found to be inactive against the human immunodeficiency virus (HIV) may be activated by simple chemical derivatisation. As part of our effort to deliver masked phosphates inside living cells we have discovered that certain phosphate triester derivatives of inactive nucleoside analogues become inhibitors of HIV replication. This discovery underlies the importance of the masked phosphate approach, and has significant implications for the future design of chemotherapeutic nucleoside analogues. If highly modified nucleoside analogues may be active without the intervention of nucleoside kinase enzymes, major advantage may accrue in terms of low toxicity and enhanced selectivity. Moreover, the increased structural freedom may have implications for dealing with the emergence of resistance. The concept herein described as ‘kinase bypass’ may thus stimulate the discovery of a new generation of antiviral agents.