Drug design for ever, from hype to hope

In its first 25 years JCAMD has been disseminating a large number of techniques aimed at finding better medicines faster. These include genetic algorithms, COMFA, QSAR, structure based techniques, homology modelling, high throughput screening, combichem, and dozens more that were a hype in their time and that now are just a useful addition to the drug-designers toolbox. Despite massive efforts throughout academic and industrial drug design research departments, the number of FDA-approved new molecular entities per year stagnates, and the pharmaceutical industry is reorganising accordingly. The recent spate of industrial consolidations and the concomitant move towards outsourcing of research activities requires better integration of all activities along the chain from bench to bedside. The next 25 years will undoubtedly show a series of translational science activities that are aimed at a better communication between all parties involved, from quantum chemistry to bedside and from academia to industry. This will above all include understanding the underlying biological problem and optimal use of all available data

Modeling of transient protein-protein interactions: a structural study of the thioredoxin system

Protein-protein interactions play a central role in most biological processes. One such biological process is the maintenance of a reducing environment inside the cell. To maintain an internal reducing environment, living cells have evolved two enzymatic systems (glutathione and thioredoxin (Trx) systems). The Trx system is composed of the enzyme TrxR and its substrate Trx. The two proteins constitute an important thiol-dependent redox system that catalyzes the reduction of many proteins that are responsible for a variety of cellular functions. The system relies on transient protein-protein interactions between Trx and TrxR for its function. Cross-reactivity of components of the Trx system between species has been shown to be medically relevant. For example, Helicobacter pylori Trx (HP Trx) is thought to mediate catalytic reduction of human immunoglobulins and thus facilitate immune evasion. It has also been proposed that Helicobacter pylori gains access to the impenetrable gastric mucous layer by using secreted HP Trx to reduce the disulfide bonds present in the cysteine-rich mucin regions that are responsible for cross-linking mucin monomers. Therefore, disruption of secreted HP Trx-host protein interaction may result in restoration of the viscoelastic and hydrophobic protective properties of mucus. Previous studies aimed at understanding the nature of cross-reactivity of Trx system components among various species have shown that Trxs have higher affinity for cognate TrxRs (same species), than for TrxRs from different species. However, the basis for this specificity is not known. A growing body of evidence suggests that most protein-protein interactions are mediated by a small number of protein-protein interface residues, referred to as “hot spot” residues or binding epitopes. Therefore, understanding the biochemical basis of the affinity of proteins for their partners usually begins by identifying the hot spot residues responsible for the protein complex interactions. In this study, the crystal structures of Deinococcus radiodurans thioredoxin reductase (DR TrxR) and Helicobacter pylori TrxR (HP TrxR) were determined at 1.9 Å and 2.4 Å respectively. Analysis of the Trx-binding sites of both structures suggests that the basis of affinity and specificity of Trx for TrxR is primarily due to the shape rather than the charge of the surface. In addition, the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (EC Trx) was used to identify residues that are responsible for TrxR-Trx interface stability. Using computational alanine scanning mutagenesis and visual inspection of the EC TrxR-Trx interface, 22 EC TrxR side chains were shown to make contact across the TrxR-Trx interface. Although more than 20 EC TrxR side chains make contact across the TrxR-Trx interface, our results suggest that only 4 residues (F81, R130, F141, and F142) account for the majority of the EC TrxR-Trx interface stability. Individual replacement of equivalent DR TrxR residues (M84, K137, F148, F149) with alanine resulted in drastic changes in binding affinity, confirming that the four residues account for most of TrxR-Trx interface stability. These hot spot residues are surrounded by less important residues (hydrophobic and hydrophilic) that are also predicted to contribute to interface stability. F148 and F149 are invariant across bacterial TrxRs, however other residues that contact Trx are less conserved including M84 and K137. When M84 and K137 were changed to match equivalent E. coli TrxR residues (K137R, M84F); D. radiodurans TrxR substrate specificity was altered from its own Trx to that of E. coli Trx. The results suggest that a small subset of the TrxR-Trx interface residues are responsible for the majority of Trx binding affinity and specificity, a property that has been shown to general to protein-protein interfaces

Designing Peptidomimetics

The concept of a peptidomimetic was coined about forty years ago. Since then, an enormous effort and interest has been devoted to mimic the properties of peptides with small molecules or pseudopeptides. The present report aims to review different approaches described in the past to succeed in this goal. Basically, there are two different approaches to design peptidomimetics: a medicinal chemistry approach, where parts of the peptide are successively replaced by non-peptide moieties until getting a non-peptide molecule and a biophysical approach, where a hypothesis of the bioactive form of the peptide is sketched and peptidomimetics are designed based on hanging the appropriate chemical moieties on diverse scaffolds. Although both approaches have been used in the past, the former has been more widely used to design peptidomimetics of secretory peptides, whereas the latter is nowadays getting momentum with the recent interest in designing protein-protein interaction inhibitors. The present report summarizes the relevance of the information gathered from structure-activity studies, together with a short review on the strategies used to design new peptide analogs and surrogates. In a following section there is a short discussion on the characterization of the bioactive conformation of a peptide, to continue describing the process of designing conformationally constrained analogs producing first and second generation peptidomimetics. Finally, there is a section devoted to review the use of organic scaffolds to design peptidomimetics based on the information available on the bioactive conformation of the peptide.Postprint (author's final draft

STRUCTURAL MODELING OF PROTEIN-PROTEIN INTERACTIONS USING MULTIPLE-CHAIN THREADING AND FRAGMENT ASSEMBLY

Since its birth, the study of protein structures has made progress with leaps and bounds. However, owing to the expenses and difficulties involved, the number of protein structures has not been able to catch up with the number of protein sequences and in fact has steadily lost ground. This necessitated the development of high-throughput but accurate computational algorithms capable of predicting the three dimensional structure of proteins from its amino acid sequence. While progress has been made in the realm of protein tertiary structure prediction, the advancement in protein quaternary structure prediction has been limited by the fact that the degree of freedom for protein complexes is even larger and even fewer number of protein complex structures are present in the PDB library. In fact, protein complex structure prediction till date has largely remained a docking problem where automated algorithms aim to predict the protein complex structure starting from the unbound crystal structure of its component subunits and thus has remained largely limited in terms of scope. Secondly, since docking essentially treats the unbound subunits as "rigid-bodies" it has limited accuracy when conformational change accompanies protein-protein interaction. In one of the first of its kind effort, this study aims for the development of protein complex structure algorithms which require only the amino acid sequence of the interacting subunits as input. The study aimed to adapt the best features of protein tertiary structure prediction including template detection and ab initio loop modeling and extend it for protein-protein complexes thus requiring simultaneous modeling of the three dimensional structure of the component subunits as well as ensuring the correct orientation of the chains at the protein-protein interface. Essentially, the algorithms are dependent on knowledge-based statistical potentials for both fold recognition and structure modeling. First, as a way to compare known structure of protein-protein complexes, a complex structure alignment program MM-align was developed. MM-align joins the chains of the complex structures to be aligned to form artificial monomers in every possible order. It then aligns them using a heuristic dynamic programming based approach using TM-score as the objective function. However, the traditional NW dynamic programming was redesigned to prevent the cross alignment of chains during the structure alignment process. Driven by the knowledge obtained from MM-align that protein complex structures share evolutionary relationships and the current protein complex structure library already contains homologous/structurally analogous protein quaternary structure families, a dimeric threading approach, COTH was designed. The new threading-recombination approach boosts the protein complex structure library by combining tertiary structure templates with complex alignments. The query sequences are first aligned to complex templates using the modified dynamic programming algorithm, guided by a number of predicted structural features including ab initio binding-site predictions. Finally, a template-based complex structure prediction approach, TACOS, was designed to build full-length protein complex structures starting from the initial templates identified by COTH. TACOS, fragments the templates aligned regions of templates and reassembles them while building the structure of the threading unaligned region ab inito using a replica-exchange monte-carlo simulation procedure. Simultaneously, TACOS also searches for the best orientation match of the component structures driven by a number of knowledge-based potential terms. Overall, TACOS presents the one of the first approach capable of predicting full length protein complex structures from sequence alone and introduces a new paradigm in the field of protein complex structure modeling

Pseudopeptides and Peptidomimetics Modulating the Proteolytic Activity of Kallikrein-related Peptidase 3

The highly prostate specific serine protease kallikrein-related peptidase 3 (KLK3, also known as prostate specific antigen, PSA) is widely used as a biomarker for prostate cancer and it has also been postulated that it may play a part in tumour growth. Especially interestesting is the antiangiogenic effect exerted by proteolytically active KLK3 in cell line models. In order to stimulate the proteolytic activity of KLK3, a series of peptides have been developed by phage display methodology. Even though the peptides are quite potent KLK3 stimulators, they are not directly suitable for in vivo studies or use as drugs. Even though there are many natural and unnatural biologically active peptides, they suffer from rapid clearance via the liver and kidneys and proteolytic degradation of the compounds both in the gastrointestinal tract and other parts of the body. This gives peptides a poor oral bioavailability meaning that they are usually administered as intravenous or intramuscular injections. Several different strategies have been developed in order to access compounds with improved bioavailability including modifications of the peptide structure, development of pseudopeptides and development of small molecular weight peptidomimetics. This thesis concentrates on the further development of the two most potent peptides known to stimulate KLK3, i.e. B-2 and C-4. The main part of the work was concentrated on the replacement of disulphide bridges in the peptides in order to both gain more information on which residues are necessary for obtaining the biological activity and at the same time also gain information on how changes to the geometry of the disulphide bridge affects the activity. A series of different disulphide bridge mimicking building blocks were designed and synthesised with the intention of using them in a protocol for solid-phase synthesis of KLK3 stimulating peptides. Unfortunately, the use of these building blocks in the synthesis of pseudopeptides based on C-4 turned out to be an unsurmountable challenge and the synthesis had to be completed using a different strategy in which the key step was the use of ring-closing metathesis (RCM) for the cyclisation of the partly completed pseudopeptide. Pleasingly, the synthesis of pseudopeptide analogues of the B-2 peptide using the building blocks was more successful. In total three pseudopeptide analogues of C-4 and four of B-2 were synthesised and shown to retain the biological activity of the parent peptides. Based on the information from the synthesised pseudopeptides and a molecular modelling study, a 4-quinolone based peptidomimetic was designed to mimic the C-4 peptide and a synthetic protocol was devised to access this compound. Even though the synthesis of the desired target compound has so far not been successful, the synthetic protocol that was designed has given access to a number of 1,2,8-trisubstituted 4-quinolone derivatives.Prostatacancer är i nuläget den vanligaste formen av cancer hos män i västvärlden, så mycket som var sjätte man har uppskattats insjukna i den under sin livstid. Även om dödligheten beräknas till endast några procent innebär den stora utbredningen att prostatacancer orsakar det näst högsta antalet dödsfall i cancer hos män efter lungcancer. Pga detta är det viktigt att hitta nya metoder både för att bota prostatacancer och för att sakta ner sjukdomsförloppet. Kallikrein 3 (human kallikrein-related peptidase 3, KLK3; även känd som prostataspecifik antigen, PSA) är ett prostataspecifikt enzym som klyver proteiner och peptider (en proteas). KLK3 utsöndras i sädesvätskan och dess biologiska uppgift är att klyva de gelbildande proteiner som utsöndras vid ejakulation. Enzymatiskt aktivt KLK3 har visat sig inhibera tillväxten av blodkärl både in vitro och in vivo vilket har antagits bidra till en långsammare tumörtillväxt. En elakartad tumör i prostatan sänker utsöndringen av KLK3 i epitelvävnaden jämfört med en frisk prostata. KLK3 inverkar på tumörtillväxten i prostatacancer genom att påverka de proteolytiska kaskaderna som bryter ned den extracellulära matrisen och därmed inhibera tumörtillväxten. Substanser som gör det möjligt att modifiera den proteolytiska aktiviteten hos KLK3 är mycket intressanta för utvecklingen av nya läkemedel mot prostatacancer. En serie peptider som stimulerar den proteolytiska aktiviteten hos KLK3 har tidigare utvecklats vid Helsingfors universitet. Även om dessa peptider uppvisar en stark stimulerande effekt på KLK3 så sönderfaller de snabbt i plasma, de har också en förhållandevis stor molekylmassa för att vara peptider vilket starkt begränsar deras användbarhet in vivo. Studier har påvisat vilka delar av dessa peptider som är viktiga för den biologiska aktiviteten, utgående från denna information har vi syntetiserat serier med modifierade peptider för att vidare söka utreda vilka modifikationer som är möjliga att göra utan att den biologiska aktiviteten sjunker avsevärt. Förhoppningen är att dessa modifikationer också förbättrar peptidernas stabilitet in vitro och in vivo. De två mest aktiva av de kända peptiderna valdes ut och de naturliga disulfidbryggorna i dessa ersattes med en kolvätekedja samtidigt som andra, mindre förändringar, gjordes i övriga delar av peptiden. Vissa av peptiderna i dessa serier visade sig ha en klar biologisk aktivitet som dock var något lägre än den hos ursprungspeptiderna. En viktig slutsats av studien var att inga omfattande förändringar i peptidens struktur är möjliga. Det slutliga målet med projektet var att utveckla småmolekyler med en biologisk effekt som motsvarar den hos de ursprungliga peptiderna. Information från studierna av peptiderna och de modifierade peptiderna i kombination med datorbaserad molekylmodellering gjorde att vi valde att använda ett 1,2,8-trisubstituerat 4-kinolonskelett som grund för dessa peptidmimetika och ett syntesprotokoll för sådana substanser utvecklades. Detta syntesprotokoll visade sig vara generellt tillämpbart och användes till att syntetisera en serie 1,2,8-trisubstituerade 4-kinoloner med variation i struktur och egenskaper. De syntetiserade substanserna studeras för tillfället för sin KLK3-stimulerande effekt

Multimethodological study of molecular recognition phenomena

The study of the bio-recognition phenomena behind a biological process is nowadays considered a useful tool to deeply understand physiological mechanisms allowing the discovery of novel biological target and the development of new lead candidates. Moreover, understanding this kind of phenomena can be helpful in characterizing absorption, distribution, metabolism, elimination and toxicity properties of a new drug (ADMET parameters). Recent estimations show that about half of all drugs in development fail to make it to the market because of ADMET deficiencies; thus a rapid determination of ADMET parameters in early stages of drug discovery would save money and time, allowing to choose the better compound and to eliminate any losers. The monitoring of drug binding to plasma proteins is becoming essential in the field of drug discovery to characterize the drug distribution in human body. Human serum albumin (HSA) is the most abundant protein in plasma playing a fundamental role in the transport of drugs, metabolites and endogenous factors; so the study of the binding mechanism to HSA has become crucial to the early characterization of the pharmacokinetic profile of new potential leads. Furthermore, most of the distribution experiments carried out in vivo are performed on animals. Hence it is interesting to determine the binding of new compounds to albumins from different species to evaluate the reliability of extrapolating the distribution data obtained in animals to humans. It is clear how the characterization of interactions between proteins and drugs determines a growing need of methodologies to study any specific molecular event. A wide variety of biochemical techniques have been applied to this purpose. High-performance liquid affinity chromatography, circular dichroism and optical biosensor represent three techniques that can be able to elucidate the interaction of a new drug with its target and with others proteins that could interfere with ADMET parameters

Polycationic multivalency: protein recognition and cell uptake via oligoguanidinium scaffolds

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 24-03-0

Computational approaches for identifying inhibitors of protein interactions

Inter-molecular interaction is at the heart of biological function. Proteins can interact with ligands, peptides, small molecules, and other proteins to serve their structural or functional purpose. With advances in combinatorial chemistry and the development of high throughput binding assays, the available inter-molecular interaction data is increasing exponentially. As the space of testable compounds increases, the complexity and cost of finding a suitable inhibitor for a protein interaction increases. Computational drug discovery plays an important role in minimizing the time and cost needed to study the space of testable compounds. This work focuses on the usage of various computational methods in identifying protein interaction inhibitors and demonstrates the ability of computational drug discovery to contribute to the ever growing field of molecular interaction. A program to predict the location of binding surfaces on proteins, STP (Mehio et al., Bioinformatics, 2010, in press), has been created based on calculating the propensity of triplet-patterns of surface protein atoms that occur in binding sites. The use of STP in predicting ligand binding sites, allosteric binding sites, enzyme classification numbers, and binding details in multi-unit complexes is demonstrated. STP has been integrated into the in-house high throughput drug discovery pipeline, allowing the identification of inhibitors for proteins whose binding sites are unknown. Another computational paradigm is introduced, creating a virtual library of -turn peptidomimetics, designed to mimic the interaction of the Baff-Receptor (Baff-R) with the B-Lymphocyte Stimulator (Blys). LIDAEUS (Taylor, et al., Br J Pharmacol, 2008; 153, p. S55-S67) is used to identify chemical groups with favorable binding to Blys. Natural and non-natural sidechains are then used to create a library of synthesizable cyclic hexapeptides that would mimic the Blys:Baff-R interaction. Finally, this work demonstrates the usage and synergy of various in-house computational resources in drug discovery. The ProPep database is a repository used to study trends, motifs, residue pairing frequencies, and aminoacid enrichment propensities in protein-peptide interaction. The LHRLL protein-peptide interaction motif is identified and used with UFSRAT (S. Shave, PhD Thesis, University of Edinburgh, 2010) to conduct ligand-based virtual screening and generate a list of possible antagonists from the EDULISS (K. Hsin, PhD Thesis, University of Edinburgh, 2010) compound repository. A high throughput version of AutoDock (Morris, et al., J Comput Chem, 1998; 19, p. 1639-62) was adapted and used for precision virtual screening of these molecules, resulting in a list of compounds that are likely to inhibit the binding of this motif to several Nuclear Receptors

2-Alquil-2-Carboxiazetidinas como inductores de giros inversos: análisis conformacional en péptidos modelo y aplicación a la sintesis de miméticos de Neurotrofinas

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Orgánica. Fecha de lectura: 19-11-200