Expression of TIMP-2 in HPV-16 infected oral squamous cell carcinoma in patients in Iraq

Aim: To determine the expression of tissue inhibitors of metalloproteinases (TIMP-2) in oralsquamous cell carcinoma (OSCC) and the difference in its expression level between positiveand negative HPV-16 (human papilloma virus- 16) OSCC patients. Methods: This study wasconducted on 33 biopsies obtained from patients with OSCC and 10 normal oral mucosa ascontrols. In situ hybridization (ISH) was used to investigate the presence of HPV-16, whileimmunohistochemistry (IHC) was used to estimate the expression level of TIMP-2. Results: TheTIMP-2 was expressed in 27 (81.8%) of OSCC sections with no significant difference betweenits expression level in HPV-16 positive and HPV-16 negative OSCC cases (p=0.058). TIMP-2was found to be highly expressed in OSCC sections, and the presence of HPV was not relatedto its overexpression. Conclusions: The percentage of samples that appeared to accommodatedetectable HPV-16 was high, but no significant difference was observed in relation to TIMP-2expression level. Future studies with a larger number of patients are highly recommended toaddress the possible association between TIMp-2 and OSCC positive HPV-16.

Aplicação de modelagem molecular e de formalismo do CAMD (Computer-Aided Molecular Design) na elucidação do mecanismo de ação de inibidores de metalopropteinases de matriz / Molecular modeling methods and computer-aided molecular design (CAMD) formalisms for elucidating the mechanism of action of matrix metalloproteinases inhibitors

As metaloproteinases de matriz (MMP) são enzimas superexpressas em quase todos os tumores humanos, sendo que os subtipos MMP-2 e MMP-9 têm sido associados ao potencial metastático e prognóstico desfavorável em neoplasias malignas como, por exemplo, melanoma metastático e glioma. Compostos capazes de inibir a atividade destas enzimas podem representar potenciais agentes terapêuticos. O composto 4-nerolidilcatecol (4-NC), isolado de plantas do gênero Pothomorphe, apresentou resultados promissores para o tratamento do melanoma e glioma e foi capaz de atuar em várias etapas bioquímicas importantes envolvidas na progressão dessas patologias, inclusive inibindo MMP-2 e MMP-9. No entanto, o mecanismo de ação do 4-NC não está completamente elucidado. O presente estudo envolveu a aplicação de métodos de modelagem molecular e de formalismos do planejamento de novas moléculas auxiliado por computador, CAMD (Computer-Aided Molecular Design) a fim de explorar a interação entre esta molécula e as enzimas MMP-2 e MMP-9, além de planejar novos inibidores para estes alvos. Análise exploratória de dados, que compreende a análise de agrupamentos hierárquicos e de componentes principais. foi desenvolvida para um conjunto de hidroxamatos (N=64) descritos como inibidores de MMP-2 e MMP-9, a fim de identificar as propriedades moleculares que mais influenciavam o processo de discriminação dos compostos. As propriedades termodinâmicas, eletrônicas e estéricas foram importantes para descrever os compostos mais ativos no conjunto de dados da MMP-2. Para a MMP-9, o coeficiente de distribuição (ClogD) em pH 1,5 foi relevante no processo de discriminação do conjunto. A presença de substituintes volumosos na porção R3 parece ser crucial para o conjunto de inibidores investigados. Esta região está envolvida em interações moleculares com a cavidade S1 de ambas as enzimas, mas há um limite de volume a ser considerado para estes substituintes. O formalismo QSAR-4D independente do receptor (IR) foi aplicado ao mesmo conjunto de dados e permitiu estabelecer o mapeamento do farmacóforo, além de explorar diferentes alinhamentos para a obtenção da hipótese de conformação bioativa prevista pelo melhor modelo de QSAR. OS modelos QSAR apresentaram boa capacidade de previsão, auxiliaram na proposição de novos inibidores e estimaram a atividade do 4-NC. Com o melhor modelo QSAR para MMP-9 (N=64), a atividade prevista para o 4-NC foi classificada na faixa dos inibidores com atividade moderada. Entretanto, o melhor modelo QSAR obtido para MMP-2 (N=38) não foi capaz de prever, de forma adequada, a atividade de compostos com arcabouço químico diferente daqueles utilizados na construção dos modelos. Estudos de ancoramento molecular foram desenvolvidos para investigar a orientação do 4-NC no sitio catalítico das duas enzimas e as interações que poderiam ser estabelecidas nestes complexos. Duas conformações favoráveis foram encontradas. Simulações computacionais de dinâmica molecular foram desenvolvidas com os complexos mais promissores selecionados nos estudos de ancoramento, a fim de obter informações mais detalhadas e de maior confiabilidade. sobre suas interações intermoleculares. O 4-NC tende a se orientar no sítio de forma a acomodar sua cadeia lateral no bolso S1 adjacente ao sítio catalítico em ambas as enzimas. Ensaios de zimografia também foram realizados com o objetivo de elucidar possíveis contribuições da cadeia lateral e do núcleo catecólico do 4-NC na atividade inibitória frente às enzimas em estudo. O núcleo catecólico parece ser o responsável por sua atividade, pois o composto 1,2dimetoxibenzeno, que possui as hidroxilas bloqueadas por grupos metil, não foi capaz de exercer atividade inibitória significante frente à MMP-2 e MMP-9. Estudos de voltametria reforçaram a hipótese de que o 4-NC tem a capacidade de quelar os íons zinco presentes no tampão de incubação

Matrix metalloproteinases (MMP) enzymes are overexpressed in almost all human tumors, and MMP-2 and MMP-9 subtypes have been associated with metastatic potential and poor prognosis in malignant tumors, such as metastatic melanoma and glioma. Compounds capable of inhibiting the activity of theses enzymes would be considered as potential therapeutic agents. The 4-nerolidylcatechol compound (4-NC), isolated from plants of genus Pothomorphe, has showed promising results in the treatment of melanoma and glioma, and was able to act in several important biochemical steps involved in the progression of these diseases, as well as inhibiting MMP-2 and MMP-9. However, the 4-NC mechanism of action is not completely understood. This study has involved the application of molecular modeling methods and formalisms of computer-aided molecular design (CAMD) in order to explore the interaction between 4-NC and MMP-2/MMP-9, and to design new inhibitors for these targets. Exploratory data analysis, which comprises hierarchical cluster analysis and principal components analysis, was performed to a set of hydroxamates (N=64). previously reported as MMP-2 and MMP-9 inhibitors, in order lo identify the molecular properties that is most critical for the discrimination process regarding the investigated compounds. The thermodynamic, electronic, and steric properties were: quite important to describe the highly active compounds in the data set of MMP-2, whereas the apparent partition coefficient (ClogD) at pH 1.5 was the property more relevant for MMP-9 data set. The presence of bulky substituents on the R3 moiety seems to be crucial for this set of inhibitors due to the molecular interaction with the S1 subsite of both enzymes. However, there is a limit regarding the substituents volume in this region. Receptor independent (RI) 4D-QSAR analysis was applied lo the same data set and it was possible to establish the pharmacophore mapping, besides to explore different alignments in order to generate the hypothesized bioactive conformation through the best QSAR model. The QSAR models have presented good predictability, assisted in proposing new inhibitors, and estimated the activity of 4-NC. Regarding the best QSAR model for MMP-9 (N=64), the 4-NC predicted activity was classified in the range of the moderate active inhibitors. The best QSAR model obtained for MMP-2 (N=38), however was not able to properly predict the activity for compounds with different chemical scaffold from those used to build up the QSAR model. Molecular docking studies have been developed to investigate the 4-NC binding mode into the catalytic site of the two enzymes and the interactions that could be established in those complexes. The results have shown two favorable conformers regarding the MMP inhibition. Molecular dynamics computational simulation were combined to molecular docking studies in order to obtain more detailed and reliable information regarding the intermolecular interactions of each complex. The 4-NC molecule tends to accommodate the side chain in the S1 pocket adjacent to the catalytic site in both enzymes. Experimental zymography assays were also performed to elucidate the possible contribution of the side chain and the catechol core in the 4-NC inhibitory activity against the MMP-2 and MMP-9 enzymes. The catechol core seems to be responsible for its activity, since the 1,2 dimethoxybenzene compound, which has the hydroxyl blocked by a methyl group, was not able to exert any significant inhibition on enzymes. Voltametric assays confirmed the hypothesis that 4-NC chelates zinc ions present in the incubation buffer

QSAR Rationale of Matrix Metalloproteinase Inhibition Activity in a Class of Carboxylic Acid Based Compounds.

The matrix metalloproteinase-13 (MMP-13) inhibitory activities of carboxylic acid based compounds, in presence and absence of bovine serum albumin (BSA), have been analyzed quantitatively in terms of chemometric descriptors. The statistically validated quantitative structure-activity relationship (QSAR) models obtained through combinatorial protocol in multiple linear regression (CP-MLR) analysis and the participated descriptors in these models provided rationales to explain the inhibitory activities of these congeners. For MMP-13 inhibition activity, the identified descriptors (BEHm1, BELm1 and BEHm8) have highlighted the role of the atomic mass in terms of the highest and lowest eigenvalues derived from Burden matrix. The positive correlation with activity suggested that their higher values are desirable in improving the activity of a compound. Additionally, the descriptor C-027 representing R-CH-X type fragment in a molecular structure advocates the absence of such type of fragment for the improved activity. On the other hand presence of RCO-N< or >N-X=X type fragment (descriptor N-072) would be beneficiary to the MMP-13 inhibitory activity. The structural features, rationalized by the descriptors MSD (Balaban’s mean square distance index), nCrHR (number of ring tertiary C (sp3), H-047 (H attached to C1(sp3)/C0(sp2)) and H-050 (H attached to heteroatom) have imparted positive impact on the MMP-13 w/BSA inhibition activity. The atomic properties such as atomic polarizability and atomic Sanderson’s electronegativity have shown their positive impact on the activity via descriptors BELp4 and GATS3e in respective eigenvalues or lag. The other descriptors, MATS1m and MATS3e, have revealed the negative influence of atomic mass and electronegativity on the of MMP-13 w/BSA inhibition activity. The results obtained from CP-MLR analysis have been supported further through partial least-squares (PLS) study.