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Development of a small-molecule antiviral targeting the HIV-1 matrix protein

By
Peter Fedichev, Quantum CTO
– March 28, 2013Posted in: collaboration, drug design, HIV, matrix proteins

Due to the emergence of drug-resistant strains and the cumulative toxicities associated with current therapies, demand remains for new inhibitors of HIV-1 replication. The HIV-1 matrix (MA) protein is an essential viral component with established roles in the assembly of the virus. Using virtual and surface plasmon resonance (SPR)-based screening, we describe the identification of the first small molecule to bind to the HIV-1 MA protein and to possess broad range anti-HIV properties.

We have identified a novel compound (7) that binds to and functions through the conserved PI(4,5)P2 binding site on HIV-MA, inhibits the production of new virus, and exhibits broad spectrum anti-HIV activity with IC50 values in the range of 7.5 to 15.6 mm for the group M isolates tested. This compound provides a novel chemical probe to investigate the roles of MA in the HIV-1 replication cycle and could serve as a good starting point for the development of a new class of HIV therapeutics through optimization by medicinal chemistry approaches.

To identify this compound, we undertook a structure-guided search for small, drug-like molecules that inhibit viral replication by binding to this highly conserved area on the HIV-1 MA protein by using  a virtual screening protocol.

First, we tested whether the identified compounds could interact with MA using surface plasmon resonance (SPR). And oone of the best compounds interacts with HIV-1 MA with an equilibrium dissociation constant of 171 μM. To identify other potential hits that were not identified in the SPR-based screen, but still represent matrix-targeted inhibitors of HIV-1 replication, we evaluated the 19 compounds identified from the initial virtual screen. This led to the discovery of the novel antiviral compounds, as well as confirmation of the first compound as a genuine hit. The lead compound from this analysis, compound 7, inhibited the replication of the primary isolate HIV-189BZ167 with the lowest IC50 value 12.3 μM, showed minimal toxicity(>100 μM), and did not inhibit the replication of a panel of non-retroviruses. Compound 7 inhibited the replication of all the group M viruses tested (panel of HIV-1 clinical isolates from different geographic locations that included HIV-1 group M subtypes A–G).

To explore the interaction of compound 7 with HIV-1 MA, we employed SPR. Compound 7 interacts with HIV-1 MA with an equilibrium dissociation constant (KD) of 22.6 μM (Figure 1) This value is in remarkable agreement with the IC50 value derived from the single-round infection assay (IC50 21.2 μM).

Figure1

Sensorgrams depicting the interaction of compound 7 with immobilized HIV-1lAI MA. Compound concentrations of 0, 6.25, 12.5, 25, 50, and 100 mm were tested and are represented as orange, magenta, green, blue, cyan and red lines, respectively. The insert shows the equilibrium plot used to derive the equilibrium dissociation constant (KD). Compound 7–MA interaction: KD=22.6 uM.

 

The effect of compound 7 on the production of both wild-type (WT) and the L21A and T81A mutant viruses was assessed by p24 content in
the supernatants after 48 h. Compound 7 decreased the amount of WT virus produced to approximately 392.5% of the control with no compound (DMSO only). However, changing residues L21 and T81 to alanine completely abrogated the effects of compound 7.

We next sought to refine the docking model and to identify the most probable binding mode of compound 7 to see whether or not the model could offer any insight into the mutational results.Exhaustive dockings to multiple conformations of HIV-1 MA resulted in the binding pose shown in Figure 2.

Figure 2

Refined predicted binding mode of compound 7 in the PI(4,5)P2 binding site of HIV-1 MA. Binding site residues (L21, R22, W36, R76, T81, and K98) are represented as dark sticks, whereas compound 7 is shown in green sticks. Residues highlighted in blue are thought to be critical for compound binding. Yellow dashed lines represent hydrogen bonds

The results were published in two papers:

  • Bioorg Med Chem Lett. 2012 Nov 29. pii: S0960-894X(12)01493-X. doi: 10.1016/j.bmcl.2012.11.041. [Epub ahead of print] Discovery of a small-molecule antiviral targeting the HIV-1 matrix protein. Zentner I, Sierra LJ, Maciunas L, Vinnik A, Fedichev P, Mankowski MK, Ptak RG, Martín-García J, Cocklin S. [PMID:23305922]
  • ChemMedChem 2013 [Epub ahead of print] Identification of a small-molecule inhibitor of HIV-1 assembly that targets the phosphatidylinositol (4,5)-bisphosphate binding site of the HIV-1 matrix protein. Zentner I, Sierra LJ, Maciunas L, Vinnik A, Fedichev P, Mankowski MK, Ptak RG, Martín-García J, Cocklin S. [Manuscript number: cmdc.201200577]
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Quantum seeks great medicinal chemist!

By
Peter Fedichev, Quantum CTO
– July 9, 2012Posted in: Uncategorized

Компания ООО “Квантум Фармасьютикалс” ищет медицинского химика на полную ставку, проживающего в Москве или в МО. Возможна работа в качестве эксперта по контракту для специалистов, проживающих в других городах.

Требования:

1. Интерпретация полученных результатов биологическимх испытаний, отбор хитов, участие в последующих этапах – hit to lead, lead optimization.

2. Проведение экспертизы, отбор молекул химических соединений для биологических испытаний (фильтрация-отбор по критериям ADME-Tox свойства, патентноспособность и др.).

3. Хорошее владение современными средствами био- хемо-информатики, компьютерной химии и химических интернет-ресурсов.

4. Наличие научных публикаций, патентов, ученой степени.

Уровень зарплаты – высокий.

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Посылайте свои резюме на andrei.vinnik@q-pharm.com,
тел.: +7(499)156 1561

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In-vitro study of DLX5 Ligands

By
Gulnara Yamanbaeva
– January 26, 2012Posted in: cancer

The manuscript entitled “Transcription Factor DLX5 As a New Target for Promising Antitumor Agents” by the Quantum Team.

The reference:

R. A. Timakhov1,2,3*, P. O. Fedichev1, A. A. Vinnik1, J. R. Testa3, O. O. Favorova2
1 Quantum Pharmaceuticals, Russia
2Pirogov Russian National Research Medical University
3 Fox Chase Cancer Centre, Philadelphia, USA
E-mail: timakhov@gmail.com
Received 11.05.2011
Copyright © 2011 Park-media, Ltd. This is an open access article distributed under the Creative Commons Attribution License,which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In the present study we used the earlier characterized line 42 of T-cell lymphoma from Akt2-transgenic mice (42-936, 42-577, and 42-588) and line 72 (wtl36). Screening for the new ligand molecules specific to DLX5 has been carried out with The QUANTUM software suite, based on the analysis of the protein crystal structure. The search was made more complicated by the absence of preliminary data on the binding of the known compounds with DLX5 protein; therefore, blind studies were performed. The best molecules and all their structural analogues from the original ENAMINE library were sorted on the basis of their predicted binding energy. According to the results of molecular docking, 100 ligands were selected; 14 of those with the best predicted binding energy of DLX5 protein were ordered and synthesized at ENAMINE company; then, they were tested on cell cultures. The cells of the earlier characterized line 42 of T-cell lymphoma from Akt2-transgenic mice were used as a model to verify the specific activity of the selected ligands. As can be seen in Fig. 1, the ligands demonstrate different efficacies of impact on the proliferation of lymphoma cells; compounds Q8, Q12, Q9, and Q13 manifested the best inhibitory activity.

Fig.1 Experimental estimation of the properties of 14 compounds, for which high binding energy for the 3D structure of DLX5 was predicted: their efficacy was estimated by the impact on proliferation of Dlx5 positive mouse lymphoma cells 42-936

The possible nonspecific cytotoxic action of the selected compounds was tested on normal human ovarian epithelial cells without DLX5 expression. When comparing with the control, it can be seen that most ligand molecules, with the exception of the compounds Q8 and Q13, manifest no significant cytotoxicity. Since Q8 and Q13 manifested a cytotoxic effect, they were eliminated from further consideration. Compounds Q12 and Q9 were selected for further studies as the most promising ones.

In order to eliminate the possibility of a nonspecific impact of compounds Q12 and Q9 on cells of the lymphoid series, their action was tested on T-cell lymphoma cells of line 72 with absent expression of Dlx5 from Akt2-transgenic mice . Than we check the effect of compound Q12 on the proliferation of an additional two subtypes of lymphoma cells expressing Dlx5 (42-577 and 42-588) , as well as the proliferation of the human lymphoma cells Jurkat and Molt16 not expressing DLX5.

It is known that the DLX5 transcription factor can directly control the expression of protooncogene c-myc. The impact of Q12 on the expression of c-myc in the lymphoma cells 42-936 expressing Dlx5 was studied by real-time RT- PCR. Figure 2 shows the levels of mRNA of c-myc with respect to the endogenous control, mRNA of TATA-binding protein (Tbp) or mRNA of Dlx5, as well as mRNA of Dlx5 with respect to mRNA of Tbp in the presence of 10 μM Q12 and without any addition of it. It can be seen that the expression of c-myc decreases considerably under the action of Q12, while the expression of Dlx5 remains intact. These results agree with the conception of the inhibitory effect of ligand Q12 on the transcription activity of the Dlx5 factor. Although these data need to be tested on a larger number of cell lines, it is tempting to make a preliminary conclusion on the specificity of binding between the transcription factor DLX5 and ligand Q12 based on the results of this study.

Fig.2 Real-time RT-PCR. The measurement of the expression of c-myc and Dlx5 in mouse lymphoma cells 42-936, after cultivation with 10 μM compound Q12 by the mRNA level. Tbp is the TATA-binding protein.
The approaches used in this study made it possible to experimentally identify the most active inhibitors of Dlx5 (DLX5) out of those that were tested. Further plans include optimizing the structure of the resulting compounds in terms of parameters such as the enhancement of efficacy, reduction of possible nonspecific toxicity, and the enhancement of the metabolic stability. The next stage of this study assumes that the activity and toxicity of the optimized compounds will be assessed in vivo, and their Kd will be measured directly. The compounds discovered are the first described low-molecular-weight ligands of DLX5 which can be used for subsequent chemical optimization and the development of highly efficient anti-tumor agents.

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High Throughput Screening of Transcription factor DLX5

By
Gulnara Yamanbaeva
– January 26, 2012Posted in: cancer

The manuscript entitled “Transcription Factor DLX5 As a New Target for Promising Antitumor Agents” by the Quantum Team

The reference:

R. A. Timakhov1,2,3*, P. O. Fedichev1, A. A. Vinnik1, J. R. Testa3, O. O. Favorova2
1 Quantum Pharmaceuticals, Russia
2Pirogov Russian National Research Medical University
3 Fox Chase Cancer Centre, Philadelphia, USA
E-mail: timakhov@gmail.com
Received 11.05.2011
Copyright © 2011 Park-media, Ltd. This is an open access article distributed under the Creative Commons Attribution License,which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Transcription factor DLX5 is a new target for promising antitumor agents.

The High Throughput Screening

The overexpression of the DLX5 gene in mammalian cells stimulates cell proliferation and can be observed in endome-trial carcinoma, non-small cell lung cancer (nScLc), and small cell lung cancer. The knockdown of the DLX5 expression using sirnA in mouse and human cancer cells results in the arrest of cell proliferation.

New data point to the fact that DLX5 has a direct effect on the expression of protooncogene c-myc. These facts allow us to regard DLX5 as a promising target for which specific ligands that have the properties of oncogenesis inhibitors can be found.

Attempts have frequently been made to use the so-called “high throughput screening” to solve the problem of the search for the ligands of a certain protein. This screening is carried out on a cell culture or on an in vitro model, using an earlier prepared compound library.

The logistics and cost of the studies required for the experimental validation of a significant number of molecules is prohibitively high in many cases. On account of these reasons, in the present study we used the algorithm earlier elaborated to search for inhibitors of new protein targets, based on the analysis of the crystal structure of a target protein. the algorithm is based on the molecular docking of chemical compounds to the known 3D model of a target protein, which predicts the possible position of a compound in the protein–ligand binding site, the calculation of the molecular dynamics being used to refine the binding energies for the best suiting compounds.

Fig. 1. Molecular docking of one compound selected for further experiments: chemical structure and position of compound in the active site the transcription factor DLX5.

Ligand preparation and molecular docking

In order to optimize the time of computational screening, the ENAMINE library consisting of 106 compounds was clustered using the Jarvis–Patrick algorithm with acceleration, which is contained in the QUANTUM software package. The so-called tanimoto metric was calculated using the Daylight molecular fingerprints, which were selected as the measure of molecular similarity. The parameters of clusterization were selected in such a manner that each cluster consisted, on average, of approximately 10 related structures; the total number of non-clustered molecules being no higher than 20% of the initial amount of the library compounds. the compounds representing the centroids of clusters were then selected for further screening. In order to enhance the speed of molecular docking, from the entire centroid library were selected the molecules with the low molecular weight.

All the selected compounds were extracted from the sdf files provided by ENAMINE and processed in the batch-mode. The library had not been additionally enriched with molecules active towards oncotargets or by any other methods. the typization of protein and ligands, as well as in silico screening, was carried out using the corresponding tools from the QUANTUM software package.

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MA/p17 HIV matrix protein is a promising target for drug discovery

By
Peter Fedichev, Quantum CTO
– December 20, 2011Posted in: Uncategorized

As a result of the analysis of HIV virus targets, the most conservative among all the possible HIV-1 virus target protein—p17/MA (see figure)—was selected. This is a matrix (structural) protein, the highly conservatism of which is determined by the so-called lego-effect: the inability of the viral capsid to to assemble from the matrix proteins even negligibly modified by medication or mutations. Other well-known targets of the human immunodeficiency virus, for example reverse transcriptase and protease, do not possess sufficient active conservative sites.

3D representation of the p17/MA protein. The red surface area is more than 99% conservative among all types of virus

To demonstrate the mechanism of action (MOA) in the United States, a direct SPR experiment was conducted to confirm the direct binding of our protein MA inhibitors with the HIV virus. Experimental research on inhibitors of all types A-F and O of the HIV virus confirmed that the high conservatism of the selected protein binding site provides the same ability to inhibit the replication of all the virus variants tested. As expected, the inhibition of the matrix protein leads to the suppression of entry (the so-called Early Event, see figure), and to the suppression of the assembly of virus particles in already infected cells (known as Late Event). This means that the inhibitors of protein p17/MA affect not one, but two major stages of the life cycle of the virus.

Schematic view of HIV virus life cycle.
The HIV matrix protein is similar to the matrix proteins of other retroviruses. The company has received experimental data on the activity of the molecules under study on the SIV, MLV, FIV, HTLV-1 and EIAIV virus.
The Company has considerable experience in developing drug candidates acting on the matrix proteins of viruses. In particular, by analogy with the HIV virus, we developed and are conducting tests on molecules acting on the M1 matrix protein of the influenza virus (the functional analogue of the HIV virus protein p17/MA). Accumulated data from our studies in vitro and in vivo confirms the high efficiency and the low toxicity of medicines (drugs) acting on matrix proteins. This lets us claim with the confidence that the proposed HIV inhibitors will provide a good therapeutic and commercial value on the market of drugs against HIV/AIDS.
The development of inhibitors of other proteins that make up the viral matrix as a treatment for HIV is already well proven in preclinical trials. The companies H-phar and Biotron have received permission and are currently conducting 2nd phase clinical studies of the molecules that bind to another matrix protein, p7 of the HIV virus, responsible for the formation of the internal (nuclear) matrix of the virus. All compounds in this group engage Zn, which is contained in protein, and as such may well interact with any other human protein containing Zn ions. This may lead to side effects and toxicity, which is well recognized by the developers of the molecules (prof Apellia, h-index> 110).
In order to identify the competition with academic groups and companies in the biotechnology sector working in the field of the inhibition of HIV p17/MA matrix proteins, a search for information in commercial databases (www.Medtrack.com) and open sources was conducted. Information on inhibitors of viral protein p17/MA undergoing preclinical and clinical trials against HIV/AIDS was not found.
The Company submitted a joint article with Simon Cocklin of Drexell Medical College (a collaborator on p17/HIV-1). In preparation for the publication, a search for literature was conducted, which revealed that a number of small molecule inhibitors of the matrix protein p17/HIV were developed in the group of M. Bukrinsky (h-index ~40). However, all inhibitors published so far were 100-1000 times weaker than those under development by the researchers at Quantum.
Reasons for the interest of consumers in the products and (or) technology description
The drug based on the inhibition of the matrix protein, developed by Quantum LLC, may be of interest to Russian consumers for several reasons:
1. By design the compounds should have the drug resistance development times substantially reduced;
2. Low level of toxicity;
3. High level of the drug-effectiveness, good efficacy against all the types of the HIV virus.

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