GD2 and GD3 gangliosides as diagnostic biomarkers for all stages and subtypes of epithelial ovarian cancer.

Background: Ovarian cancer (OC) is the deadliest gynecological cancer, often diagnosed at advanced stages. A fast and accurate diagnostic method for early-stage OC is needed. The tumor marker gangliosides, GD2 and GD3, exhibit properties that make them ideal potential diagnostic biomarkers, but they have never before been quantified in OC. We investigated the diagnostic utility of GD2 and GD3 for diagnosis of all subtypes and stages of OC. Methods: This retrospective study evaluated GD2 and GD3 expression in biobanked tissue and serum samples from patients with invasive epithelial OC, healthy donors, non-malignant gynecological conditions, and other cancers. GD2 and GD3 levels were evaluated in tissue samples by immunohistochemistry (n=299) and in two cohorts of serum samples by quantitative ELISA. A discovery cohort (n=379) showed feasibility of GD2 and GD3 quantitative ELISA for diagnosing OC, and a subsequent model cohort (n=200) was used to train and cross-validate a diagnostic model. Results: GD2 and GD3 were expressed in tissues of all OC subtypes and FIGO stages but not in surrounding healthy tissue or other controls. In serum, GD2 and GD3 were elevated in patients with OC. A diagnostic model that included serum levels of GD2+GD3+age was superior to the standard of care (CA125, p<0.001) in diagnosing OC and early-stage (I/II) OC. Conclusion: GD2 and GD3 expression was associated with high rates of selectivity and specificity for OC. A diagnostic model combining GD2 and GD3 quantification in serum had diagnostic power for all subtypes and all stages of OC, including early stage. Further research exploring the utility of GD2 and GD3 for diagnosis of OC is warranted.

Therapeutic Neuroprotection by an Engineered Neurotrophin that Selectively Activates Tropomyosin Receptor Kinase (Trk) Family Neurotrophin Receptors but Not the p75 Neurotrophin Receptor.

The neurotrophin growth factors bind and activate two types of cell surface receptors: the tropomyosin receptor kinase (Trk) family and p75. TrkA, TrkB, and TrkC are bound preferentially by nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 (NT3), respectively, to activate neuroprotective signals. The p75 receptors are activated by all neurotrophins, and paradoxically in neurodegenerative disease p75 is upregulated and mediates neurotoxic signals. To test neuroprotection strategies, we engineered NT3 to broadly activate Trk receptors (mutant D) or to reduce p75 binding (mutant RK). We also combined these features in a molecule that activates TrkA, TrkB, and TrkC but has reduced p75 binding (mutant DRK). In neurodegenerative disease mouse models in vivo, the DRK protein is a superior therapeutic agent compared with mutant D, mutant RK, and wild-type neurotrophins and protects a broader range of stressed neurons. This work rationalizes a therapeutic strategy based on the biology of each type of receptor, avoiding activation of p75 toxicity while broadly activating neuroprotection in stressed neuronal populations expressing different Trk receptors. SIGNIFICANCE STATEMENT: The neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 each can activate a tropomyosin receptor kinase (Trk) A, TrkB, or TrkC receptor, respectively, and all can activate a p75 receptor. Trks and p75 mediate opposite signals. We report the engineering of a protein that activates all Trks, combined with low p75 binding, as an effective therapeutic agent in vivo.

Rapid High-Yield Production of Functional SARS-CoV-2 Receptor Binding Domain by Viral and Non-Viral Transient Expression for Pre-Clinical Evaluation.

Vaccine design strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are focused on the Spike protein or its subunits as the main antigen target of neutralizing antibodies. In this work, we propose rapid production methods of an extended segment of the Spike Receptor Binding Domain (RBD) in HEK293SF cells cultured in suspension, in serum-free media, as a major component of a COVID-19 subunit vaccine under development. The expression of RBD, engineered with a sortase-recognition motif for protein-based carrier coupling, was achieved at high yields by plasmid transient transfection or human type-5-adenoviral infection of the cells, in a period of only two and three weeks, respectively. Both production methods were evaluated in 3L-controlled bioreactors with upstream and downstream bioprocess improvements, resulting in a product recovery with over 95% purity. Adenoviral infection led to over 100 µg/mL of RBD in culture supernatants, which was around 7-fold higher than levels obtained in transfected cultures. The monosaccharide and sialic acid content was similar in the RBD protein from the two production approaches. It also exhibited a proper conformational structure as recognized by monoclonal antibodies directed against key native Spike epitopes. Efficient direct binding to ACE2 was also demonstrated at similar levels in RBD obtained from both methods and from different production lots. Overall, we provide bioprocess-related data for the rapid, scalable manufacturing of low cost RBD based vaccines against SARS-CoV-2, with the added value of making a functional antigen available to support further research on uncovering mechanisms of virus binding and entry as well as screening for potential COVID-19 therapeutics.

Alternative Splicing of a Receptor Intracellular Domain Yields Different Ectodomain Conformations, Enabling Isoform-Selective Functional Ligands.

Events at a receptor ectodomain affect the intracellular domain conformation, activating signal transduction (out-to-in conformational effects). We investigated the reverse direction (in-to-out) where the intracellular domain may impact on ectodomain conformation. The primary sequences of naturally occurring TrkC receptor isoforms (TrkC-FL and TrkC.T1) only differ at the intracellular domain. However, owing to their differential association with Protein Disulfide Isomerase the isoforms have different disulfide bonding and conformations at the ectodomain. Conformations were exploited to develop artificial ligands, mAbs, and small molecules, with isoform-specific binding and biased activation. Consistent, the physiological ligands NT-3 and PTP-sigma bind both isoforms, but NT-3 activates all signaling pathways, whereas PTP-sigma activates biased signals. Our data support an "in-to-out" model controlling receptor ectodomain conformation, a strategy that enables heterogeneity in receptors, ligands, and bioactivity. These concepts may be extended to the many wild-type or oncogenic receptors with known isoforms.

Vaccination with Tumor-Ganglioside Glycomimetics Activates a Selective Immunity that Affords Cancer Therapy.

Immune targeting of (glyco)protein tumor markers has been useful to develop cancer and virus vaccines. However, the ganglioside family of tumor-associated glycolipids remains intractable to vaccine approaches. Here we show that synthetic antigens mimicking the carbohydrate moiety of GD2 or GD3 gangliosides can be used as vaccines to activate a selective humoral and cellular immunity that is therapeutic against several cancers expressing GD2 or GD3. Adoptive transfer of T cells generated after vaccination elicits tumor-infiltrating lymphocytes of the γδ T cell receptor and CD8+ phenotypes; and affords a high therapeutic index. The glycomimetic vaccine principles can be expanded to target the family of tumor gangliosides and other carbohydrates expressed primarily in pathological states.

In retinitis pigmentosa TrkC.T1-dependent vectorial Erk activity upregulates glial TNF-α, causing selective neuronal death.

In some diseases the TrkC.T1 isoform is upregulated in glia, associated with glial TNF-α production and neuronal death. What remains unknown are the activating signals in glia, and how paracrine signals may be selective for a targeted neuron while sparing other proximate neurons. We studied these questions in the retina, where Müller glia contacts photoreceptors on one side and retinal ganglion cells on the other. In a mutant Rhodopsin mouse model of retinitis pigmentosa (RP) causing progressive photoreceptor death-but sparing retinal ganglion cells-TrkC.T1 and NT-3 ligand are upregulated in Müller glia. TrkC.T1 activity generates p-Erk, which causes increased TNF-α. These sequential events take place predominantly in Müller fibers contacting stressed photoreceptors, and culminate in selective death. Each event and photoreceptor death can be prevented by reduction of TrkC.T1 expression, by pharmacological antagonism of TrkC or by pharmacological inhibition Erk. Unmasking the sequence of non-cell autologous events and mechanisms causing selective neuronal death may help rationalize therapies.

The Paradoxical Signals of Two TrkC Receptor Isoforms Supports a Rationale for Novel Therapeutic Strategies in ALS.

Full length TrkC (TrkC-FL) is a receptor tyrosine kinase whose mRNA can be spliced to a truncated TrkC.T1 isoform lacking the kinase domain. Neurotrophin-3 (NT-3) activates TrkC-FL to maintain motor neuron health and function and TrkC.T1 to produce neurotoxic TNF-α; hence resulting in opposing pathways. In mouse and human ALS spinal cord, the reduction of miR-128 that destabilizes TrkC.T1 mRNA results in up-regulated TrkC.T1 and TNF-α in astrocytes. We exploited conformational differences to develop an agonistic mAb 2B7 that selectively activates TrkC-FL, to circumvent TrkC.T1 activation. In mouse ALS, 2B7 activates spinal cord TrkC-FL signals, improves spinal cord motor neuron phenotype and function, and significantly prolongs life-span. Our results elucidate biological paradoxes of receptor isoforms and their role in disease progression, validate the concept of selectively targeting conformational epitopes in naturally occurring isoforms, and may guide the development of pro-neuroprotective (TrkC-FL) and anti-neurotoxic (TrkC.T1) therapeutic strategies.

Combinatorial assembly of small molecules into bivalent antagonists of TrkC or TrkA receptors.

A library of peptidomimetics was assembled combinatorially into dimers on a triazine-based core. The pharmacophore corresponds to ß-turns of the neurotrophin polypeptides neurotrophin-3 (NT-3), nerve growth factor (NGF), or brain-derived neurotrophic factor (BDNF). These are the natural ligands for TrkC, TrkA, and TrkB receptors, respectively. The linker length and the side-chain orientation of each monomer within the bivalent mimics were systematically altered, and the impact of these changes on the function of each ligand was evaluated. While the monovalent peptidomimetics had no detectable binding or bioactivity, four bivalent peptidomimetics (2c, 2d, 2e, 3f) are selective TrkC ligands with antagonistic activity, and two bivalent peptidomimetics (1a, 1b) are TrkC and TrkA ligands with antagonistic activity. All these bivalent compounds block ligand-dependent receptor activation and cell survival, without affecting neuritogenic differentiation. This work adds to our understanding of how the neurotrophins function through Trk receptors, and demonstrates that peptidomimetics can be designed to selectively disturb specific biological signals, and may be used as pharmacological probes or as therapeutic leads. The concept of altering side-chain, linker length, and sequence orientation of a subunit within a pharmacophore provides an easy modular approach to generate larger libraries with diversified bioactivity.

Enhancement of the cytotoxic potential of the mixed EGFR and DNA-targeting 'combi-molecule' ZRBA1 against human solid tumour cells by a bis-quinazoline-based drug design approach.

ZRBA1 is a quinazoline-based molecule termed 'combi-molecule' designed to block the epidermal growth factor receptor (EGFR) and further degrade to FD105, an EGFR inhibitor plus a DNA-alkylating agent. To augment the potency of ZRBA1, we designed JDE52, a bistriazene that, following degradation, was 'programmed' to yield higher concentrations of the free inhibitor FD105 and a more cytotoxic bifunctional DNA-damaging species. The results indicated that JDE52 was capable of inducing significant blockade of EGFR phosphorylation, DNA strand breaks and interstrand cross-links in human cells. The fluorescent property of FD105, the secondary inhibitor that both JDE52 and ZRBA1 are capable of releasing, has permitted the analysis of its levels in tumour cells by ultraviolet flow cytometry. It was found that JDE52 was indeed capable of significantly releasing higher levels of fluorescence (P<0.05) in human tumour cells when compared with ZRBA1. Apoptosis was triggered by JDE52 at a faster rate than ZRBA1 and led to higher levels of cell killing. The results in toto suggest that the superior potency of JDE52, when compared with ZRBA1, may be imputed to mechanisms associated with the generation of higher intracellular concentrations of FD105 and to the induction of DNA cross-links. These combined mechanisms (blockade of EGFR-tyrosine kinase and induction of cross-links) contributed to an accelerated rate of apoptosis by JDE52. This study conclusively demonstrated that designing molecules as prodrugs of high levels of quinazoline inhibitors of EGFR and bifunctional DNA cross-linking species is a valid strategy to enhance the potency of mixed EGFR-DNA-targeting combi-molecules.

A bioanalytical investigation on the exquisitely strong in vitro potency of the EGFR-DNA targeting type II combi-molecule ZR2003 and its mitigated in vivo antitumour activity.

ZR2003 is a type II of combi-molecule designed to target DNA and the epidermal growth factor receptor (EGFR) without requirement for hydrolysis. In human tumour cell lines cultured as monolayers, it showed 6.5-35 fold greater activity than Iressa. Further evaluation in 3D organ-like multilayer aggregates showed that it could block proliferation at submicromolar level. However, despite the superior potency of ZR2003 over Iressa in vitro, its activity xenograft models was not significantly different from that of Iressa. To rationalize these results, we determined the tumour concentration of both ZR2003 and Iressa in vivo and more importantly in vitro in multicellular aggregates. The results showed that in A431 and 4T1 xenografts, the level of ZR2003 absorbed in the tumours were consistently 2-fold less than those generated by Iressa. Likewise, in the multicellular aggregates model, the penetration of ZR2003 was consistently lower than Iressa. In serum containing media, the level of extractable or free ZR2003 was also inferior to those of Iressa. The results from this bioanalytical study, suggest that the discrepancy between the in vitro and in vivo potency of ZR2003 when compared with Iressa, may be imputed to its significantly lower tumour concentration.

Ligand-dependent TrkA activity in brain differentially affects spatial learning and long-term memory.

In the central nervous system, the nerve growth factor (NGF) receptor TrkA is expressed primarily in cholinergic neurons that are implicated in spatial learning and memory, whereas the NGF receptor p75(NTR) is expressed in many neuronal populations and glia. We asked whether selective TrkA activation may have a different impact on learning, short-term memory, and long-term memory. We also asked whether TrkA activation might affect cognition differently in wild-type mice versus mice with cognitive deficits due to transgenic overexpression of mutant amyloid-precursor protein (APP mice). Mice were treated with wild-type NGF (a ligand of TrkA and p75(NTR)) or with selective pharmacological agonists of TrkA that do not bind to p75(NTR). In APP mice, the selective TrkA agonists significantly improved learning and short-term memory. These improvements are associated with a reduction of soluble Aß levels in the cortex and AKT activation in the cortex and hippocampus. However, this improved phenotype did not translate into improved long-term memory. In normal wild-type mice, none of the treatments affected learning or short-term memory, but a TrkA-selective agonist caused persistent deficits in long-term memory. The deficit in wild-type mice was associated temporally, in the hippocampus, with increased AKT activity, increased brain-derived neurotrophic factor precursor, increased neurotrophin receptor homolog-2 (p75-related protein), and long-term depression. Together, these data indicate that selective TrkA activation affects cognition but does so differently in impaired APP mice versus normal wild-type mice. Understanding mechanisms that govern learning and memory is important for better treatment of cognitive disorders.

A monovalent agonist of TrkA tyrosine kinase receptors can be converted into a bivalent antagonist.

BACKGROUND: Receptor tyrosine kinases (RTK) act through dimerization. Previously it was thought that only bivalent ligands could be agonistic, whereas monovalent ligands should be antagonistic. This notion changed after the demonstration that monovalent ligands can be agonistic, including our report of a small molecule monovalent ligand "D3" that is a partial agonist of the NGF receptor TrkA. A bivalent "D3-linker-D3" was expected to increase agonism. METHODS: Dimeric analogs were synthesized and tested in binding, biochemical, and biological assays. RESULTS: One analog, 1-ss, binds TrkA with higher affinity than D3 and induces or stabilizes receptor dimers. However, 1-ss exhibited antagonistic activity, through two mechanisms. One mechanism is that 1-ss blocks NGF binding, unlike D3 which is non-competitive. Inhibition of NGF binding may be due to the linker of 1-ss filling the inter-receptor space that NGF traverses before docking. In a second mechanism, 1-ss acts as a pure antagonist, inhibiting NGF-independent TrkA activity in cells over-expressing receptors. Inhibition is likely due to 1-ss "freezing" the TrkA dimer in the inactive state. CONCLUSIONS: Dimerization of an RTK can result in antagonism, through two independent mechanisms. GENERAL SIGNIFICANCE: we report a small molecule monovalent agonist being converted to a bivalent antagonist.

Bivalent diketopiperazine-based tropomysin receptor kinase C (TrkC) antagonists.

Bivalent molecules containing two beta-turn mimics with side chains that correspond to hot-spots on the neurotrophin NT-3 were prepared. Binding assays showed the mimetics to be selective TrkC ligands, and biological assays showed one mimetic to be an antagonist of the TrkC receptor.

An agonistic TrkB mAb causes sustained TrkB activation, delays RGC death, and protects the retinal structure in optic nerve axotomy and in glaucoma.

PURPOSE: Brain-derived neurotrophic factor (BDNF) receptors TrkB and p75(NTR) are expressed in the retina. However, exogenous BDNF does not provide retinal ganglion cells (RGCs) with long-lasting neuroprotection in vivo during optic nerve axotomy or in glaucoma rat models of neurodegeneration. The authors set out to answer the hypothesis that a selective TrkB agonist might afford more efficient neuroprotection. METHODS: Animal models of acute neurodegeneration (complete optic nerve axotomy) and chronic neurodegeneration (ocular hypertension, glaucoma) were used. After intravitreal delivery of test agents or controls, surviving RGCs were quantified. Transient or sustained activation of TrkB receptors in vivo was quantified by Western blot analysis retinal samples for TrkB-phosphotyrosine. Time-dependent changes to the neuronal retinal layers were quantified longitudinally by Fourier domain-optical coherence tomography. RESULTS: The authors show that a selective TrkB agonist caused long-lived TrkB activation and significantly delayed RGC death in these models of acute and chronic retinal injury in vivo. Importantly, using noninvasive retinal imaging, they also show that a selective TrkB agonist caused preservation of the retinal structure in both animal models, with maintenance of the layers comprising neurons and neuronal fibers. CONCLUSIONS: In animal models of acute and chronic neurodegeneration, a TrkB agonist affords long-lasting neuroprotection by causing sustained TrkB activation. The use of structural end points could have prognostic value to evaluate neuroprotection. This work contributes to the understanding of neurotrophic mechanisms underlying RGC death in glaucoma and optic nerve axotomy.

Bivalent peptidomimetic ligands of TrkC are biased agonists and selectively induce neuritogenesis or potentiate neurotrophin-3 trophic signals.

This study was initiated to find small molecule ligands that would induce a functional response when docked with neurotrophin Trk receptors. "Minimalist" mimics of beta-turns were designed for this purpose. These mimics are (i) rigid, yet easily folded into turn-like conformations, and (ii) readily accessible from amino acids bearing most of the natural side chains. Gram quantities of 16 of these turn mimics were prepared and then assembled into 152 fluorescein-labeled bivalent peptidomimetics via a solution-phase combinatorial method. Fluorescence-based screening of these molecules using cells transfected with the Trk receptors identified 10 potential ligands of TrkC, the receptor for neurotrophin-3. Analogues of these bivalent peptidomimetics with biotin replacing the fluorescein label were then prepared and tested to confirm that binding was not due to the fluorescein. Several assays were conducted to find the mode of action of these biotinylated compounds. Thus, direct binding, survival and neuritogenic, and biochemical signal transduction assays showed 8 of the original 10 hits were agonistic ligands binding to the ectodomain of TrkC. Remarkably, some peptidomimetics afford discrete signals leading to either cell survival or neuritogenic differentiation. The significance of this work is three-fold. First, we succeeded in finding small, selective, proteolytically stable ligands for the TrkC receptor; there are very few of these in the literature. Second, we show that it is possible to activate distinct and biased signaling pathways with ligands binding at the ectodomain of wild-type receptors. Third, the discovery that some peptidomimetics initiate different modes of cell signaling increases their potential as pharmacological probes and therapeutic leads.

A peptidomimetic of NT-3 acts as a TrkC antagonist.

Neurotrophins are a family of growth factors that regulate the peripheral and central nervous system. We designed and tested a mini-library of small molecules peptidomimetics based on beta-turns of the neurotrophin growth factor polypeptides NT-3, which is the natural ligand for TrkC receptors. Biological studies identified a peptidomimetic 2Cl that exhibited selective antagonism of TrkC. 2Cl reduces TrkC activation and signaling promoted by NT-3, and selectively blocks ligand-dependent cell survival. 2Cl also blocks ligand-independent TrkC activation and signals that take place when the receptor is over-expressed. This work adds to our understanding of how the neurotrophins function through Trk receptors, and demonstrates that peptidomimetics can be designed to selectively disturb neurotrophin-receptor interactions, and receptor activation.

A combinatorial method for solution-phase synthesis of labeled bivalent beta-turn mimics.

Piperidine-functionalized, 1,4-disubstituted-1,2,3-triazoles of generic structure 1 were conceived as "minimalist" mimics of peptidic beta-turn structures. Key features of these molecules include (i) the possibility of incorporating amino acid side chains corresponding to many of the protein amino acids; (ii) a close correspondence of separations of these side chains to i + 1 to i + 2 residues in turns; (iii) facile adjustment of the side-chain vectors on docking while only influencing two critical degrees of freedom; and (iv) some electrostatic polarity. Fifteen monomers of this type were made via copper-mediated cycloaddition reactions. Solution-phase methodologies were devised to assemble these monomers into bivalent compounds in high purity states (typically >85%) so that they could be used in first-pass biological assays without further purification. The skeleton for forming these bivalent compounds is triazine-based. There is a third site which allowed for introduction of a fluorescent label (library of compounds 2) or an alkyne-functionalized triethylene glycol chain (library of compounds 3) included to promote water-solubility and to allow incorporation of probes via copper-mediated cycloaddition reactions. In the event, two 135-membered libraries were prepared, one consisting of compounds 2 and the other of 3. No protecting groups or coupling agents were required; these attributes of the method were important to allow most of the products to be obtained in over 85% purities. The fluorescein-tagged library of compounds 2 was screened in a fluorescence-activated cell sorting (FACS) assay using cells transfected to overexpress one of the following neurotrophin receptors: TrkA, TrkC, and p75. Preliminary findings indicate four compounds 2gm, 2gn, 2gi, and 2gj bound the TrkA receptor selectively; all of these contain a threonine-lysine turn mimic. Thus, a pharmacological probe for the TrkA receptor has been developed.

Novel nitrogen mustard-armed combi-molecules for the selective targeting of epidermal growth factor receptor overexperessing solid tumors: discovery of an unusual structure-activity relationship.

To enhance the potency of "combi-molecules", we designed 6a-d and 18 to release an inhibitor of EGFR TK and a bifunctional alkylator. The combi-molecules blocked EGFR TK with potency increasing with the basicity of the mustard moiety. They selectively killed cells transfected with EGFR and were potent against the DU145 prostate cancer cells. Combi-molecule 6a blocked EGFR phosphorylation in an irreversible manner, induced DNA-cross-links, and arrested the cells in mid-S.

The combi-targeting concept: in vitro and in vivo fragmentation of a stable combi-nitrosourea engineered to interact with the epidermal growth factor receptor while remaining DNA reactive.

PURPOSE: JDA58 (NSC 741282), a "combi-molecule" optimized in the context of the "combi-targeting concept," is a nitrosourea moiety tethered to an anilinoquinazoline. Here, we sought to show its binary epidermal growth factor receptor (EGFR)/DNA targeting property and to study its fragmentation in vitro and in vivo. EXPERIMENTAL DESIGN: The fragmentation of JDA58 was detected in cells in vitro and in vivo by fluorescence microscopy and tandem mass spectrometry. EGFR phosphorylation and DNA damage were determined by Western blotting and comet assay, respectively. Tumor data were examined for statistical significance using the Student's t test. RESULTS: JDA58 inhibited EGFR tyrosine kinase (IC(50), 0.2 micromol/L) and blocked EGFR phosphorylation in human DU145 prostate cancer cells. It induced significant levels of DNA damage in DU145 cells in vitro or in vivo and showed potent antiproliferative activity both in vitro and in a DU145 xenograft model. In cell-free medium, JDA58 was hydrolyzed to JDA35, a fluorescent amine that could be observed in tumor cells both in vitro and in vivo. In tumor cells in vitro or in vivo, or in plasma collected from mice, the denitrosated species JDA41 was the predominant metabolite. However, mass spectrometric analysis revealed detectable levels of the hydrolytic product JDA35 in tumor cells both in vitro and in vivo. CONCLUSIONS: The results in toto suggest that growth inhibition in vitro and in vivo may be sustained by the intact combi-molecule plus JDA35 plus JDA41, three inhibitors of EGFR, and the concomitantly released DNA-damaging species. This leads to a model wherein a single molecule carries a complex multitargeted-multidrug combination.

The combi-targeting concept: synthesis of stable nitrosoureas designed to inhibit the epidermal growth factor receptor (EGFR).

According to the "combi-targeting" concept, the EGFR tyrosine kinase (TK) inhibitory potency of compounds termed "combi-molecules" is critical for selective growth inhibition of tumor cells with disordered expression of EGFR or its closest family member erbB2. Here we report on the optimization of the EGFR TK inhibitory potency of the combi-molecules of the nitrosourea class by comparison with their aminoquinazoline and ureidoquinazoline precursors. This led to the discovery of a new structural parameter that influences their EGFR TK inhibitory potency, i.e., the torsion angle between the plane of the quinazoline ring and the ureido or the nitrosoureido moiety of the synthesized drugs. Compounds (3'-Cl and Br series) with small angles (0.5-3 degrees ) were generally stronger EGFR TK inhibitors than those with large angles (18-21 degrees ). This was further corroborated by ligand-receptor van der Waals interaction calculations that showed significant binding hindrance imposed by large torsion angles in the narrow ATP cleft of EGFR. Selective antiproliferative studies in a pair of mouse fibroblast NIH3T3 cells, one of which NIH3T3/neu being transfected with the erbB2 oncogene, showed that IC(50) values for inhibition of EGFR TK could be good predictors of their selective potency against the serum-stimulated growth of the erbB2-tranfected cell line (Pearson r = 0.8). On the basis of stability (t(1/2)), EGFR TK inhibitory potency (IC(50)), and selective erbB2 targeting, compound 23, a stable nitrosourea, was considered to have the structural requirements for further development.