RESUMEN
Starting from our in-house library of pyrazolo[3,4-d]pyrimidines, a cross-docking simulation was conducted on Bcr-Abl T315I mutant. Among the selected compounds (2a-e), the 4-bromo derivative 2b showed the best activity against the Bcr-Abl T315I mutant. Deeper computational studies highlighted the importance of the bromine atom in the para position of the N1 side chain phenyl ring for the interaction with the T315I mutant. A series of 4-bromo derivatives was thus synthesized and biologically evaluated. Compound 2j showed a good balance of different ADME properties, high activity in cell-free assays, and a submicromolar potency against T315I Bcr-Abl expressing cells. In addition, it was converted into a water-soluble formulation by liposome encapsulation, preserving a good activity on leukemic T315I cells and avoiding the use of DMSO as solubilizing agent. In vivo studies on mice inoculated with 32D-T315I cells and treated with 2j showed a more than 50% reduction in tumor volumes.
Asunto(s)
Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Diseño de Fármacos , Femenino , Proteínas de Fusión bcr-abl/química , Proteínas de Fusión bcr-abl/genética , Humanos , Ratones , Ratones Desnudos , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Mutación Puntual , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Estructura Terciaria de Proteína , Pirazoles/síntesis química , Pirimidinas/síntesis química , Transfección , Carga Tumoral/efectos de los fármacosRESUMEN
We investigated some pyrrolobenzoxazepinone (PBOs, 3e-i) analogues of early described effective non-nucleoside inhibitors of HIV-1 reverse transcriptase (RT). Enzymological studies of 3e-i enantiomers, with wild type (wt) RT and some drug-resistant mutants, revealed a stereoselective mode of action and selectivity for RT ternary complex. Unexpectedly (+)-3g was found more potent towards the L100I mutant than towards the wt RT, whereas (+)-3h inhibited the K103N mutant and RT wt with comparable potency.
Asunto(s)
Fármacos Anti-VIH/química , Transcriptasa Inversa del VIH/química , VIH-1 , Oxazepinas/química , Inhibidores de la Transcriptasa Inversa/química , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral/efectos de los fármacos , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Mutación , Oxazepinas/metabolismo , Oxazepinas/farmacología , Pirroles/química , Pirroles/metabolismo , Pirroles/farmacología , Inhibidores de la Transcriptasa Inversa/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A hit optimization protocol applied to the first nonnucleoside inhibitor of the ATPase activity of human DEAD-box RNA helicase DDX3 led to the design and synthesis of second-generation rhodanine derivatives with better inhibitory activity toward cellular DDX3 and HIV-1 replication. Additional DDX3 inhibitors were identified among triazine compounds. Biological data were rationalized in terms of structure-activity relationships and docking simulations. Antiviral activity and cytotoxicity of selected DDX3 inhibitors are reported and discussed. A thorough analysis confirmed human DDX3 as a valid anti-HIV target. The compounds described herein represent a significant advance in the pursuit of novel drugs that target HIV-1 host cofactors.
Asunto(s)
Fármacos Anti-VIH/síntesis química , ARN Helicasas DEAD-box/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/toxicidad , Línea Celular Tumoral , Simulación por Computador , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/toxicidad , Técnicas de Silenciamiento del Gen , VIH-1/efectos de los fármacos , VIH-1/enzimología , Humanos , MicroARNs/metabolismo , Rodanina/síntesis química , Rodanina/química , Rodanina/toxicidad , Relación Estructura-Actividad , Triazinas/síntesis química , Triazinas/química , Triazinas/toxicidad , Replicación Viral/efectos de los fármacosRESUMEN
Among the enzymes involved in the life cycle of HCV, the non-structural protein NS3, with its double function of protease and NTPase/helicase, is essential for the virus replication. Exploiting our previous knowledge in the development of nucleotide-mimicking NS3 helicase (NS3h) inhibitors endowed with key structural and electronic features necessary for an optimal ligand-enzyme interaction, we developed the tetrahydroacridinyl derivative 3a as the most potent NS3h competitive inhibitor reported to date (HCV NS3h K(i)=20 nM).
Asunto(s)
Descubrimiento de Drogas , Hepacivirus/enzimología , Hidrazinas/química , Hidrazinas/farmacología , Pirazinas/química , Pirazinas/farmacología , Quinolinas/química , Quinolinas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Unión Competitiva/efectos de los fármacos , Hidrazinas/síntesis química , Unión Proteica , Pirazinas/síntesis química , Quinolinas/síntesis químicaAsunto(s)
Benzamidas/síntesis química , Proteínas de Fusión bcr-abl/química , Tiadiazoles/síntesis química , Tiazoles/química , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Sustitución de Aminoácidos , Benzamidas/química , Benzamidas/farmacología , Sitios de Unión , Simulación por Computador , Diseño de Fármacos , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Mutagénesis Sitio-Dirigida , Tiadiazoles/química , Tiadiazoles/farmacología , Tiazoles/síntesis química , Tiazoles/farmacologíaRESUMEN
The tyrosine kinase Src and its close homolog Abl, both play important roles in chronic myelogenous leukemia (CML) progression and Imatinib resistance. No clinically approved inhibitors of the drug-resistant AblT315I exist to date. Here, we present a thorough kinetic analysis of two potent dual Src-Abl inhibitors towards wild type Src and Abl, and the AblT315I mutant. Our results show that the most potent compound BO1 shows only a modest loss of potency (fourfold) towards the AblT315I mutant in vitro and was an ATP-competitive inhibitor of wild type Abl but it acted as a non-competitive inhibitor in the case of AblT315I.
Asunto(s)
Proteínas Mutantes/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Familia-src Quinasas/antagonistas & inhibidores , Adenosina Trifosfato , Benzamidas , Resistencia a Antineoplásicos , Humanos , Mesilato de Imatinib , Cinética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Mutación Missense , Piperazinas/farmacología , Pirimidinas/farmacologíaRESUMEN
We report the synthesis and structure-activity relationship (SAR) of a large series of acridones and acridone-fragment derivatives designed on the basis of the selective antihepatitis C virus (HCV) activity shown by acridone 2, previously studied as a potential antibovine viral diarrhea virus (BVDV) compound. The evaluation of their ability to inhibit the HCV replication in Huh-5-2 cells led to the identification of new, selective inhibitors. This indicates that the acridone skeleton, when properly functionalized, is a suitable scaffold to obtain potential anti-HCV agents. Interestingly, during identification of possible cellular and viral targets, it was discovered that compound 23 exerts inhibitory activity on the HCV NS3 helicase, a very promising target for the development of anti-HCV drugs.
Asunto(s)
Acridinas/síntesis química , Hepacivirus/genética , Proteínas no Estructurales Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Acridinas/farmacología , Acridonas , Antivirales/síntesis química , Antivirales/farmacología , Hepacivirus/fisiología , Hepatitis C/tratamiento farmacológico , Humanos , ARN Helicasas/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
New potent indolylarylsulfone (IAS) HIV-1 NNRTIs were obtained by coupling natural and unnatural amino acids to the 2-carboxamide and introducing different electron-withdrawing substituents at position 4 and 5 of the indole nucleus. The new IASs inhibited the HIV-1 replication in human T-lymphocyte (CEM) cells at low/subnanomolar concentration and were weakly cytostatic. Against the mutant L100I, K103N, and Y181C RT HIV-1 strains in CEM cells, sulfones 3, 4, 19, 27, and 31 were comparable to EFV. The new IASs were inhibitors to Coxsackie B4 virus at low micromolar (2-9 microM) concentrations. Superimposition of PLANTS docked conformations of IASs 19 and 9 revealed different hydrophobic interactions of the 3,5-dimethylphenyl group, for which a staking interaction with Tyr181 aromatic side chain was observed. The binding mode of 19 was not affected by the L100I mutation and was consistent with the interactions reported for the WT strain.
Asunto(s)
Aminoácidos/química , Antivirales/química , Enterovirus Humano B/efectos de los fármacos , VIH-1/efectos de los fármacos , Indoles/química , Inhibidores de la Transcriptasa Inversa/química , Sulfonas/química , Animales , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Antivirales/síntesis química , Antivirales/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citostáticos/síntesis química , Citostáticos/química , Citostáticos/farmacología , Farmacorresistencia Viral , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , VIH-1/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Indoles/síntesis química , Indoles/farmacología , Linfocitos/efectos de los fármacos , Linfocitos/virología , Ratones , Modelos Moleculares , Conformación Molecular , Mutación , Unión Proteica , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Sulfonas/síntesis química , Sulfonas/farmacología , Replicación ViralRESUMEN
Starting from the prototypic compound 4, we describe new, potent, and broad-spectrum pyrrolobenzo(pyrido)oxazepinones antivirals. A biochemical and enzymological investigation was performed for defining their mechanism of inhibition at either recombinant HIV-1 RT wild type and non-nucleoside reverse transcriptase inhibitors (NNRTIs)-resistant mutants. For the novel compounds (S)-(+)-5 and (S)-(-)-7, a clear-cut stereoselective mechanism of enzyme inhibition was found. Molecular modeling studies were performed for revealing the underpinnings of this behavior.
Asunto(s)
Fármacos Anti-VIH/química , Resistencia a Medicamentos/efectos de los fármacos , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa/química , Fármacos Anti-VIH/farmacología , Secuencia Conservada , Resistencia a Medicamentos/genética , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/enzimología , VIH-1/genética , Humanos , Modelos Moleculares , Mutación , Inhibidores de la Transcriptasa Inversa/farmacología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The role played by stereochemistry in the C2-substituent (left part) on the S-DABO scaffold for anti-HIV-1 activity has been investigated for the first time. A series of S-DABO analogues, where the double bond in the C2-substituent is replaced by an enantiopure isosteric cyclopropyl moiety, has been synthesized, leading to the identification of a potent lead compound endowed with picomolar activity against RT (wt) and nanomolar activity against selected drug-resistant mutants. Molecular modeling calculation, enzymatic studies, and surface plasmon resonance experiments allowed us to rationalize the biological behavior of the synthesized compounds, which act as mixed-type inhibitors of HIV-1 RT K103N, with a preferential association to the enzyme-substrate complex. Taken together, our data show that the right combination of stereochemistry on the left and right parts (C6-substituent) of the S-DABO scaffold plays a key role in the inhibition of both wild-type and drug-resistant enzymes, especially the K103N mutant.
Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/metabolismo , Pirimidinonas/síntesis química , Pirimidinonas/farmacología , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/farmacología , Sulfuros/síntesis química , Sulfuros/farmacología , Línea Celular Tumoral , Simulación por Computador , Diseño de Fármacos , Farmacorresistencia Viral , Humanos , Cinética , Modelos Moleculares , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras , Estereoisomerismo , Resonancia por Plasmón de SuperficieRESUMEN
Indolyl aryl sulfone (IAS) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) have been previously shown to effectively inhibit wild-type (wt) and drug-resistant human immunodeficiency virus type 1 (HIV-1) replication. IASs proved to act through different mechanisms of action, depending on the nature and position of their chemical substituents. Here we describe selected novel IAS derivatives (di-halo-IASs). Our results show that these compounds are selective for the enzyme-substrate complex. The molecular basis for this selectivity was a different dissociation rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the resistant enzymes carrying the single mutations Lys103Asn, Leu100Ile, and Tyr181Ile (K103N, L100I, and Y181I), we found that one compound (RS1914) dissociated from the mutated enzymes almost 10-fold slower than from the wild type RT. These results demonstrate that IASs are very flexible molecules, interacting dynamically with the viral RT, and that this property can be successfully exploited to design inhibitors endowed with an enhanced binding to common NNRTI-resistant mutants.
Asunto(s)
Fármacos Anti-VIH/química , Farmacorresistencia Viral , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH/efectos de los fármacos , VIH/genética , Inhibidores de la Transcriptasa Inversa/química , Sulfonas/química , Fármacos Anti-VIH/farmacología , VIH/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/metabolismo , Humanos , Hidantoínas/química , Hidantoínas/farmacología , Cinética , Mutación , Unión Proteica/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad , Sulfonas/farmacologíaRESUMEN
A small family of S-DABO cytosine analogs (S-DABOCs) has been synthesized and biologically evaluated as HIV-1 inhibitor both on wild type (wt) and drug-resistant mutants leading to the identification of an interesting compound (5d). Molecular modeling studies have been finally performed in order to rationalize the results.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Citosina/análogos & derivados , Fármacos Anti-VIH/química , Citosina/síntesis química , Citosina/química , Citosina/farmacología , VIH-1/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Modelos MolecularesRESUMEN
A series of dihydro-alkylthio-benzyl-oxopyrimidines (S-DABOs) bearing a 2-aryl-2-oxoethylsulfanyl chain at pyrimidine C2, an alkyl group at C5, and a 2,6-dichloro-, 2-chloro-6-fluoro-, and 2,6-difluoro-benzyl substitution at C6 (oxophenethyl- S-DABOs, 6-8) is here described. The new compounds showed low micromolar to low nanomolar (in one case subnanomolar) inhibitory activity against wt HIV-1. Against clinically relevant HIV-1 mutants (K103N, Y181C, and Y188L) as well as in enzyme (wt and K103N, Y181I, and L100I mutated RTs) assays, compounds carrying an ethyl/ iso-propyl group at C5 and a 2,6-dichloro-/2-chloro-6-fluoro-benzyl moiety at C6 were the most potent derivatives, also characterized by low fold resistance ratio. Interestingly, the structure-activity relationship (SAR) data drawn from this DABO series are more related to HEPT than to DABO derivatives. These findings were at least in part rationalized by the description of a fair superimposition between the 6-8 and TNK-651 (a HEPT analogue) binding modes in both WT and Y181C RTs.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Benceno/química , Pirimidinonas/síntesis química , Pirimidinonas/farmacología , Compuestos de Azufre/síntesis química , Compuestos de Azufre/farmacología , Alquilación , Fármacos Anti-VIH/química , Fenómenos Químicos , Química Física , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , VIH-1/efectos de los fármacos , VIH-1/enzimología , VIH-1/genética , Hidrógeno/química , Modelos Moleculares , Estructura Molecular , Mutación/genética , Oxígeno/química , Unión Proteica , Pirimidinonas/química , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Relación Estructura-Actividad , Compuestos de Azufre/químicaRESUMEN
PBO (pyrrolobenzoxazepinone) derivatives are non-nucleoside reverse transcriptase inhibitors (NNRTIs), which display a selective interaction with the catalytic ternary complex of HIV-1 reverse transcriptase (RT) and its substrates. In order to develop novel PBOs with improved resistance profiles, we synthesised additional PBO derivatives, specifically designed to target highly conserved residues in the beta12-beta13 hairpin, the so-called "primer grip" region of HIV-1 RT. Here, we investigated the biochemical and enzymological mechanism of inhibition of HIV-1 RT wild type and carrying NNRTIs-resistance mutations, by these derivatives. Our kinetic analysis indicates that the ability of PBOs to selectively target the catalytic ternary complex of RT with its substrates directly correlates with greatly reduced sensitivity to NNRTIs-resistance mutations, particularly the K103N substitution. Molecular modeling and docking studies provided an explanation for this correlation at the structural level.
Asunto(s)
Azepinas/farmacología , Farmacorresistencia Viral , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa/farmacología , Células 3T3 , Alquinos , Animales , Azepinas/síntesis química , Benzoxazinas/farmacología , Catálisis , Línea Celular , Células Cultivadas , Ciclopropanos , ADN Polimerasa Dirigida por ADN/metabolismo , VIH-1/genética , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Ratones , Modelos Moleculares , Mutación , Nevirapina/farmacología , Inhibidores de la Transcriptasa Inversa/síntesis químicaRESUMEN
Among the FDA approved drugs for the treatment of AIDS, non-nucleoside reverse transcriptase inhibitors (NNRTIs) are essential components of first-line anti-HIV-1 therapy because of the less-severe adverse effects associated with NNRTIs administration in comparison to therapies based on other anti-HIV-1 agents. In this contest, 3,4-dihydro-2-alkoxy-6-benzyl-4-oxypyrimidines (DABOs) have been the object of many studies aimed at identifying novel analogues endowed with potent inhibitory activity towards HIV-1 wild type and especially drug-resistant mutants. Accordingly, based on the encouraging results obtained from the biological screening of our internal collection of S-DABO derivatives, we started with the systematic functionalization of the pyrimidine scaffold to identify the minimal required structural features for RT inhibition. Herein, we describe how the combination of synthetic, biological, and molecular modeling studies led to the identification of two novel subclasses of S-DABO analogues: S-DABO cytosine analogues (S-DABOCs) and 4-dimethyamino-6-vinylpyrimidines (DAVPs).
Asunto(s)
Diseño de Fármacos , VIH-1/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/síntesis química , Transcriptasa Inversa del VIH/genética , VIH-1/genética , Humanos , Mutación , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-ActividadRESUMEN
Indolyl aryl sulfones bearing the 4,5-difluoro (10) or 5-chloro-4-fluoro (16) substitution pattern at the indole ring were potent inhibitors of HIV-1 WT and the NNRTI-resistant strains Y181C and K103N-Y181C. These compounds were highly effective against the 112 and the AB1 strains in lymphocytes and inhibited at nanomolar concentration the multiplication of the IIIBBa-L strain in macrophages. Compound 16 was exceptionally potent against RT WT and RTs carrying the K103N, Y181I, and L100I mutations.
Asunto(s)
Transcriptasa Inversa del VIH/metabolismo , VIH-1/efectos de los fármacos , Indoles/síntesis química , Inhibidores de la Transcriptasa Inversa/síntesis química , Sulfonas/síntesis química , Línea Celular , Farmacorresistencia Viral , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , Humanos , Técnicas In Vitro , Indoles/química , Indoles/farmacología , Linfocitos/efectos de los fármacos , Linfocitos/virología , Macrófagos/efectos de los fármacos , Macrófagos/virología , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad , Sulfonas/química , Sulfonas/farmacologíaAsunto(s)
Acetatos/farmacología , Farmacorresistencia Viral/genética , Guanina/análogos & derivados , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/farmacología , Acetatos/química , Guanina/química , Guanina/farmacología , VIH-1/genética , Cinética , Inhibidores de la Transcriptasa Inversa/químicaAsunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Nucleótidos/metabolismo , Pirimidinas/síntesis química , Pirimidinas/farmacología , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/farmacología , Cristalografía por Rayos X , Transcriptasa Inversa del VIH/genética , Modelos Moleculares , Mutación , Conformación Proteica , Inhibidores de la Transcriptasa Inversa/metabolismoAsunto(s)
Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral , VIH-1/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/genética , Estructura Molecular , Mutación , Relación Estructura-ActividadRESUMEN
Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of action has not been elucidated yet. Here, we describe the mechanism of inhibition of HIV-1 reverse transcriptase (RT) by selected IAS derivatives. Our results showed that, depending on the substitutions introduced in the IAS common pharmacophore, these compounds can be made selective for different enzyme-substrate complexes. Moreover, we showed that the molecular basis for this selectivity was a different association rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the nonnucleoside reverse transcriptase inhibitor-resistant mutant Lys103Asn, it was possible to hypothesize, on the basis of their mechanism of action, a rationale for the design of drugs which could overcome the steric barrier imposed by the Lys103Asn mutation.